Trans-lycopene is a functional phytochemical abundant in red-fleshed watermelons, and its contents vary among cultivars. In this study, the genetic basis of high trans-lycopene contents in scarlet red flesh was evaluated. Three near-isogenic lines (NILs) with high trans-lycopene contents were derived from the scarlet red-fleshed donor parent DRD and three coral red-fleshed (low trans-lycopene contents) recurrent parents. The lycopene contents of DRD (589.4 ± 71.8 µg/g) were two times higher than that of the recurrent parents, and values for NILs were intermediate between those of the parents. Coral red-fleshed lines and F1 cultivars showed low trans-lycopene contents (135.7 ± 18.0 µg/g to 213.7 ± 39.5 µg/g). Whole-genome resequencing of two NILs and their parents and an analysis of genome-wide single-nucleotide polymorphisms revealed three common introgressed regions (CIRs) on chromosomes 6, 9, and 10. Twenty-eight gene-based cleaved amplified polymorphic sequence (CAPS) markers were developed from the CIRs. The CAPS markers derived from CIR6 on chromosome 6, spanning approximately 1 Mb, were associated (R2 = 0.45–0.72) with the trans-lycopene contents, particularly CIR6-M1 and CIR6-M4. Our results imply that CIR6 is a major genomic region associated with variation in the trans-lycopene contents in red-fleshed watermelon, and CIR6-M1 and CIR6-M4 may be useful for marker-assisted selection. Citrullus lanatus var. lanatus; 2n = 2x = 22) is a desert plant native to Africa and a widely cultivated vegetable fruit crop in temperate climates. Watermelon production in 2019 was more than 32 tons per hectare, and more than 5790 kilotons were produced worldwide, making it an economically important crop . The flesh of watermelon is rich in sugars, such as glucose, fructose, and sucrose, and various nutrients, including carotenoid components, vitamins , and minerals , along with a large amount of water. In addition to hydration, it has great nutritional value [Watermelon . Differences in flesh color correspond to the differences in the composition of carotenoids and the relative amount of each carotenoid component ,5. In reotenoids ,7,8. Redotenoids ,5,10. Fotermelon .CRISTO) gene on chromosome 4 [LCYB) gene on chromosome 2 are responsible for the accumulation of trans-lycopene in red flesh [LCYB is detected in yellow flesh. However, to the best of our knowledge, the genetic mechanisms or genes that regulate trans-lycopene levels in scarlet- and coral red-fleshed watermelons have not been reported.Variation in the flesh color and carotenoid components is directly regulated by variation in genes encoding the enzymes involved in the carotenoid biosynthetic pathway. The accumulation of pro-lycopene in orange flesh is related to mutations in the carotenoid isomerase  are often used in identifying trait-related genes using genetic association studies in various crops, such as rice, soybean, and wheat ,14,15. Ntrans-lycopene contents between coral red- and scarlet red-fleshed watermelon varieties. In particular, an introgressed region associated with high trans-lycopene contents was identified based on full-length genome analysis of NILs. A set of molecular markers within this region was developed for marker-assisted selection for high lycopene (HL) contents.Therefore, in this study, genetic data were obtained to clarify the molecular mechanism underlying variation in 1 hybrids also showed lycopene contents of between 135.7 ± 18.0 and 213.7 ± 39.5 µg/g. Overall, the lycopene contents of scarlet red-fleshed cultivars, NILs, and coral red-fleshed cultivars differed significantly  were subjected to next-generation sequencing (NGS) . The numA total of 213,920 SNPs and 11,695 InDels were detected in the comparison between DRD and SBA. Among these variants, 12,750 SNPs and 498 InDels alleles for DRD were detected in DRDSBA. In addition, among the 216,329 SNPs and 11,606 InDels detected between DRD and 45NC, 13,864 SNPs and 665 InDels alleles for DRD were detected in DRD45NC.The number and genomic distribution of these SNPs on each chromosome are shown in The CIRs in the DRD genome, which were found in both DRDSBA and DRD45NC NILs, were detected at approximately 24.0–24.9 Mb on Chr. 6 (CIR-6), 1.6–3.9 Mb on Chr. 9 (CIR-9), and 29.4–30.0 Mb on Chr. 10 (CIR-10) . In CIR-R2) = 0.46–072 at p < 0.05), with the highest values for CIR6-M1 and CIR6-M4  responsible for high trans-lycopene contents in DRD may be located on CIR6.SNPs in genes in the CIRs were selected to ensure an even genomic distribution, and CAPS markers were designed. A total of 17 SNPs within CIR-6, 18 SNPs in CIR-9, and 6 SNPs in CIR-10 were converted to CAPS markers and evaluated using four parents of the NILs. Unambiguously polymorphic PCR bands were observed for 26 CAPS markers , althoug CIR6-M4 . The R2 GGPP), phytoene synthase (PSY1), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene β-cyclase (LCYB-cyclase), lycopene α-cyclase (LCYE), nine-cis-epoxycarotenoid dioxygenases (NCED), β-carotene hydroxylase (CHYb), zeaxanthin epoxidase (ZEP), and violaxanthin de-epoxidase (VDE), were compared among DRD, SBA, and 45NC. The CDS sequences of these genes were highly conserved among the cultivars, and only the following SNPs were found: one nonsynonymous SNP in PSY1 (Cla97C01G024630), one nonsynonymous and two synonymous SNPs in PDS1 (Cla97C017G142100), one nonsynonymous and one synonymous SNP in PDS2 (Cla97C08G151960), and one synonymous SNPs in NCED (Cla97C07G137260) (data not shown). Among the three nonsynonymous SNPs, only one in PDS1 discerned DRD from SBA and 45NC. None of these 11 genes were located in the CIRs, except PDS (Cla97C09G165530), which was found in CIR9.The CDS of 16 genes  in the wR2 values in CIR6 included genes related to ion movement, endopeptidase activity, and lipid and fatty acid biosynthesis.The CIR6 region harbored 106 annotated genes but did not include watermelon lycopene biosynthesis genes. Genes located in the vicinity of two CAPS markers with the highest Lycopenes are major functional compounds in red-fleshed watermelons and have a major influence on fruit quality, such as glucosinolate in Chinese cabbage ,25, capscis-lycopene) and trans-lycopene, which are abundant in orange-colored flesh and red flesh, respectively [CRTISO are a limiting factor for the conversion of pro-lycopene to trans-lycopene and cause the accumulation of pro-lycopene in orange-fleshed watermelon fruits [LCYB and CHYB. In the early development stage, the expression levels of these two genes increase significantly in scarlet red fruits and remain stable in coral red watermelons [The main lycopene components in watermelon are pro-lycopene  developed from an introgressed region at 24–25 Mb on Chr. 6 were correlated with the trait.In this study, we identified a genomic region associated with high scrY) responsible for scarlet red and coral red flesh was mapped to approximately 16.75–23.79 Mb on Chr. 6, which is approximately 300 kb upstream of CIR6. Two InDel markers (InDel27_fc6 and InDel28_fc6) were developed for the candidate genes within the major QTL region [trans-lycopene in DRD.In a previous study , a majorL region . In our Although NILs have the target trait of the donor, they show a nearly identical genomic composition to that of the recurrent parent. Therefore, NILs are suitable for discovering genes or QTLs involved in the target trait by analyses of regions of introgression of the donor’s chromosomal segment ,42. Comptrans-lycopene contents than those of the coral red-fleshed cultivars [trans-lycopene contents were intermediate between those of the donor and recurrent parents. Lower trans-lycopene contents in NILs than in the donor parent may be because of the fact that genetic factors involved in lycopene accumulation were not fully recovered in these NILs. In addition, we observed that all F1 plants from crosses between scarlet red- and coral red-fleshed cultivars showed substantially lower lycopene contents than that of the HL parental line, indicating that the HL contents of DRD are not controlled by a single dominant or recessive allele. Therefore, differences in the accumulation of trans-lycopene between scarlet red and coral red flesh may be determined by a complex genetic regulatory mechanism. Dou et al. [Of the 20 cultivars in this study, the scarlet red-fleshed cultivars showed higher ultivars ,38. Inteu et al.  revealedtrans-lycopene contents). Our results indicated that a 3 Mb introgressed region on Chr. 6 is associated with high trans-lycopene contents in scarlet red-fleshed watermelon. The CAPS markers developed from this region of introgression were correlated with the accumulation of trans-lycopene and can be useful for marker-assisted selection.In conclusion, our WGRS analysis of two NILs and the identification of CIRs revealed the genomic location responsible for the target trait in this study  using the conventional greenhouse cultivation method to set a single fruit per individual plant. Seeds were sown on a plastic 50-cell tray filled with the bed soil. Seedlings were grafted at the 3–4 true leaf stage with the rootstock Sintoza. At 15 days after grafting, 5–7 healthy seedlings per cultivar were transplanted at 30 cm intervals in the field soil of a plastic film greenhouse. Before transplanting, 2 tons of organic compost and 60 kg of N/P/K were applied to the field soil as a basic fertilizer. During the cultivation period, additional fertilization was performed by applying nitrogen fertilizer and liquid compound fertilizer three times, potassium twice, and phosphoric acid once. Water management was carried out by supplying three tons of water per 500 mFive fruits were harvested for each cultivar and used for the lycopene contents analysis using high-performance liquid chromatography (HPLC). The central part of the cross-section of the harvested fruit was cut at a thickness of approximately 3 cm, the flesh part was cut into 3 × 3 cm cubes, and the seeds were removed. Then, the flesh samples were put in a zip-lock bag, immediately frozen at −80 °C, and dried with a freeze dryer .2SO4 was added and passed through a filter  to prepare the final analysis sample.For sample pretreatment, the freeze-dried pulp was crushed finely, and 0.1 g was added to a 50 mL screw-top tube with beads at the volume ratio of 1:1. Then, 1 mL of ethanol containing 0.5 mM butylated hydroxytoluene was added and shaken vigorously for 2 min and 30 s. The ethanol, samples, and beads in the tube were all transferred to a 15 mL tube, and the tube was washed three times with 1 mL of acetone and transferred to a 15 mL tube. Then, 3 mL of 15 mL petroleum ether was added and vortexed, followed by the addition of 8 mL of 20% NaCl and vortexing. After centrifugation at 3000 rpm for 10 min, only the supernatant was picked up, and after mass-up, NaTrans-lycopene was used as a standard product for quantification , and the contents of lycopene were quantified by measuring absorbance at 289 nm, 450 nm, and 660 nm. The statistical analysis of the lycopene contents was performed using SPSS version 25.0  to verify the differences among the varieties at a 5% significance level using one-way ANOVA and Duncan’s multiple range test.For the quantitative analysis of lycopene, an HPLC system  equipped with a reverse-phase column  was used. For mobile phase A, 78% methanol was used, and 100% ethyl acetate was used for phase B. Separation conditions were as follows: 0–8 min, 70% B; 8–10 min, 60% B; 10–12 min, 100% B; 12–14.01 min, 0% B; 14.01–20 min, 100% B, and the flow rate was 1 mL per min. http://cucurbitgenomics.org) using BWA (0.6.1-r104) [Genomic DNA was extracted from the donor parent DRD, recurrent parents SBA and 45NC, and their two NILs using CTAB from young leaves collected during the seedling period, following the methods described by Hwang et al. .The extr.1-r104) . Raw SNP.1-r104) , and onl.1-r104) . The filThe introgressed regions in each NIL were detected based on SNPs and InDel loci derived from the donor parent DRD. Only homozygous SNPs satisfying depth ≥ 3 were used for the analysis. The full-length genome sequence of each NIL was divided by a window size of 1 Mb, and the SNP/InDel loci derived from the donor were plotted using GGT2 Graphical Genotypes . The introgressed regions commonly detected in both NILs were identified as candidate regions harboring gene(s) related to HL contents for further analyses.Homozygous SNPs detected in CIRs and distributed at relatively regular intervals were selected. These SNPs were converted into CAPS markers using their flanking sequences. Primer3 (version. 0.4.0) and NEB Cutter (version 2.0) were used for CAPS marker design.For CAPS marker genotyping, gDNAs of 20 cultivars were extracted as described in R2 between the marker genotype and lycopene contents was determined using a simple linear regression analysis at the 5% significance level with SPSS (version 25.0).The The coding sequences of 16 genes involved in the lycopene biosynthesis pathway were compared between the donor parent DRD and recurrent parents SBA and 45NC. Multiple sequence alignment and allele variant detection were performed using ClustalW. The list of genes located, the region of introgression was annotated based on information for the reference assembly 97103 v2.


