Morella rubra (Sieb. et Zucc.). A total of 3,151,123 SNPs, 371,757 small indels, and 15,904 SVs were detected in 52 accessions. Verification by Sanger sequencing demonstrated that the positive rate of the SNPs was approximately 97.3%. Search for more genetic variations was expanded to 141 red bayberry accessions, most of which were cultivars, by sequencing 19 selected genomic segments (SEG1-19). The results showed that each segment harbored, on average, 7.8 alleles (haplotypes), a haplotype diversity of 0.42, and a polymorphic information content (PIC) of 0.40. Seventy-two different genotypes were identified from the 141 accessions, and statistical analysis showed that the accessions with duplicated genotypes were either somatic mutants or simply synonyms. Core set selection results showed that a minimum of 34 genotypes could already have covered all the alleles on the segments. A DNA fingerprinting system was developed for red bayberry, which used the diversity information of only 8 DNA segments yet still achieved a very high efficiency without losing robustness. No large clade was robustly supported by hierarchical clustering, and well-supported small clusters mainly included close relatives. These results should lead to an improved understanding of the genetic diversity of red bayberry and be valuable for future molecular breeding and variety protection.High throughput sequencing was used to reveal the distribution of whole-genome variations in cultivated  Morella rubra Sieb. et Zucc.), also known as Chinese bayberry, is an economically important fruit crop that has been cultivated in China for more than two thousand years. Its fruit is not only very tasty but also rich in anthocyanins, vitamin C, and other antioxidant compounds (Red bayberry (ompounds . Red bayompounds . Red bayompounds .M. rubra) of Morella and detected 8,360 single nucleotide polymorphisms (SNPs). However, there is still limited knowledge about the abundance of genetic variations in red bayberry at the whole-genome level.Hybridization and molecular breeding are believed to play more important roles in the improvement of red bayberry germplasm in the future. In this respect, much effort has been made to identify molecular markers associated with its important traits such as sex determination . DiffereAlthough a total of more than 300 red bayberry accessions have been recorded, including no less than 268 named cultivars , their gCitrus maxima, and the results showed that each segment contained multiple SNPs and harbored no less genetic diversity than SSRs (Cucumis sativus L.), multiplex PCR amplification and GBS were applied for genotyping multiple genomic segments in the variety fingerprinting (Camellia sinensis L.) (Theobroma cacao L.) (Solanum L.) (Ananas comosus L.)  per marker . Howeverhan SSRs . Taking han SSRs . In cucuprinting . SNP-basnsis L.) , cacao (acao L.) , eggplananum L.) , and pinosus L.) .In this study, we report whole-genome genetic variations in red bayberry based on NGS of DNA mixture of 52 cultivars. The genotypes of 141 red bayberry accessions were revealed on 19 genomic segments. These data allowed us to perform genetic diversity, clustering, and pedigree relationship analysis. We also selected a core set of genotypes from the accessions to facilitate genetic diversity preservation and developed an efficient red bayberry DNA fingerprinting system.The leaves of 141 red bayberry accessions  of DNA solutions from 52 of the 141 red bayberry accessions  were mapped to the female red bayberry reference genome (GCA_003952965.1)  using BWWhole-genome SVs were detected using Manta v1.6.0 . Five diThe circular graphs , and theFifteen of the segments SEG1-19  were selected based on the “Linhaizaodamei” assembly. The DNA mixture sequencing reads were mapped to the assembly, and regions with >0.5 and <1.5 average whole-genome sequencing depth were filtered. The scaffolds of “Linhaizaodamei” assembly were ranged in descending order of scaffold size. To minimize the influence of linkage, the segments had better be physically distant