Homozygous recessive deleterious mutations can cause embryo/fetal or neonatal lethality, or genetic defects that affect female fertility and animal welfare. In livestock populations under selection, the frequency of such lethal mutations may increase due to inbreeding, genetic drift, and/or the positive pleiotropic effects of heterozygous carriers on selected traits.By scanning the genome of 19,102 Lacaune sheep using 50 k single nucleotide polymorphism (SNP) phased genotypes and pedigree data, we identified 11 Lacaune deficient homozygous haplotypes (LDHH1 to LDHH11) showing a highly significant deficit of homozygous animals ranging from 79 to 100%. These haplotypes located on chromosomes 3, 4, 13, 17 and 18, spanned regions from 1.2 to 3.0 Mb long with a frequency of heterozygous carriers between 3.7 and 12.1%. When we compared at-risk matings (between carrier rams and daughters of carrier rams) and safe matings, seven of the 11 haplotypes were associated with a significant alteration of two fertility traits, a reduced success of artificial insemination , and/or an increased stillbirth rate . The 11 haplotypes were also tested for a putative selective advantage of heterozygous carrier rams based on their daughter yield deviation for six dairy traits . LDHH1, 3, 4, 5, 7, 9 and 11 were associated with positive effects on at least one selected dairy trait, in particular milk yield. For each haplotype, the most probable candidate genes were identified based on their roles in lethality of mouse knock-out models and in mammalian genetic disorders.Based on a reverse genetic strategy, we identified at least 11 haplotypes with homozygous deficiency segregating in French Lacaune dairy sheep. This strategy represents a first tool to limit at-risk matings in the Lacaune dairy selection scheme. We assume that most of the identified LDHH are in strong linkage disequilibrium with a recessive lethal mutation that affects embryonic or juvenile survival in sheep but is yet to be identified.The online version contains supplementary material available at 10.1186/s12711-021-00634-1. Most of the individuals in a population are likely to be heterozygous for several loss-of-function mutations. When these mutations are homozygous, they very often lead to early embryo death , or to developmental defects that affect fetuses and subsequently young individuals with varying degrees of severity . AdvanceIn livestock under selection, the effective size of populations (Ne), which is used as an indicator of genetic diversity, is limited Ne ~ 100–300) compared with that of the human population ; and as a result, the number of reproducers is rather small, particularly given the widespread use of artificial insemination (AI) , 4. Even00–300 coIn recent decades, several genomic tools have been developed to help improve fertility in dairy cattle . Among tCompared to cattle, the management of genetic diversity in dairy sheep takes advantage of their more local selection and breed management and of the use of a wider range of rams to produce fresh semen during a short reproductive campaign (May to August) . For exaIn order to discover such mutations, a reverse genetic screen method was applied to the large genome-wide SNP dataset available from a genomic selection program in Lacaune dairy sheep. The specific objectives of this study were to identify haplotypes with a deficit of homozygous animals, to test the hypothesis of a negative impact of these haplotypes on fertility traits in the case of at-risk matings, to test their putative pleiotropic effects on milk production traits, and to propose candidate genes that could harbor the causal mutations.Confédération Générale de Roquefort . Table The genotyped dairy Lacaune animals . The second pattern corresponds to LDHH that have an impact on both AIS and SBR and that we associate with embryo/fetal loss throughout the gestation period, i.e. LDHH8, 9 and 10 on OAR18. In this case, we suspect a unique lethal mutation that affects one of the four following candidate genes FAH, ARNT2, MPI and STRA6, which are reported to be homozygous lethal in knock-out mouse models . The third pattern groups LDHH3 and LDHH6 on OAR3 and affects only SBR. We assume that these LDHH harbor lethal mutations with effects that occur at the end of gestation and/or very soon after birth. Within the LDHH3 region in complete homozygous deficiency, the CAD gene is the most obvious candidate. Indeed, invalidation of the Cad gene in mouse causes preweaning lethality with complete penetrance [MGI:1916969], and a missense mutation in CAD  associated with the NH7 homozygous deficient haplotype [OMIA 002201-9913] causes late abortion during the last months of gestation in Normande cattle . Among these, a mutation in CCDC65 encoding a nexin-dynein regulatory complex, is reported to cause “ciliary dyskinesia, primary 27” , which is a neonatal respiratory distress syndrome in humans   and Strae OMIA 0001-9913 cn humans . Interesay cilia . Finallyay cilia  and in hsuppressor of cytokine signaling 2 (SOCS2) gene in Lacaune dairy sheep and is associated with sensitivity to mastitis [SOCS2, had positive effects on milk production, on fat and protein yields, but negative effects on LSCS and fat content, as already observed for the C variant of the p.R96C mutation. Since 2017, genotypes for OAR3:129722200C>T are available for all animals genotyped on the LD chip when candidate rams are selected to enter the breeding center. Although LDHH4 and LDHH5 are not in linkage disequilibrium with LDHH11, all three are associated with the SOCS2 mutation. Indeed, 69% of the LDHH4, 92% of the LDHH5 and 97% of the LDHH11 heterozygous carriers were C/T for the SOCS2 mutation but only 16%, 18% and 23% of the heterozygotes for the SOCS2 mutation carried one of these haplotypes, respectively . This could be explained by the fact that the SOCS2 mutation is carried by two different haplotypes, one in strong linkage disequilibrium with LDHH4–5 and the other with LDHH11. Thus, linkage disequilibrium with the SOCS2 mutation may explain the observed effect of LDHH4–5 and LDHH11 on milk traits. However, OAR3:129722200C>T in SOCS2 is not a candidate lethal mutation associated with these haplotypes that could explain their deficit of homozygotes. We hypothesize that a recessive lethal allele located in one of those haplotypes is in partial linkage disequilibrium with the mutant allele of SOCS2. Maintaining frequencies of LDHH4–5 (4%) and LDHH11 (7%) in the Lacaune population would then not be explained by a heterozygous advantage of deleterious mutations but by genetic hitchhiking due to SOCS2 and its association with selected dairy traits [Particular attention should also be focused on OAR3 and the region spanned by LDHH4–5 and LDHH11. These haplotypes are located in the region of the p.R96C mutation  that has been previously detected in the mastitis . InteresIn this study, we demonstrated the possible segregation of recessive lethal alleles in a sheep population by applying a reverse genetic screen approach on a large genotyping dataset and searching for homozygous haplotype deficiency. Among the 11 LDHH genomic regions detected, we hypothesize that at least eight independent recessive mutations cause early embryonic loss, peri/neonatal lethality or severe health defects in young lambs. We identified the most obvious candidate genes that are assumed to be altered by these mutations, and provide strong working hypotheses to identify them by whole-genome sequencing of heterozygous carriers of the different LDHH. Identification of causal mutations, and of the corresponding altered genes, is important to accurately identify the phenotype they control and to improve our knowledge of the fundamental mechanisms underlying the phenotype. As already observed for haplotypes associated with deleterious recessive mutations, particularly in dairy cattle, most of the LDHH evidenced in dairy sheep were associated with decreased fertility, but also had positive pleiotropic effects on milk production. Management of these haplotypes—or of their causal mutations once they are discovered—in the Lacaune dairy sheep selection scheme through reasoned mating of carrier rams and putative carrier ewes could improve overall fertility and lamb viability. Moreover, if these mutations segregate more widely in other ovine populations, the consequences would apply more broadly to sheep breeding in general.Additional file 1: Figure S1. Distribution of genotyped animals. Figure S1 shows the number of genotyped animals according to sex and year of birth.Additional file 2: Table S1. Clustering HHD in LDHH regions. Table S1 shows all significant haplotypes of 20 markers . As described in the “Table S2. SNPs defining the LDHH regions. Table S2 gives the position of each SNP within LDHH regions according to the sheep reference genome v2.0 and v3.1, and the phased alleles of each deficient haplotype.Additional file 3: Table S3. Positional candidate genes within LDHH regions. Table S3 summarizes all the protein coding genes that are located within the 11 LDHH regions extended by 1 Mb on each side. Genomic coordinates refer to the sheep reference genome v3.1. When available, gene information and association with autosomal recessive disorders in mammals are reported for each protein coding gene based on the following databases: NCBI Entrez: https://www.ncbi.nlm.nih.gov/Web/Search/entrezfs.html; MGI: www.informatics.jax.org; IMPC: https://www.mousephenotype.org); OMIM: Online Mendelian Inheritance in Man (https://omim.org) and OMIA: Online Mendelian Inheritance in Animal (https://omia.org).Additional file 4: Figure S2. Manhattan plot of HHD. Each point represents one haplotype of 20 markers with a frequency > 1% in the maternal phase. The red line represents the P-value threshold (1.9 × 10−4) to consider a haplotype significantly deficient in homozygotes. Only HHD with a deficit in homozygotes ≥ 75% were selected and resulted in the identification of 266 significant HHD (represented by green dots).Additional file 5: Table S4. Contingency table between LDHH4, 5 and 11 status and genotypes for the SOCS2 mutation OAR3:129722200C>T. Ram genotypes at both SOCS2 OAR3:129722200C>T and LDHH loci.


Heilongjiang Province is a high-quality japonica rice cultivation area in China. One in ten bowls of Chinese rice is produced here. Increasing yield is one of the main aims of rice production in this area. However, yield is a complex quantitative trait composed of many factors. The purpose of this study was to determine how many genetic loci are associated with yield-related traits. Genome-wide association studies (GWAS) were performed on 450 accessions collected from northeast Asia, including Russia, Korea, Japan and Heilongjiang Province of China. These accessions consist of elite varieties and landraces introduced into Heilongjiang Province decade ago.After resequencing of the 450 accessions, 189,019 single nucleotide polymorphisms (SNPs) were used for association studies by two different models, a general linear model (GLM) and a mixed linear model (MLM), examining four traits: days to heading (DH), plant height (PH), panicle weight (PW) and tiller number (TI). Over 25 SNPs were found to be associated with each trait. Among them, 22 SNPs were selected to identify candidate genes, and 2, 8, 1 and 11 SNPs were found to be located in 3′ UTR region, intron region, coding region and intergenic region, respectively.All SNPs detected in this research may become candidates for further fine mapping and may be used in the molecular breeding of high-latitude rice.The online version contains supplementary material available at 10.1186/s12863-021-00995-y. However, evidence suggests that they may have been domesticated separately from the ancestral species approximately 18 and 12 thousand years ago [O. sativa japonica, temperate, subtropical and tropical japonica, which grow in diverse environments with different climate characteristics [Rice cultivated in Asia is the staple food for most of the population worldwide. Research on its genetic variation, population structure and diversity has advanced greatly in recent decades –3. Cultiears ago . Genomiceristics .Every subspecies may have distinctive signatures or alleles that are formed during domestication or artificial selection. People in a specific area selected particular traits for their consumption needs . Many stQuantitative trait locus (QTL) analysis has turned out to be a very effective tool for gene and locus discovery in recent years. A large number of genes have been cloned based on QTLs from different species around the world –12. The Yield is one of the main traits that rice breeders focus on because of its relevance to worldwide food security. Nearly half of the world population consumes rice as a staple food. Yield is also known to be a multigene controlled trait, and many genes and loci have been found that could account for yield differentiation. Furthermore, yield is a complicated trait that is affected by many other traits, such as the tiller number, plant height, grain number, grain weight and number of primary branches , 32. AssRice cultivated at high latitudes in Asia shows many good characteristics, such as cold tolerance and high quality. There might be a large number of effective alleles that would be useful for further breeding for these traits. Few studies have focused on high-latitude natural populations and their effective alleles. In this research, we collected hundreds of cultivars and landraces as a natural population from high-latitude areas, including Northeast China, South Korea, Russia and Japan, and performed GWASs to examine their PH, PW, TI and DH in four different environments in Heilongjiang Province, China, with the aim of discovering genes or associated SNPs that could account for the differentiation of phenotypes and are expected to be used in further breeding programs.The plant height of 450 accessions ranged from 51.33 cm to 146.67 cm, with an average of 93.67 cm , intergenic (24.17%) or upstream of genes (31.37%)  decay analysis across all chromosomes. The LD decay distance ranged from 15 kb to 27 kb, with an average of 20 kb in the four groups predicted by population structure analysis , mostly breeding varieties. Group 2 contained 54 (12.0%) accessions, mostly cultivar introductions  was performed. When the first and second eigenvectors were used, all accessions could be divided with four subgroups Fig. A. HoweveA kinship matrix was calculated to detect the genetic relationship within the population. The coefficient of relatedness ranged from − 0.25 to 2.01, and a kinship heat map was drawn to visualize the relationships. It is clear that only the upper left corner has a relatively close relationship, and the other accessions have a lower coefficient of relatedness Fig. , indicatThe GLM found 597 SNPs to be significantly associated with four traits in total, but the MLM found only 322 Table D. Among O. sativa japonica group, according to the Rice Genome Annotation Project database.A total of 317 SNPs detected either by two models or in more than two locations were selected for candidate gene identification. Of them, 223 SNPs were used for PW, and 19 missense variants, 1 splice region variant and 1 stop gained locus were identified Table . All theMany models have been reported for use in GWAS, , 34, 35 Association analysis was first used on populations of unrelated human individuals,  but it iPrincipal component analysis has been shown to be a substitute for population structure in GWAS . TherefoA total of 144 DH-related SNPs were detected in four locations by GLM, and 25 of them were detected in more than two locations, which implied that even at different latitudes, heading date was functionally affected by the same genes. Little attention has been given to the associations of PW in cereal crops, but relationships with grain yield and rice quality have been reported . The PW From the Manhattan plots, it is obvious that DH shows significant peak values by GLM: 2 peak values on chromosome 2, 1 on chromosome 4 and 2 on chromosome 7  ≥ 6.58. In total, 597 and 322 significantly associated loci were detected for the four traits by GLM and MLM, respectively. After filtered, 22 larger effect or pleiotropic SNPs for the four traits were used to identify candidate genes. Eleven SNPs were identified within coding regions, two of them were located in 3′ UTR, eight in intron region and one in coding region. The rest of 11 SNPs were found to be located in intergenic region.In this study, 450 accessions were used to perform whole-genome resequencing, and 189,019 SNPs were used for GWAS after filtering according to the MAF, missing genotype rate and All these candidate genes associated with the four yield traits could be used for further gene identification or fine mapping, and related SNPs will also provide guidance for rice breeding in high-latitude areas.A collection of 450 temperate japonica rice varieties was used as a GWAS panel, including landraces and cultivars collected from Japan, North Korea, Russia, Heilongjiang Province, Jilin Province and Liaoning Province in China, and other unknown origins (Supplementary Table 1). Many landraces and foreign varieties have been introduced into Heilongjiang Province of China in recent decades. Moreover, a number of intermediate varieties were added to the panel for further analyses.All materials were planted in the field in 2015. Four experimental fields were located at Wuchang , Harbin , Jiamusi  and Heihe  in Heilongjiang Province, and all these locations were used as paddy fields for successive years. Wuchang is the most fertile black land of China, with approximately 145 days above the minimal temperature. At higher latitudes, Harbin and Jiamusi have fewer days above the minimal temperature. Heihe is not only the highest latitude of the Chinese temperate zone but also the highest latitude of the world where rice is cultivated. The growth season in Heihe is less than 120 days. Experiments were constructed with a complete randomized design. Ten plants of each accession were used for a single row with 13 cm spacing within the plants and 30 cm spacing between the rows. Field management was conducted normally for a local paddy field.https://cran.r-project.org/web/packages/lmerTest/index.html). Lines and locations were treated as random effects, and traits were treated as fixed effects with the formula Trait ~ (1|line) + (1|location) by lme4/R (https://github.com/lme4/lme4). The broad-sense heritability (H2) of the four traits was calculated by the following equation: H2 = Vg/(Vg + Ve/L), where Vg is the variance of genotypes, Ve is the variance of environments, and L is the number of locations. Statistical plots were drawn with ggplot2/R (http://had.co.nz/ggplot2/).The PH was evaluated before harvest and was measured from ground to the highest panicle tip. The TI was counted in each plant with panicles. The main panicle in each plant was collected and weighed from the rachis internode to obtain the PW. The mean of three plants was calculated as the final value. The heading date was noted when over 50% of plants in a row were heading, and the number of days from sowing to heading was used as DH for further analysis. Phenotype statistics and distribution analyses were performed with the R/base package. Analyses of variance for the four traits were performed using the lmerTest package in R by Student’s t test with a confidence level of α < 0.001  was isolated by a rapid method to obtain high-quality total DNA (DOI:10.21769/BioProtoc.1010106). Paired-end libraries were constructed and sequenced on an Illumina HiSeq sequencing system . The Nipponbare genome (MSU version 7.0) was used as a reference genome. All reads were aligned to the reference genome with the Burrows-Wheeler Alignment (BWA) tool . After thttp://www.cog-genomics.org/plink2/). After genotype imputation, SNPs were filtered by Plink with thresholds of MAF greater than 0.05 and a missing genotype rate greater than 0.2. Whole-genome LD decay was estimated by pairwise squared correlation coefficients (r2) between SNPs in PopLDdecay [r2 value was calculated when all accessions were divided into four groups by population genetic analysis. The LD decay distances were determined where the average r2 dropped to half of its maximum value. The SNPs were further filtered according to the r2 value in Plink with parameters --indep-pairwise 50 10 0.2.The MAF was calculated with Plink . The clustering distance of rows and columns used correlation as its parameter, and the complete parameter was used for the clustering method.Population structure analysis, including group estimation, best K value selection and population structure plotting, was performed with fastSTRUCTURE (version 1.0) by the structure, chooseK and distruct plugins, respectively . The PCA10(P) ≥ −Log10 ≈ 6.58. Manhattan plots and QQ plots were drawn by CMplot/R .The GWAS analysis was carried out with TASSEL 5, and GLM and MLM were used to detect significantly associated loci. After filtering by LD value, 189,019 SNPs were used for association analysis with a threshold LOD value of 6.58. Genotypes and phenotypes were used in the GLM model with the first five components of PCA as the population structure matrix. However, in the MLM, a kinship matrix was also added as a relatedness covariation. The threshold values for associated SNPs were obtained by the Bonferroni correction, which was calculated as follows: -Loghttp://rice.plantbiology.msu.edu/) annotation database was used as a reference database. SnpEff (Version 4.3 T) was used to annotate significantly associated SNPs [Loci that were detected in more than two locations or by both methods were used for candidate gene identification. The Nipponbare genome  Linkage disequilibrium of all accessions across 12 chromosomes. (B) Linkage disequilibrium differences between four groups predicted by population structure analysis when K = 4. Supplementary Fig. 2 Plots of the first two principal components in PCA with data points divided into 3 (A) and 5 to 9 (B-F) groups by faststructure. Supplementary Fig. 3. Manhattan plots and QQ plots for the four traits in Harbin by MLM. (A) Days to heading. (B) Plant height. (C) Panicle weight. (D) Tiller number. Supplementary Fig. 4. Manhattan plots and QQ plots for the four traits in Jiamusi by MLM. (A) Days to heading. (B) Plant height. (C) Panicle weight. (D) Tiller number. Supplementary Fig. 5. Manhattan plots and QQ plots for the four traits in Wuchang by MLM. (A) Days to heading. (B) Plant height. (C) Panicle weight. (D) Tiller number. Supplementary Fig. 6. Line plot of eigenvalues with the first 20 principal components. A significant downward trend was shown for the first 5 principal components. Supplementary Fig. 7. Manhattan plots and QQ plots for Days to heading in four locations by GLM. (A) Heihe. (B) Jiamusi. (C) Harbin. (D) Wuchang.