from each other on the chromosomes, thus only one segment was designed on each of the largest 15 scaffolds. Primer3 was used to design the primers , and thePCR amplifications were carried out for the 141 accessions using primer pairs SEG1-19  implemented in DnaSP v6.12.03 , with 10The statistical power of SEG1-19 in parentage analysis under different conditions was calculated using Cervus v3.0.7  P1, P2, http://darwin.cirad.fr/). A 1,000 bootstraps of UPGMA clustering was carried out in MEGA X  and NJ clustering  by DARwin v6.0.021   with allsib were calculated for the segments individually and cumulatively using GenAlEx v6.51b2  was used to identify the whole-genome DNA variations in red bayberry. DNAs extracted from leaves of a total of 52 red bayberry (Yangmei) accessions were mixed and sequenced. A total of ~175 × sequencing reads were obtained and mapped to the female red bayberry reference genome (GCA_003952965.1). Genomic regions with abnormally high  and low  sequencing depth were excluded from SNP and indel detection  were identified from the sequencing data. Among the variations, 2,883,745 SNPs and 339,513 indels were located on the 8 pseudo-chromsomes Chr 1–8, while the rest were located on 32,244 scaffolds which failed to be assigned to any chromosomes. The SNP density of the whole genome was thus averaged at 10.3 ± 9.2 SNPs/kbp. Comparatively, Chr 1 and Chr 6 had the highest (12.0 SNPs/kbp) and the lowest (9.1 SNPs/kbp) SNP densities, respectively. The transition/transversion ratio of all the SNPs was 2.04, and the most abundant SNP substitution types were C>T and G>A  were identified from the sequencing data   and hierarchical clustering (UPGMA) methods were applied to the 72 genotypes based on the 176 SNPs in SEG1-19  and the probability of identity between two siblings sharing both parents (PIsib) were 6.4e-10 and 1.5e-4, respectively (13.6) random individuals and 26 (24.7) siblings. Therefore, it was possible to identify all the 14 members of the largest putative sibling group associated with the genotype J (To investigate the power of using the diversity of SEG1-19 in identifying 14.5) random individuals, and 33 (25.05) siblings (par/off (probability of identity between a genotype and its parents or offspring), and PIsib were calculated for each of the 72 genotypes. It was shown that SEG1-19 was very powerful in distinguishing an individual genotype from other randomly selected genotypes, and from its parents, offspring, or its siblings (10.1) randomly sampled individuals, a number that greatly exceeded the total number of existing red bayberry cultivars.The discrimination capacity of the SEG1-19 could even be increased to 23,873 , C (“Dingaomei”), and B (“Dongkui”) not only possessed the most duplicated genotypes but also the largest numbers of putative parent-offspring links.Our results also showed that different cultivars may have played different roles in the history. Some cultivars were more widely cultivated and some have played more important roles than others in breeding. The number of duplicated genotypes observed should be a good indicator of cultivar popularity. For instance, the two most widely grown cultivars Dongkui (B) and Biqi (F) had the largest number of accessions that shared their genotypes. Three other genotypes, C, O, and V, represented by three popular cultivars, Dingaomei, Chise, and Shangyubaiyangmei, were the second most observed in the 141 accessions. The links for putative parent-offspring relationship varied significantly among different genotypes  can be found in the article/BW analyzed the data and wrote the manuscript. YZ, QW, and YC carried out the experiments. FC and GZ collected the plant materials. All authors contributed to the article and approved the submitted version.This work was supported by the 13th Five Year Plan of Chinese bayberry Breeding Special Project (2016C02052-2), the discipline construction of Zhejiang Academy of Agricultural Sciences , and the Taizhou Science and Technology Project (2020.03).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.


Option a) allows for introgressing genes from other individuals in one or both of these nearly-inbred lines. Peach, a woody-perennial, clonally-reproduced species, was chosen as a model for a proof of concept of the Resynthesis process due to its biological characteristics: self-compatibility, compact and genetically well-known genome, low recombination rates and relatively short intergeneration time (3–4 years). From 416 F2 seedlings from cultivar Sweet Dream (SD), we obtained seven individuals with 76–94% identity with SD, and selected five pairs of complementary lines with average homozygosity of the two parents ≥0.70 such that crossing would produce some individuals highly similar to SD. The application of this scheme to other species with more complex genomes or biological features, including its generalization to F1 hybrids, is discussed.We propose a method for marker-based selection of cultivars of clonally-reproducing plant species which keeps the basic genetic architecture of a top-performing cultivar , with the addition of some agronomically relevant differences , providing added value to the product or cultivation process. The method is based on selecting a) two complementary nearly-inbred lines from successive selfing generations (ideally only F A few high-quality cultivars dominate the market of many clonally propagated species that are often highly, or at least partly, heterozygous individuals. Sometimes they are recognized by their name and their main features by the consumers . Reproducing the genotype of a top performing cultivar with a minor but significant improvement may have a high market value . With classical approaches  this is extremely unlikely, and to our knowledge, it has never been tried. Other approaches have difficult-to-overcome regulatory issues and sometimes a negative social perception , or are rare and occur unpredictably (somatic mutations). Resynthesis is an approach based on conventional breeding technologies that, with the help of molecular markers, allows the selection of two homozygous (or nearly homozygous) complementary individuals that, when crossed, produce offspring with a similar, albeit different, genetic composition from the original cultivar. Homozygous fragments of either of the two parents in specific genome regions that were heterozygous in the original cultivar could result in phenotypic changes for traits of interest in the context of the performance of a well-defined variety. In addition, the development of two parental lines allows for the incorporation of an exotic gene  in one of them and the Resynthesis of the improved cultivar in a predictable time frame (two generations after the first cross).in vitro culture, two methods that are well established in a limited number of crops. Additionally, Reverse Breeding was proposed to generate homozygous parents to obtain a hybrid identical to one already existing, while our goal is the generation of individuals with a similar genetic background but with different characteristics with respect to a top performing clonally-reproducing cultivar.The number of crossovers per chromosome and meiosis is limited, usually between one and three in plants , and thex=8) , self-compatibility, and a compact, diploid and sequenced genome  , with a x=8) . In thisThis method consists of selecting two highly homozygous and complementary individuals in the selfed offspring of the original variety that, when crossed, reproduce most of its heterozygous make-up along with additional homozygous regions 8≈6.0×10−8, or 1.2×10−3 for five or more homozygous chromosome pairs  are monomorphic , identitectively . We have2 progeny of N=2,000, with 1.2 co/cs and 50% homozygosity, an average of nine plants with six or more homozygous chromosome pairs would be expected, or 50 plants with five or more homozygous chromosomes. In this case, the objective would be to select 1–5 pairs of plants with four or more chromosomes in homozygosis and complementary to each other. This is achievable because the number of individuals needed to find two complementary lines from a set of lines with four homozygous chromosomes is n=10 with a 0.95 probability. This is calculated with the formula n(n−1)/2=log0.05/log[(2x−1)/2x], where n is the number of individuals with x (four in this case) homozygous chromosomes. And approximately one in four of the possible pairs of these 50 plants with five homozygous chromosomes would have four homozygous chromosomes in common. It is also important to be sure that the three non-homozygous chromosomes of each pair of lines allow construction of two complementary lines without homozygous fragments of the same haplotype in both lines. By selfing these individuals, sets of genotypes could be obtained that are homozygous for six or more chromosomes and complementary in a sample of N=1,000. From these we would expect to obtain at least one pair of lines that, crossed, would produce a plant ≥ 95% genetically identical to SD with >10% probability.Considering as an example a peach F2 population of N=2,000 to test the Resynthesis B approach by selecting for heterozygous individuals with chromosomal compositions identical to SD, with the same probabilities as those calculated for finding a homozygous genotype  in July of 2015, 2016 and 2017 . Seeds were extracted, cleaned and kept in vacuum bags until November, when they were stratified at 4˚ C for three months, after which they were germinated in trays in the greenhouse. Germinated plants were transplanted to the field of the IRTA Experimental Station of Gimenells, and grown following standard agricultural practices.