ST23 isolated in Italy are analysed by cgMLST and SNP approaches and they are also compared with ST23 from other countries. They are found to be phylogenetically related independently on year, town, or country of isolation. Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks. Legionella genus with the most cases due to Legionella pneumophila serogroup 1 (Lp1) (https://atlas.ecdc.europa.eu/public/index.aspx).Legionnaires’ disease (LD) is a severe pneumonia caused by bacteria belonging to the  1 (Lp1) . The ill 1 (Lp1) . In Euro 1 (Lp1) . AccordiflaA, pilE, asd, mip, mompS, proA and neuA), is the gold standard for genotyping Lp isolates . However, SBT alone has not been discriminatory enough in complex outbreak investigations linked to multiple clades or clones of the same ST that were only identified by whole-genome sequencing (WGS) (The ESCMID Study Group for Legionella Infection (ESGLI) has produced a standardized protocol for SBT and many clinical and environmental isolates have been sequence typed and there are currently 14,054 entries and 3,026 STs (ng (WGS) .WGS is now used to investigate LD outbreaks . MitchelIn Bresso, Italy, two outbreaks associated with ST23 were identified in 2014 (6 cases) and in 2018 (52 cases). The only suspected, yet unlikely, source of infection was identified as a public fountain, where Lp1 Philadelphia and Lp1 France/Allentown ST23 matching the clinical isolates was found .The SBT database was queried and ST23 was found to be widespread but in a restricted number of countries including Italy, where it caused all up to now described outbreaks and also several sporadic cases . It alsoThe monoclonal subgroup of all the Lp 1 isolates analysed by WGS from Italy were MAb 3/1 positive, and they are listed in All the 63 Italian ST23/ST2695 were de novo assembled and phylogenetic relationships inferred. First, based on the previously published cgMLST scheme , cgMLST neuA allele 37 of ST 2695 differed by only eight nucleotides compared with neuA allele 6 found in ST23.cgMLST targets differing between ST23 and the ST2695 variant identified in Bresso outbreak in 2018.Table S1 L. pneumophila strains ST2695, isolated during Bresso 2018 outbreak.Loci of difference found in Table S2 The differences observed in clade C (Table S3) were in loci adjacent to each other and by Genious analysis, they showed an identity of 90–99%, with missense nucleotidic substitutions ranged from 0 to 24 and the highest number of substitutions was found in the lpg2530 locus, encoding a 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (Table S4).cgMLST targets differing between ST23 isolated in Cesano Maderno 2008 and Bresso 2018 LD outbreaks.Table S3 L. pneumophila isolates during Cesano Maderno outbreak.Loci of difference found in Table S4 Second, a minimum spanning tree was constructed based on the subset of 1,346 core genes shared by all ST23/ST2695 independently on the year and town of isolation and few loci differences were found . Indeed,cgMLST loci of difference found in 299C genome isolated in Verona.Table S5 cgMLST loci of difference found in 1C genome isolated in Rome.Table S6 cgMLST loci of difference found in 325C genome isolated in Bolzano.Table S7 To establish if the phylogenetic relationships were found elsewhere, ST23 isolated in other countries were added to the analysis. Danish isolates were sequenced during this study and isolates from Scottish patients that had already been sequenced as part of an ongoing study and others previously reported were included .The cgMLST based on the subset of 1,216 cgMLST shared by all ST23, as listed in Table S8, were determined . This anList of the sequenced genomes submitted to the GenBank.Table S8 ST23 isolated in Scotland from patients who had travelled to Southern Europe differed by between 1 and 33 cg loci from ST23 isolated in Italy and France. Generally, the loci differences found in ST23 genomes mainly corresponded to genes encoding proteins related to the carbohydrate, amino acids, and lipid metabolism, and several for transport and binding (Table S9). As expected, independently on the country of isolation, all the ST23 were quite distant from other common STs, such as ST1, ST37, and ST62 .Loci of difference between Italian and not Italian ST23 highlighted by PHYLOViZ representation.Table S9 SNP analysis was used to provide phylogenetic information about ST23. SNP analysis performed on the 63 ST23  and otheST23 from other countries and other STs used in this study.Table S10 Genomes with single-nucleotide polymorphisms ranging 1–40.Table S11 Genomes with single-nucleotide polymorphisms ranging 171–802.Table S12 The origin of these variations determined by Gubbins analysis showed that 99.7% of the SNP were a result of recombination. After removing the recombined regions, corresponding to a mean of 3.13% of the 3.3 Mb genomes, to leave only the SNP vertically transmitted, the maximum number of pairwise SNP differences was 48 (found in 299C) with a mean of 6.7 for all ST23.A 10,421 core SNP of all 120 genomes, including this entire collection (ST23/ST2695) compared with ST1, ST37, and ST62 showed 2,602, 7,265, and 1,745 SNP differences, respectively, whereas among the ST23, the highest number of SNPs (392) was found in the genome 299C .Overall SNP analysis provided greater resolution than cgMLST. The minimum spanning tree generated from core genome SNP showed the central node, consisting of five strains, further separated into five different nodes. SNP analysis identified six polymorphisms  among strains undistinguishable by cgMLST.To verify the origin of SNP in this collection, ST23 were aligned against a reference genome using snippy and the maximum pairwise SNP difference calculated for the entire ST23 collection was 4,799 and was found in the HL05063005 genome from France (Table S8). As determined by Gubbins, a range of 37.5–99.9% of SNP differences were due to recombination. The genomes that showed least recombinations were those from (DK10) and Southern Europe (4697). After removal of the SNP due to recombination, which represented a mean of 4.62%, the maximum pairwise differences was 46 and again it was found in 299C genome .As determined by Roary pipeline, the pangenome consisted of 2,662 core genes and 1,511 accessory genes out of a total of 4,675 genes (including 280 soft-core genes and 222 shell not counted in the core and accessory genome). As shown in Lp ST23 are a well-known cause of both sporadic and epidemic LD cases, representing 4.4% of the ST found in Italy and 6.6% of the ST reported to the SBT database, although reporting countries are only France and Italy, with France having the most abundant number of ST23 cases .L. pneumophila and among diverse Legionella species , and the geographical distribution of cases would not represent the real areal circulation of this linage. On the contrary, it could be speculated that the genomes DK04 and DK05, both associated with a resort in south Sweden and indistinguishable from Italian strains, could be believed as a recent introduction (year of isolation 2017) in the north Europe, incoming from Italy via different spreading mechanisms, such as wind transport.Legionella infection, only the environment can exert a selective pressure. Therefore, it appears surprising that genomes isolated in different countries could be so phylogenetically close. The pangenome data highlighted the presence of accessory genes shared by unrelated ST23, but also specifically present in ST23 closely related in terms of country of origin. This pool of accessory genes could have been determined by plasmids and other mobile genetic elements that have not contributed to divergence of the ST23 genomes. Understanding the origin of these genes will be the subject of further investigations, possibly including a larger number of genomes from other countries. However, it could be speculated that the recombination events could have introduced some advantages for Legionella pathogenesis, as determined by the major spread of ST23 among community- and travel-acquired cases.At intra-ST23 level, most loci differences were located adjacent to each other and upon removing of horizontally transmitted SNP due to recombination events, the distances among the ST23 were significantly reduced, suggesting that the SNP differences observed were mainly due to recombination events irrespective of the year or country of isolation. This behaviour has already been observed in ST23 and in ST1, ST37, and ST62 but not in ST47, where recombinations events were not found . It has This study demonstrated that the ST23 responsible for the two outbreaks occurred in Bresso in 2014 and 2018 were genetically very similar. In 2018, during the outbreak, epidemiological and microbiological data identified a decorative fountain located in a public garden as a probable source of infection . cgMLST In Bresso, ST2695, a single locus variant of ST23 was also isolated and this also showed nucleotide variations in neighbouring loci, attributable to recombination events and when SNP due to horizontal transfer was removed, the divergence was further reduced. Therefore, it is possible that the newer ST2695 could have originated from ST23 and belongs to the same linage. Additional information about the ST23 phylogeny could be provided by other strains with single allele differences from ST23, which were unfortunately not isolated.mviN and enhA/C involved in uptake into host cells , a region of about 20 Kb was identified encoding the putative virulence gene st cells . These gst cells  were alsIt is noteworthy that, apart the ST23 isolated in France, most of the ST23s of other countries were from travel-associated cases and therefore not representative of an evolutive pressure determined by a specific geographic niche. ST23 isolated in both Denmark and Scotland, both countries with a low incidence of ST23 LD cases, were all from abroad travel-associated cases. On the contrary, French ST23, mostly from national community-acquired cases, were a good example of a genomic similarity from a large, single geographical area. In the Italian ST23 LD cases, southern Italy was not represented and this is could be probably due to a lower notification rate compared with the North of the country. Therefore, the Italian ST23 are from an even smaller geographic region of a country with high ST23 incidence and high genomic similarity.Looking at the ST23 isolated in Italy and those entered the SBT database, it is noteworthy that they are prevalently associated to travel- and community-acquired LD cases. The evidence that the ST23 strains are able to cause disease in a population not at high risk of infection deserves more in-depth studies, with particular regard to its virulence features. It would also be interesting to further investigate the presence of environmental ST23 strains, to identify specific water ecological niches and adopt appropriate prevention and control measures to limit future outbreaks.This is the first study focusing on Lp ST23 showing that it is highly conserved although distant temporally and geographically. It also highlights the importance of combining both epidemiological and genomic data, adopting typing methods with highly discriminatory power, to identify new clusters and the possible source of infection for future LD incidents caused by this ST.L. pneumophila ST23/ST2695 from the collection of the National reference laboratory for Legionella, Rome, Italy were analysed: 29 of them were from the two Bresso outbreaks in 2014 and 2018, 15 were from outbreaks occurred in Rome, Cesano Maderno, and Lazise, whereas 19 were from sporadic cases. These last included 15 clinical and four environmental strains, isolated between 1995 and 2018 in different Italian towns. They were all ST23 except five that were ST2695, differing from ST23 for just the neuA allele, and they were all isolated following either community- or travel-associated outbreaks and sporadic LD cases, as described in PRJNA606275; accession numbers listed in Table S8).In total, 63 To better understand the phylogenies of ST23 isolated in Italy, further 41 ST23 isolates already published, sequenced elsewhere or as part of this study were included. In addition, 14 