® Plant Mini Kit (Qiagen) following the manufacturer’s instructions. A paired-end sequencing library with an average insert size of 315 bp was prepared following the manufacturer’s instructions (Illumina). The library was sequenced at CNAG  using the Illumina HiSeq2500 platform at a paired-end mode with 2x250 cycles. Adapter removal and read trimming was done using Trimmomatic v0.36 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for quality control of the reads before and after adapter removal and trimming. With a good quality dataset, reads were mapped onto the peach genome downloaded from GDR (ftp://ftp.bioinfo.wsu.edu/species/Prunus_persica/Prunus_persica-genome.v2.0.a1/assembly/Prunus_persica_v2.0.a1_scaffolds.fasta.gz) using BWA-MEM (v0.7.5a) (Young leaves were collected in the spring of 2015 and DNA extracted for resequencing using DNeasyic v0.36 . FastQC v0.7.5a) . Alignmev0.7.5a) , taking 2 seedlings, SNPs covering the whole genome were selected. We chose 87 known to be heterozygous in SD and previously validated in other peach materials from the peach 9k array , germinated and produced viable plants. This is a low germination rate (~10%), considering that we used the standard methods of IRTA’s peach breeding program, where the average germination rate is 20–30%, although it is known that certain parents and crosses produce very poor yields, and that there is an extremely wide range of success (0–90%), depending on the cross. One of the reasons we selected SD was because, being a mid-season producing cultivar, no need for “Illumina sequencing of SD DNA generated 91,441,354 reads with a total output of 22.8Gbp. After quality filtering we were left with 81,866,684. Of these, 66,950,399 (81.8%) were mapped onto the peach genome, covering 94% of its length with at least five reads. The resulting average depth was determined to be 36X. In total, we detected 94,451 heterozygous SNPs, with chromosomes Pp02 having the highest and Pp08 the lowest number, and with an average of 11,806 heterozygous SNPs per chromosome .Prunus chromosomes. The order of the markers within the linkage groups was identical to their physical order in the chromosomes  were dominant, with only two genotypes. A map was constructed that detected all eight linkage groups corresponding to the expected eight For Resynthesis A, we selected four pairs of complementary lines with average heterozygosity of the two parents ≤0.29  that contained the full genome of SD and a few DNA fragments that were fixed  and complementary individuals to be selected.We found seven individuals nearly identical to SD (Resynthesis B), which gives us an opportunity to look for the effects of specific regions of the genome on key agronomic characters, although to a limited extent . More individuals will be produced with appropriate compositions from the crosses between the complementary pairs of Resynthesis A that, once genotyped, will allow for additional genome coverage. It would not be surprising if some of these individuals have characteristics that may already make them commercially interesting for selection as new cultivars.de novo’ sequencing.The two completely homozygous lines were an unexpected result, showing that these individuals can be recovered with a limited number of offspring. One of them was selected as one of the parents for Resynthesis A, but beyond that, these lines are of biological interest as a relatively rare event. The self-pollinating nature of peach provides the opportunity to produce inbreds, and certain traditional cultivars of Spain are highly homozygous  as a conx=8, 1.0 co/cs) Ri=16.0  Ri=16.1  Ri= 24.3 (x=17 1.5 co/cs) Ri=42.5  Ri= 47.5 (2 selected couples), and plants with valuable phenotypes selected at this stage. Selfing generations to fix specific lines could be done in parallel. Backcrossing of selected lines to obtain parents with genes from other materials (disease resistance or others), could also be started at early stages.Massive linkage disequilibrium is a favorable circumstance for Resynthesis and species with low chromosome numbers and low recombination rates, usually associated with small genomes , are goo Ri=16.0 , raspber Ri=16.1  or citruRi= 24.3  are exam Ri=42.5  or grapeRi= 47.5 , would rIt is important that the genotype to be resynthesized is self-compatible as the complementary lines are obtained from successive selfing generations. For self-incompatible genotypes, a possible way to circumvent the problem is crossing first