genomes, ST1 (n = 5), ST37 (n = 4), and ST62 (n = 5) were also included (Table S10).Lp 1 strains stored at −80°C were thawed and cultured on Buffered Charcoal Yeast Extract  agar at 37°C for 48 h. A suspension on 1% formalin was used for MAb typing by indirect immune-fluorescence according to Dresden method , 2002. Ihttps://aries.iss.it/; Sequencing libraries were prepared using the NextEra XT library prep kit (Illumina) and then run on two different Illumina platforms: a 150-bp paired-end sequencing run was performed on the NextSeq-500 (Illumina) using a Mid output Kit v2, whereas a 250-bp paired-end sequencing run was performed on the Miseq (Illumina). The fastq files were trimmed and assembled using the fully automated pipeline INNUca by Galaxy ARIES platform (PRJEB48072). A further seven ST23, originated from Scottish patients who had travelled to the Southern Europe, were sequenced at the University of Birmingham by MicrobesNG, using Illumina 2 × 250 bp paired-end short read sequencing and deposited in the SRA database (Submission ERA1765603: Bioproject PRJEB31628).Eleven ST23 from Danish patients were sequenced at the Statens Serum Institut, as described above, and the reads were de novo assembled using SPAdes v3.12.1 with default parameters  (BioprojPreprint).chewBBACA software (24-25) was used to convert the cgMLST scheme defined by Geneious 9.1.8 (Biomatters Ltd.) was used for nucleotide sequence alignment of loci to identify differences against corresponding nucleotide reference sequences.The kSNP program, based on k-mer analysis, was used to identify core genome SNP on WGS data for each isolate. After the kSNP analysis, the core SNP matrix output file, containing only SNP loci common to all isolates, was used to construct minimum spanning tree by PHYLOViZ online software.https://aries.iss.it/; To investigate if SNP occurrence was due to recombination events, sequence reads were mapped to the LT632615.1 ST23 reference genome using Snippy v4.5.0, and horizontally transmitted SNP were removed by Gubbins analysis (Genomes were annotated using Prokka  and panghttps://www.ncbi.nlm.nih.gov/) Bioproject database PRJNA606275 and accession numbers are also listed in Table S10 in Supplemental material. All the other isolates, sequenced in this study, were submitted to bioprojects PRJEB48072 and PRJEB31628.All raw and processed sequencing data generated in this study have been submitted to the National Center for Biotechnology Information (NCBI;


Gene set enrichment analysis based on the GWAS results at p < 0.05 (1680 SNPs) showed that 32 Gene Ontology terms and two Kyoto Encyclopedia of Genes and Genomes pathways, including regulation of transcription, motor activity, the mitogen-activated protein kinase signaling pathway, and tight junction, were significantly enriched (p < 0.05) for BW-associated genes. These pathways are involved in cell growth and development, related to BW gain. The identified SNPs are potential biomarkers in KNC breeding.Meat from Korean native chickens (KNCs) has high consumer demand; however, slow growth performance and high variation in body weight (BW) of KNCs remain an issue. Genome-wide association study (GWAS) is a powerful method to identify quantitative trait-associated genomic loci. A GWAS, based on a large-scale KNC population, is needed to identify underlying genetic mechanisms related to its growth traits. To identify BW-associated genomic regions, we performed a GWAS using the chicken 60K single nucleotide polymorphism (SNP) panel for 1328 KNCs. BW was measured at 8 weeks of age, from 2018 to 2020. Twelve SNPs were associated with BW at the suggestive significance level ( Growth traits are one of the most economically important characteristics in the chicken industry and are greatly associated with both the breeding value and retail meat value . Therefohttp://dad.fao.org/, accessed on 7 November 2020) [Korean native chickens (KNCs) are known to have originated in the Korean Peninsula more than 1400 years ago. Under the Korean government-led KNC restoration project, five distinct lines have been established based on their feather color. These lines, namely, White (Baeksaek Jaerae-jong), Black (Heuksaek Jaerae-jong), Grey , Yellow , and Red , have been registered with the Domestic Animal Diversity Information System of the Food and Agriculture Organization (er 2020) . Owing ter 2020) .With recent improvements in high-throughput next-generation sequencing technologies, it has become possible to uncover genomic regions that are highly associated with specific traits at the genome-wide level. In particular, a genome-wide association study (GWAS) is the most effective method to determine genomic loci significantly associated with quantitative traits. A previous GWAS has uncovered important genomic regions affecting the growth, reproduction, and disease-resistance traits in chickens . FurtherSeveral studies have already been carried out on QTL that affect economic traits, such as meat quality and BW, in KNCs ,12; howen = 732) and Yellow (n = 596), were collected from 2018 to 2020. All the chickens were provided by the Poultry Research Institute of the National Institute of Animal Science (NIAS). Chickens (n = 10) were randomly assigned to windowless pens (size: 0.5 × 1.0 m) under standard conditions of temperature, humidity, and ventilation, with automatic ventilation and temperature control during the experimental period. Feeds were provided separately from 0 to 6 weeks of age  and 7 to 8 weeks of age  according to the nutritional requirements of each growth stage. Chickens had free access to water during all growth stages. All phenotypic data related to BW were measured at age 8 weeks. In this study, BW at age 8 weeks was chosen as the only major target trait owing to the Korean Native Chicken (KNC) breeding strategy. Our poultry breeding program has been focused mainly on improvement of market-weight in KNCs. Currently, KNCs have been shipped to the market at age 10 weeks; therefore, it is important to develop an efficient breeding strategy that can ensure that individuals with good growth capability can be selected in advance before reaching market-weight. The related research to determine the age at which to select birds for breeding programs showed that BW at age 8 weeks had the highest correlation among early growth periods with market-weight [A total of 1328 birds from two KNC breeds, namely, Red . After measuring the DNA concentration and purity using a NanoDrop 1000 spectrophotometer , the total DNA samples were genotyped using the 60 K chicken SNP chip  involving 57,636 SNPs. SNPs located on the sex chromosomes (Z and W) and those with unknown positions were excluded from this analysis. The Plink software was used to perform SNP filtering for the genotypic data based on the following criteria : minor aPrinciple component analysis (PCA) was performed to estimate the genetic relatedness between individuals and to account for potential population stratification prior to the GWAS. PCA based on the genomic relationship matrix (GRM) was performed to calculate the eigenvectors for all genotypic data using the genome-wide complex trait analysis (GCTA) tool .y is the vector of phenotypic values for the BW; X and Z are incidence matrices for the vectors for parameters b and µ, respectively; b is a vector of fixed effects including the breed, sex, and year of hatching; µ is the vector of the additive genetic effect of the candidate SNP to be tested for association; g is the accumulated effect of all the SNPs captured by the GRM; e is the vector of the residual effect. The significance threshold for the GWAS was defined using the Bonferroni correction method. We set the following two thresholds for our data: p < 1/N for suggestive significance and p < 0.05/N for a 5% genome-wide significance level, where N is the number of the SNPs remaining after quality control. The genomic inflation factor (λ) was calculated using the CMplot package in R with the median option. Manhattan and quantile–quantile (QQ) plots were created using the CMplot package in the R software [Genome-wide association study (GWAS) was carried out between all the genotyped SNPs and BW using a mixed linear model (MLM). The MLM for BW was performed using the breed (Red or Yellow), sex , and year of hatching  as fixed effects and the top two PCs as covariates. All association tests were performed using the MLMA option in GCTA, based on the following model (Equation (1)):software .h2) was calculated as the ratio of the additive genetic variance (Vg) to the phenotypic variance (Vg + Ve).Estimation of variance components was calculated using the restricted maximum-likelihood analysis option in GCTA, whereas genomic heritability  genome assembly 6 was used as the reference genome. When we first performed the GSEA based on the GWAS data, we referred to several published studies that have reported relevant similar analyses [p-value threshold for filtering GWAS SNPs to be used in GSEA differed between 0.05 and 0.005 among reports. Furthermore, the criteria for setting the flanking region when performing gene annotation, which also differed among reports, were either 5 or 15 kb. Thus, we adjusted the criteria for selecting GWAS SNPs to be used in GSEA to include approximately 1000 unique genes in the final dataset, resulting in the final SNP filtering criteria being determined as p < 0.05 and a 5 kb flanking region. Annotated genes were submitted to the Database for Annotation, Visualization and Integrated Discovery (DAVID) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses [p-value and is calculated by penalizing (removing) one gene within the given category from the list and calculating the resulting Fisher exact probability for that category [p-value in the Functional Annotation chart was calculated, and we defined the p value threshold (p < 0.05) for significantly enriched GO/KEGG term [Gene set enrichment analysis (GSEA) was performed based on the GWAS result to identify the biological pathways related to a group of genes harboring significant BW-associated SNPs. First, SNPs to be used for functional analysis were detected by filtering software , the filanalyses ,22,23,24analyses ,26. We dcategory . Based oEGG term ,21.A descriptive statistical summary for BW in the 8-week-old KNC population is provided in p < 2.95 × 10−5)  are located in the genes MYOCD and MAP2K4, respectively.The GWAS was performed for 33,878 SNPs to find genomic regions associated with the BW of KNCs at 8 weeks of age. The MLM-based GWAS revealed that 12 SNPs were associated with the BW and reached the suggestive significance level ( × 10−5) . Manhattp < 0.05, and these SNPs were mapped to 931 unique genes (p < 0.05) GO terms and KEGG pathways are shown in p < 0.05) in two pathways, which included the mitogen-activated protein kinase (MAPK) signaling pathway and tight junction.To further investigate significant GO/KEGG pathway terms associated with BW, gene set enrichment analysis was performed for the group of genes to which the SNPs were mapped. A total of 1680 SNPs showed an association with BW at a nominal threshold of ue genes . Subsequue genes . The mosIn this study, we performed a GWAS to detect significant SNPs associated with BW in a population of 1328 KNCs and explored genomic regions around these SNPs for candidate genes.Several studies ,29,30 haThe moderate heritability estimate of 0.47 was quite similar to those in various studies that have reported heritability estimates of BW at different growth stages in a range from 0.15 to 0.55 in different native chicken breeds . Moreovep < 2.95 × 10−5) and were located near or within 11 candidate genes, including WDR37, KCNIP4, SLIT2, PPARGC1A, MYOCD, and ADGRA3. BW is closely related to the growth of various tissues, including muscle, fat, and bone. The candidate genes identified in this study are correlated to the growth and development of these tissues. Of the top SNPs detected, the most significant, Gga_rs15062501, is located on chromosome 2 and is an intron variant of the WDR37 gene, encoding a member of the WD-repeat protein family, which is involved in the growth-related processes, including cell cycle progression and gene regulation. Although this variant is a noble SNP that has not yet been reported in the chicken QTL database, Khatri et al. [WDR37 was differentially expressed between broilers selected for rapid and slow growth.The GWAS result showed that 12 SNPs were associated with BW at the suggestive significance level . The MAPK signaling pathway is an important regulator of skeletal muscle development and was found to be activated during differentiation of a myogenic cell line . MAPK siWDR37, KCNIP4, SLIT2, PPARGC1A, MYOCD, and ADGRA3. The main biological role of the identified candidate genes was found to be the regulation of cell growth and development, which can affect BW. Our findings provide insights into BW-associated genes and pathways. Furthermore, the identified SNPs can potentially be used as biomarkers in KNC breeding.To the best of our knowledge, this is the first study to explore genetic markers and biological pathways associated with BW using 60K SNP chip data in a population of more than 1000 KNCs. This GWAS identified a total of 12 BW-associated SNPs, which were located near or within 11 candidate genes, including


Pennisetum glaucum (L.) R Br.] is an important staple food crop in the semi-arid tropics of Asia and Africa. It is a cereal grain that has the prospect to be used as a substitute for wheat flour for celiac patients. It is an important antioxidant food resource present with a wide range of phenolic compounds that are good sources of natural antioxidants. The present study aimed to identify the total antioxidant content of pearl millet flour and apply it to evaluate the antioxidant activity of its 222 genotypes drawn randomly from the pearl millet inbred germplasm association panel (PMiGAP), a world diversity panel of this crop. The total phenolic content (TPC) significantly correlated with DPPH  radical scavenging activity (% inhibition), which ranged from 2.32 to 112.45% and ferric-reducing antioxidant power (FRAP) activity ranging from 21.68 to 179.66 (mg ascorbic acid eq./100 g). Genome-wide association studies (GWAS) were conducted using 222 diverse accessions and 67 K SNPs distributed across all the seven pearl millet chromosomes. Approximately, 218 SNPs were found to be strongly associated with DPPH and FRAP activity at high confidence [–log (p) > 3.0–7.4]. Furthermore, flanking regions of significantly associated SNPs were explored for candidate gene harvesting. This identified 18 candidate genes related to antioxidant pathway genes  residing within or near the association signal that can be selected for further functional characterization. Patterns of genetic variability and the associated genes reported in this study are useful findings, which would need further validation before their utilization in molecular breeding for high antioxidant-containing pearl millet cultivars.Pearl millet [ Pennisetum glaucum (L.) R Br.] provides nutritious staple food grains worldwide especially to the poorest rural households in some of the hottest and driest rainfed farming regions of Africa and Asia. Global cereal production has been estimated to increase by ~1 billion tons over the last 50 years  was included in this study for antioxidant analysis . The PMig for 10 min at room temperature, and the supernatant was transferred to clean tubes. The pellets were then washed with 2.0 ml of 70% ethanol and centrifuged again at 10,000 × g for 10 min. The second supernatant fraction was added to the first to maximize recovery of target compounds. The ethanol was then removed using a heated centrifugal evaporator  set to 70°C. The dried extract was re-dissolved with 70% ethanol to an equivalent concentration of 15 mg/ml of the original sample and stored at 4°C until analysis. All biochemical analyses (see below) were carried out in triplicate, which allowed us to calculate the repeatability of these measurements.Pearl millet flour was extracted for antioxidant analysis according to a modification of the method described in Akpanika et al. . A quantDPPH is a stable free radical for the determination of antioxidant or radical scavenging capabilities. The DPPH radical portion of the molecule is a centralized nitrogen atom that gives rise to a maximum absorbance at 515 nm in methanol in its oxidized form. When a solution of DPPH in its radical form is mixed with a proton-donating substance such as antioxidants, the radicals are scavenged, and DPPH=H is formed with a concomitant decrease in absorbance. The ability of the pearl millet ethanolic extracts to scavenge free radicals was determined against a very stable free radical DPPH . DPPH is violet in color, while the reduced product is colorless, and the loss of color was determined in a plate reader Hauck, . Aliquot3+/tripyridyl-s-triazine complex present in stoichiometric excess to the blue ferrous form  analysis , spray voltage −4.0 kV in negative and 4.8 kV in positive ionization mode, capillary temperature 320°C, capillary voltage −1.0 and 45 V, respectively, and tube lens voltage −68 and 110 V, respectively.The analyses were carried out on a Thermo Finnigan LC-MS system  with a Finnigan PDA plus detector, a Finnigan LTQ linear ion trap with ESI source, and a Waters C18 Nova-Pak column . The column temperature was maintained at 30°C and equilibrated with 95% solvent A (water/formic acid 99.9:0.1 v/v) and 5% solvent B (methanol/formic acid 99.9:0.1 v/v) at a flow rate of 1 ml minAglycones were identified by direct comparison with relevant flavonoid standards, and glycosylated flavonoid compounds were identified by comparison of UV spectra and MS2 fragmentation patterns with standard flavonoid compounds. Standards were purchased from Carbosynth Ltd. .viz., FRAP and DPPH expressed in mg/ml was subjected to one factor analysis of variance (ANOVA) using the statistical software OPSTAT to find out the significant differences among the PMiGAP lines for both FRAP and DPPH activity . Low-quality SNPs and SNPs with missing data were removed using the protocols described in Iquira et al. . The “correct” K from the Ln probability of data [Ln P(D)], the delta-K values were estimated, as per the procedure suggested by Evanno et al.  as per the instructions of the user manual. LD was estimated using the squared allele frequency correlations (r2), which is a measurement of the correlation between a pair of variables  explained in percentage was also recorded for each individual marker. MTA analyses were also performed with an additional statistical method using the principal component analysis (PCA) matrix using the GLM  analysis was performed using TASSEL program, ver. 5.2.64 by two different models, generalized linear model (GLM) and mixed linear model (MLM)  using the CLC Genomics Workbench v.6.5 . Upstream and downstream regions surrounding each significantly associated SNP were searched in order to find and propose possible candidate gene. Furthermore, BLAST search of the NCBI database was conducted on each region of interest against the NCBI nr database.Significant SNPs from the GWAS were located by position and chromosome number on the DPPH free radical scavenging activities were assessed for the pearl millet flour extracts. In this study, FRAP was calculated using the equation Y = 0.2453x, where x is the OD of the sample. The genotypic differences between the 222 genotypes were significant for FRAP activity . Overalln is presented in C-glucoside , apigenin-C-pentoside-C-pentoside (ADP), and apigenin-O-hexoside-C-hexoside (ADH). Luteolin-glycosides were also relatively abundant with luteolin-C-O-dihexosidedihex (LDH), and its caffeic acid conjugate was detected in extracts. The phenolamide, dicaffeoyl spermidine, was also observed in the sample extracts. An interesting pattern is seen in The total phenolics and flavonoid content that resulted from the HPLC-PDA-ESI/MSr2 = 0.88*), and FRAP (r2 = 0.64**) indicating a strong association between phenolic compounds and antioxidant activity. FRAP also showed significant and positive correlation with LHDC (r2 = 0.97**), and (LDH) (r2 = 0.96**). THL has also shown positive correlation with LDH (r2 = 0.92**)  compared with total apigenin content   . The resctively) . Multivactively) . The pai as 0.24 . This inA total of 67,979 high-quality SNPs distributed all over the seven chromosomes of pearl millet genome were identified . In geneK = 6) (The clustering program has estimated the membership probability (Q-matrix) of each PMiGAP accession to combine into a number of hypothetical subpopulations (K3–K10), and ΔK value was generated for subsequent runs. The optimum K at run 6 showed maximum peak during cluster analysis. In this way, Bayesian model-based cluster analysis revealed that the 222 individual genotypes were clustered into six groups (K = 6) . These iP < 0.01). The average of r2 for all pairs was 0.108, which may be attributable to low levels of markers. Counts for individual r2 values were also analyzed by Varshney et al.  was observed.The extent of LD was assessed among 67,979 pairs of loci . Across y et al. , where tp-value ≤ 0.001  to remark the highly significant SNP markers associated with a trait. Thus, higher stringent threshold values were considered to filter out the highly associated markers with a specific trait –log 10  to minimize the effects of moderate size of the association panel and background noise of the Manhattan plots. Twenty-one SNP markers were found to be highly associated with the DPPH trait at p-value = 0.001, and Manhattan plot visualization revealed the high –log 10 p-value ranging from 3.0 to 3.76  plots displayed linear distribution when plotted against the observed and expected distribution of p-values for both the traits (DPPH and FRAP) (Marker trait association (MTA) analysis was performed for DPPH and FRAP activity-related trait using TASSEL. The MLM model-based association mapping showed 65 SNP markers associated with DPPH, whereas 153 SNPs showed association with FRAP trait at a  ≤ 0.001 , 5. The  to 3.76 . The per6325E−08 . The strnd FRAP) .p-value ≤ 0.001, but only common markers present in MLM-based analysis have been considered for further downstream analysis    . QQ (quand FRAP) , 9.Pennisetum glaucum by reference genome assembly (https://cegresources.icrisat.org/data_public/PearlMillet_Genome/). A total of 436 gene surrounding each SNP was identified using the GFF file of the reference genome. A BLAST search against the NCBI database using Blast2go revealed a large number of hits, representing a vast number of candidate genes of which the functions included defense against biotic/abiotic stress factors, growth, development, and regulation of bioactive metabolites (‴-hydroxycinnamoyltransferase) (Pgl_GLEAN_10025805), GDSL esterase/lipase (Pgl_GLEAN_10037404), purple acid phosphatase 15 (Pgl_GLEAN_10028864), DUF3755 family protein (Pgl_GLEAN_10026569), and ATP binding cassette (Pgl_GLEAN_10005798), which were deemed to be the most significant (P = 6.84E−06). These candidate genes will be taken forward for further verification by haplotyping and characterization of their functions in future studies.Furthermore, we investigated the candidate gene falling in the association region, which showed strong association with the trait of interest. The significant SNPs identified as a result of the GWAS were located by position on their respective chromosome of abolites . Out of abolites . FunctioPenisetum glaucum (L) R. Br.] is widely cultivated as a dietary staple food in the arid and semi-arid regions of the world, particularly in India and Africa and known to be a good source of natural antioxidants. In the present study, we explored the antioxidant activity in the pearl millet germplasm (PMiGAP) set of 222 genotypes, by screening it for DPPH and FRAP assays. These assays are widely applied in numerous studies to investigate the free radical scavenging ability and for measuring antioxidant activity of natural extracts [Alam et al.  and play a significant role in plants and human health conditions such as cancer, diabetes, and heart disease . This is consistent with the theory that the dihydroxy functionality present in the luteolin molecule increases, reducing power in comparison with flavonoids with a single hydroxy group on the B ring as found with apigenin  were further searched for LD status and for candidate gene searching. GWAS of these was performed by using widely used statistical models GLM and MLM , GDSL esterase/lipase, purple acid phosphatase 15, DUF3755 family protein, ATP binding cassette, and putative DUF594 domain-containing protein were found to be directly associated with antioxidant biosynthetic pathways across all selected SNP datasets. These candidates were found in close vicinity of the SNPs identified in this study but would require further validation in the future before they can be deployed in breeding programs. For validation, one can use candidate gene-based association mapping using large populations, or functional characterization through RNAi, VIGS, etc. Identification of desirable alleles of these MTAs will further confirm their roles in antioxidant synthesis and ensure their efficient utilization in crop improvement programs. Similar approaches as we report here were also adopted by Luo et al. (r2 > 0.6), while 185 of the other SNPs were not mapped in LD blocks. Thus, they identified 88 genes, which were predicted to be transcription factors, including NAC domain transcript factors, and Myb domain transcripts. However, they selected EgHGGT (homogentisate geranylgeranyl transferase) for further analysis, which is involved in biosynthesis of tocotrienols and had higher expression levels in the mesocarp compared with other tissues. Antioxidant-associated candidate gene identification has previously been attempted by many researchers in maize and other cereal crops. In maize, association analysis was performed by Li et al. (A total of 436 genes were identified around each SNP found to be associated with DPPH and FRAP activity. Eighteen candidate genes including UDP-glycosyltransferase, agmatine coumaroyltransferase-2, anthocyanin 5-(6o et al.  for assoi et al.  to identi et al.  to identi et al.  in pearli et al.  in pearli et al.  and Sehgi et al.  for manyIn conclusion, the present study explored the genetic architecture of antioxidant content in pearl millet for the first time using LD-based GWAS exploiting historical recombination in a natural germplasm population. The population used showed a wide range of variability for traits studied. Furthermore, resequencing data and quality phenotyping of DPPH and FRAP led us to the identification of highly significant SNP markers associated with antioxidant content. Such associations provided better insights into the genetic architecture of this very important trait in pearl millet. Candidate gene analysis in the significantly associated SNP region has identified potential candidates, which on further validation, will provide a great resource to select these important traits in pearl millet breeding programs.The original contributions presented in the study are included in the article/RSY conceived and supervised the completed study. JT, DY, PG, AW, and RY contributed to the phenotyping experiments. CY analyzed the results and wrote the manuscript. RKS and RBS developed the HapMap used for GWAS in this study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum merozoites involves the interplay of several proteins that are also targets for vaccine development. The proteins PfRh5-PfRipr-PfCyRPA-Pfp113 assemble into a complex at the apical end of the merozoite and are together essential for erythrocyte invasion. They have also been shown to induce neutralizing antibodies and appear to be less polymorphic than other invasion-associated proteins, making them high priority blood-stage vaccine candidates. Using available whole genome sequencing data (WGS) and new capillary sequencing data (CS), this study describes the genetic polymorphism in the Rh5 complex in P. falciparum isolates obtained from Kilifi, Kenya.The invasion of the red blood cells by P. falciparum samples obtained from a drug trial conducted from 2005 to 2007. The frequency of polymorphisms in the merozoite invasion proteins, PfRh5, PfRipr, PfCyRPA and PfP113 were examined and where possible polymorphisms co-occurring in the same isolates.162 samples collected in 2013 and 2014 were genotyped by capillary sequencing (CS) and re-analysed WGS from 68 culture-adapted From a total 70 variants, including 2 indels, 19 SNPs [27.1%] were identified by both CS and WGS, while an additional 15 [21.4%] and 36 [51.4%] SNPs were identified only by either CS or WGS, respectively. All the SNPs identified by CS were non-synonymous, whereas WGS identified 8 synonymous and 47 non-synonymous SNPs. CS identified indels in repeat regions in the p113 gene in codons 275 and 859 that were not identified in the WGS data. The minor allele frequencies of the SNPs ranged between 0.7 and 34.9% for WGS and 1.1–29.6% for CS. Collectively, 12 high frequency SNPs (> 5%) were identified: four in Rh5 codon 147, 148, 203 and 429, two in p113 at codons 7 and 267 and six in Ripr codons 190, 259, 524, 985, 1003 and 1039.This study reveals that the majority of the polymorphisms are rare variants and confirms a low level of genetic polymorphisms in all proteins within the Rh5 complex.The online version contains supplementary material available at 10.1186/s12936-021-03815-x. Plasmodium falciparum Reticulocyte Binding homologue 5 , which is currently advancing through clinical trials [Despite some progress over the last decade, malaria continues to be a significant global health burden with a vaccine deemed essential to effectively control the disease in high malaria transmission zones . The RTSAotus monkeys, demonstrated protection from heterologous P. falciparum challenge [P. falciparum infections develop anti-PfRH5 antibodies at a relatively low prevalence, the presence of these antibodies have been associated with protection from symptomatic malaria in Papua New Guinea, and Mali [Rh5 is the smallest in the Reticulocyte Binding Protein homolog (Rh) family that includes Rh1, Rh2a, Rh2b and Rh4 . Furtherhallenge , while nand Mali –15. Base2+ concentration and alters the erythrocyte cytoskeleton [Rh5 does not function in isolation during erythrocyte invasion, but acts as part of a multi-protein complex with Rh5 interacting protein  , cysteinskeleton . Rh5 undskeleton  and CyRPskeleton , while Rskeleton . Therefoskeleton –18, 21.P. falciparum strains [Similar to Rh5, the genes encoding CyRPA and Ripr cannot be knocked out, suggesting that they are essential for parasite growth , 18, and strains , 17, 22. strains , 23. AllPolymorphisms are a particular barrier for the development of blood-stage vaccines, as proteins that are exposed to the immune system during invasion are often very diverse, presumably the result of pressure from the immune system . This prCyRPA (PF3D7_0423800), P113 (PF3D7_1420700), Ripr (PF3D7_0323400) and Rh5 (PF3D7_0424100) genes were examined. Genomic DNA was previously extracted from packed frozen erythrocytes using the QIAcube (Qiagen), according to the manufacturer’s instructions . All four genes were amplified using High Fidelity Taq polymerase (Roche) , parasite DNA was extracted from 162 blood samples from children below 11 years admitted and attended to at the Kilifi County Hospital in 2013 and 2014. The children had variable parasitaemia ranging from 160 to 705,600 parasites/µl, with a median of 7440 parasites/µl. This study was reviewed and approved by the Centre Scientific Committee and the Scientific Ethical Review Unit (SERU) of the Kenya Medical Research Institute, on SERU protocol number 3149. . [P. falciparum Community Project [For whole genome sequencing, parasite DNA was previously extracted from 68 blood samples obtained from children recruited into an artemisinin-based combination therapy (ACT) drug trial of dihydroartemisinin-piperaquine and artemether-lumefantrine conducted in Pingilikani dispensary, Kilifi from 2005 to 2007 . Additio. . The gen Project . The selRh5, Ripr, CyRPA and P113 was filtered, by using a bed file containing the chromosome numbers and genomic positions, to generate one VCF file. Using PLINK [A VCF file containing 68 samples obtained from Kilifi, Kenya, were used as the input file in the downstream analysis. Using VCFtools (v. 0.1.13) a targeted analysis of four genes: ng PLINK , the VCFPlasmodium falciparum community project version 4.0 was used. This data was generated through an analysis of 3488 P. falciparum samples collected at 43 different locations in West Africa (WAF), Central Africa (CAF), East Africa (EAF), South Asia (SAS), West South East Asia (WSEA), East South East Asia (ESEA), Oceanic (OCE) and South America (SAM). A total 930,000 exonic SNPs and their frequencies were obtained. The method used to generate the data are described in Amato et al. [CyRPA (PF3D7_0423800), P113 (PF3D7_1420700), Ripr (PF3D7_0323400) and Rh5 (PF3D7_0424100). The pool of SNPs identified were filtered to obtain their frequencies.To further validate the SNPs, we identified through CS and WGS, data from the MalariaGEN o et al. . The dplo et al.  in R v4.The allele frequency distribution indices, Tajima’s D and Fu and Li’s D* and F*, were computed using DnaSP v5.10 software  for the Rh5, Ripr, CyRPA and P113 was determined within and between genes using R v3.6.0. The statistical significance of LD was tested, at the 5% level, using χ2 tests.For each of the four genes obtained from the whole genome data, the minor and major allele frequencies of all the SNPs were computed using PLINK. Only SNPs with a > 5% minor allele frequency were included in the analysis. The extent of linkage disequilibrium (LD) between pairs of SNPs in http://www.rcsb.org/). Wong et al. [The cryo-electron microscopy structure of Rh5-CyRPA-Ripr (PDB ID: 6MPV) was downloaded from the Protein Data Bank . Indels were only found in p113, with variation in repeat regions at codon 275 with asparagine (N) (ranging from 3 to 9 N) and at codon 859 with glutamic acid (E) (ranging from 2 to 3E). The CyRPA analysis was conducted in two fragments from the N and C-terminal ends. The N-terminal end fragment, codons 1 to 170, contained only one non-synonymous SNP, at codon 165 and found in only a single infection, while the C-terminal end though shorter in comparison contained three polymorphic sites.Capillary sequencing data was attempted from 162 samples taken from children admitted to Kilifi Hospital. Data on all four genes was not obtained by capillary sequencing for any single sample, but data was obtained for multiple pairs of genes from individual isolates: Rh5 gene complex: 10 in CyRPA, 14 in P113, 21 in Ripr and 10 in Rh5 as shown in Table CyRPA, 5 in p113 and 2 in Ripr and forty-seven ns polymorphisms with CyRPA, p113, Rh5 and Ripr containing 9, 9, 10 and 19, respectively  SNP data for all the four genes were obtained from 68 independent samples from a previous drug trial  for Rh5, P113 and Ripr were also found in the global MalariaGEN dataset, arguing against any systematic missing SNP identification issues. In addition, more than two thirds of the rare variant SNPs were identified in these samples had also been identified previously in the global MalariaGEN data, giving us confidence in the polymorphisms identified in this study using CS and WGS. Combining our data with global MalariaGEN data confirmed that the majority of variants in the genes encoding the Rh5 complex are rare mutations  were located within the CyRPA-Ripr interacting region [Ripr α- helix, Fig. CyRPA β-propeller, amino acids 281 to 311 [Mutations within known protein interacting regions were mapped onto published structures for the Rh5 protein complex. The structure of the ublished  and the p region . This Rhg region . The Rip1 to 311 . All theP. falciparum populations in Africa, which revealed that the majority of genes were associated with a negative Tajima’s D value. Therefore, suggesting there was a historical parasite population expansion in Africa [p113 and Ripr, which may be due to the parasite’s need to preserve their function. These proteins are involved in a critical step during the invasion of erythrocytes and this polymorphism data reinforces the fact that they are likely to make good vaccine candidates to inhibit invasion and prevent disease [The Rh5 complex is a relatively conserved set of proteins with few polymorphisms. They are not highly immunogenic, as previously shown , 23. Then Africa –41. The  disease .P. falciparum parasites have been known to differ significantly from source populations due to adaptation to environments that exclude the host immune responses [Sequence data was obtained using two different methods and resulted in the identification of more SNPs using whole genome sequencing (WGS) analysis than Capillary Sequencing (CS), but there are pros and cons to both approaches. In CS, each read is accompanied by a long (on average 500 bp) chromatogram, which makes it easy to assemble and align to a reference genome in order to manually identify variants, but the process as a whole is low-throughput. In WGS, millions of short reads are produced with each read being accompanied by a quality score. It is thus not feasible to manually check the quality of each nucleotide and quality score cut-offs are set in the bioinformatic pipelines to confidently call a nucleotide. This presents a challenge in identifying indels within repeat regions—because the assembly and alignment of these regions to reference genomes is based on short reads, confidence is often low in these regions, making it difficult to unambiguously determine the numbers of repeat nucleotides . Howeveresponses . There aThe majority of the polymorphisms in this complex or merozoite invasion antigens were rare, which is in contrast to previous findings from surface exposed and abundant merozoite antigens such as apical membrane antigen 1 (AMA1) , merozoiThis study identified only one Rh5 mutation C203Y at the Rh5-Basigin interface. It has been shown that the Rh5 protein variant with the 203Y mutant binds to recombinant basigin with the same affinity as the Rh5 C203 wild type . It is tPlasmodium translocon of exported proteins (PTEX), which is known to be a mechanism of immune evasion [The low immunogenicity of Rh5 complex members in field studies , 15, 22  evasion . Further evasion .Most of the observed SNPs were not in statistically significant LD with the exception of codons 147 and 148 for Rh5 and 985 and 1003 in CyRPA, which are 3 bp and 54 bp apart respectively. The limited LD is likely due to a combination of the fact that most of the SNPs are rare variants and therefore occur at a low frequency, and the limited sample size in this study. Rh5 codons, 147 and 148 are included in the protein structure  on the uThe development of new tools and adaptation of existing tools for use in malaria elimination and eradication remains a priority, and deeper understanding of polymorphism(s) in vaccine candidate genes is particularly important. This study highlights pros and cons to both CS and WGS approaches to identifying vaccine-relevant polymorphisms. The ideal molecular tool should be able to provide quality and high-throughput sequence reads capable of detecting low frequency variants including indels. One such approach would be amplicon deep sequencing, where longer fragment amplicons can be generated and sequenced using an NGS platform, focussing analysis on the regions of interest rather than the whole genome, but producing deeper and higher quality data than CS. Low frequency mutations should be assessed by functional assays to ascertain their biological and immunological relevance. One of the main obstacles in the development of effective vaccines for malaria is the occurrence of polymorphisms on candidate vaccine targets that result in strain-specific immunity. Among the members of the Rh5 complex, Rh5 is the most advanced in vaccine development. The identification of a limited number of high frequency polymorphisms on Rh5 shows promising prospects of Rh5 based vaccines in this region, but it is still possible that low frequency variants may lead to immune evasion—this needs to be systematically investigated.One gene does not appear to conceal the other genes in the complex, by being more polymorphic and acting as a decoy to direct the immune pressure away from the rest of the genes in the complex. Thus, the limited polymorphisms are potentially a result of their hidden location in the apical end of the merozoite and their limited exposure to host immune responses. Due to the minimal acquisition of mutations, Rh5, CyRPA, Ripr and P113 proteins are potentially a good next-generation multi-antigen vaccine formulation.Additional file 1: Table S1. List of primers for PCR and capillary sequencing.Additional file 2: Table S2. The expected PCR region amplified and product size for each gene.Additional file 3: Table S3. List of SNPs identified by capillary electrophoresis method.Additional file 4: Table S4. List of SNPs identified by whole genome sequencing method.


Next-generation sequencing (NGS) is an efficient tool used for identifying pathogenic variants that cause Mendelian disorders. However, the lack of bioinformatics training of researchers makes the interpretation of identified variants a challenge in terms of precision and efficiency. In addition, the non-standardized phenotypic description of human diseases also makes it difficult to establish an integrated analysis pathway for variant annotation and interpretation. Solutions to these bottlenecks are urgently needed.We develop a tool named “Cruxome” to automatically annotate and interpret single nucleotide variants (SNVs) and small insertions and deletions (InDels). Our approach greatly simplifies the current burdensome task of clinical geneticists and scientists to identify the causative pathogenic variants and build personal knowledge reference bases. The integrated architecture of Cruxome offers key advantages such as an interactive and user-friendly interface and the assimilation of electronic health records of the patient. By combining a natural language processing algorithm, Cruxome can efficiently process the clinical description of diseases to HPO standardized vocabularies. By using machine learning, in silico predictive algorithms, integrated multiple databases and supplementary tools, Cruxome can automatically process SNVs and InDels variants (trio-family or proband-only cases) and clinical diagnosis records, then annotate, score, identify and interpret pathogenic variants to finally generate a standardized clinical report following American College of Medical Genetics and Genomics/ Association for Molecular Pathology (ACMG/AMP) guidelines. Cruxome also provides supplementary tools to examine and visualize the genes or variations in historical cases, which can help to better understand the genetic basis of the disease.http://114.251.61.49:10024/cruxome/.Cruxome is an efficient tool for annotation and interpretation of variations and dramatically reduces the workload for clinical geneticists and researchers to interpret NGS results, simplifying their decision-making processes. We present an online version of Cruxome, which is freely available to academics and clinical researchers. The site is accessible at The online version contains supplementary material available at 10.1186/s12864-021-07728-6. Curreach year . Of all ach year , 6 whereach year –9. Thus,ach year .Next-generation sequencing (NGS) has emerged as an innovative tool for medical genetics, and has led to a paradigm shift in medical research and clinical practice –13. WithSeveral open-source analysis tools for variant annotation and functional effect prediction have been reported including spliceAI , ANNOVARWhen a set of candidate variants are identified, the aim of follow-up analysis is to establish a strong relationship between the candidate genes and known diseases by using information in the published literature and databases. However, this information is sometimes incomplete or fragmented and distributed differently across many databases, which makes this step very time-consuming and inefficient. There are several reported tools that integrate the various databases and simplify the search. These tools include IPAD  , SIDD (shttps://hpo.jax.org/) [Electronic health records (EHRs) have been widely implemented by clinical geneticists and include the patient’s information such as name, age, gender, laboratory test results, phenotypic description, diagnosis and medication details. Almost all tools or databases adopt Human Phenotype Ontology (HPO) as the reference. HPO uses standardized vocabulary for describing phenotypic abnormalities in human disease, drawing on over 13,000 terms and over 156,000 annotations to hereditary diseases (ax.org/) , 32. Forax.org/) . Benefitax.org/) –36. SincBased on these challenges, we develop Cruxome, an automated and user-friendly tool for variant interpretation which is designed to efficiently and precisely handle the Variant Call Format (VCF) file (either from WES or gene panel data) and generate standardized clinical reports. By mining the hundreds of thousands of literature accounts and integrating appropriate databases, Cruxome harbors a comprehensive and regularly updated biomedical knowledge base to keep pace with precise variant interpretation. Cruxome uses a natural language processing algorithm (NER) to transform colloquial descriptions of phenotype to standard HPO vocabulary. Cruxome also supports building a personal knowledge base to efficiently manage patient’s information and interpret results with traceable evidence record of interpretation decisions. Above all, Cruxome provides an overall solution for variant interpretation, dramatically reducing workload and facilitating better decision-making processes.https://www.docker.com/) which is a standard unit of software that packages up code and all its dependencies so the application runs quickly and reliably from one computing environment to another. Thus, Cruxome can easily be deployed on either a cloud server  or on a local server. To enhance the functionality of Cruxome, improve efficiency and simplify code maintenance, a layered pattern was used in the basic architecture of Cruxome  and for storage of the information.Cruxome was designed with a user-friendly interface and developed based on a Browser/Server style to facilitate easy access and to minimize incompatibility with different computer operating systems. Cruxome runs in Docker mode :A modern browser .A 24-core server with 64G memory, 1T hard disk.An internet or intranet connection of 10Mbit.The overall workflow of Cruxome is shown in Fig. After login to Cruxome, the user first creates the patient’s record with detailed information about phenotype, age, family relationship or directly imports the records from existing patient databases. Secondly, the VCF files are uploaded. Cruxome supports all of the VCF file formats that meet VCF 4.2 standard or higher, and supports both the GRCh37 (hg19) and GRCh38 reference genomes. Cruxome then automatically checks file formats and standardizes the files.After checking the patient information and VCF file format, Cruxome then launches its annotation module. For the most comprehensive evaluation and interpretation of variants, Cruxome integrates multiple databases, including sequence databases for gene functional information, population databases for calculation of variants allele frequencies and disease databases to define clinical significance and phenotype relationships relevant to disease phenotype. Multiple tools are then applied to evaluate the effect of variants on protein function and to finally generate a variant score .Next, Cruxome uses a natural language processing algorithm NER to transform clinical diagnosis records to HPO standard format Fig. . By usinOnce variant interpretation is complete, Cruxome automatically updates your personal knowledge base and stores all the information generated during the interpretation process, including candidate variants, ACMG evidences, literature and versions of modules and databases, thus making the interpretation decisions traceable Fig. . PersonaAHDC1 gene in this case), users should mark the corresponding variants as “Positive” in the conclusion column  and a pathogenic (NM_015506.2: c.217 C > T: p.R73X) variant in the stinuria  : Platform independent.Programming language: Java.Other requirements: Java (version > = 1.8.1), Tomcat (version > = 8.0), Docker (version > = 18.03.1-ce), MySQL (version > = 5.7).License: Free for academic and research use.Additional file 1.