with a self-compatible genotype, select the self-compatible individual in this progeny with the least number of recombination events in the original cultivar gamete, and start the selection from the BC1 between the hybrid line and the target genotype. Then only self-compatible lines having as much as possible of their genome fixed for one of the alleles of the target genotype (looking for pairs of complementary lines following a model similar to Resynthesis A) or that are highly similar to its genetic makeup (Resynthesis B), should be selected.2 and identifying genomic regions with highly distorted segregation frequencies. No regions of this type have been detected in our F2 population of SD.Another aspect to consider is the existence of lethal alleles in heterozygosis in the original cultivar. This cannot be discarded in highly heterozygous genomes of typically outcrossing species, such as those of almond and grape, especially in old cultivars of unknown origin. In this case, one of the two complementary lines must be kept in heterozygosis for the smallest possible genomic region around the gene causing the lethal effect, and selection should be for heterozygous individuals in the final cross between the two nearly-homozygous lines in the last step of Resynthesis A. Knowing whether a certain cultivar has lethal or deleterious alleles in heterozygosis could be tested by constructing a linkage map with markers in the F1 hybrids produced between them: whenever the evaluation of single genotypes  from segregating progenies can be done with sufficient accuracy, highly performing individuals could be selected and their genotype A resynthesized, following a strategy that somewhat mimics the clonally-propagated breeding scheme.Resynthesis could be expanded to other crops, particularly those that are seed propagated and where F1 hybrids are the usual cultivar type. In this case, there is an alternative approach to the usual generation of inbred lines and selection of the best performing FNovel resequencing data are deposited in ENA (European Nucleotide Archive) with sample ID: SAMEA5704508. SNPs for the genotyping are all described in detail in IE performed the proof of concept experiment, obtained and analyzed the SNP data and revised the manuscript. SA developed the population for the experiment and revised the manuscript. KGA, analyzed the DNA resequence data and revised the manuscript. PA designed the method, supervised the experiment and drafted and revised the manuscript.This research was supported in part by grants from: The Spanish Ministry of Economy and Competitiveness (MINECO/FEDER projects AGL2012-40228-C02-01 and RTA2015-00050-00-00), the project RIS3CAT (COTPA-FRUIT3CAT) financed by the European Regional Development Fund through the FEDER frame of Catalonia 2014-2020, the Severo Ochoa Program for Centres of Excellence in R&D 201-2019 (SEV-2015-0533) and CERCA Programme-Generalitat de Catalunya. This manuscript has been released as a pre-print at BioRxiv .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.


Bactrian camels can adapt to harsh natural environments. This unique tolerance of camels is tightly linked to their hematological traits, which are related to their immune, metabolic, and disease status. Therefore, mapping genomic regions that affect blood cell traits can help identify genomic characteristics that can be used as biomarkers of immune, metabolic, and disease states. This knowledge will further our understanding of the camel’s tolerance mechanisms.Camelus bactrianus) are one of the few large livestock species that can survive in the Gobi Desert. Animal immunity and disease resistance are related to hematological traits, which are also associated with tolerance observed in Bactrian camels. However, no genome-wide association studies have examined the genetic mechanism of the immune capability of Bactrian camels. In the present study, we used genotyping-by-sequencing data generated from 366 Bactrian camel accessions to perform a genome-wide association study for 17 hematological traits. Of the 256,616 single-nucleotide polymorphisms (SNPs) obtained, 1,635 trait–SNP associations were among the top quantitative trait locus candidates. Lastly, 664 candidate genes associated with 13 blood traits were identified. The most significant were ZNF772, MTX2, ESRRG, MEI4, IL11, FRMPD4, GABPA, NTF4, CRYBG3, ENPP5, COL16A1, and CD207. The results of our genome-wide association study provide a list of significant SNPs and candidate genes, which offer valuable information for further dissection of the molecular mechanisms that regulate the camel’s hematological traits to ultimately reveal their tolerance mechanisms.Bactrian camels ( Animal metabolism and immune status can be directly reflected by their hematological traits . TherefoCamelus bactrianus) and the one-humped dromedary (Camelus dromedarius) [Camelus ferus) are only distributed in these two countries [Camelus bactrianus is one of the few large livestock animals that can survive in the Gobi Desert because of their ability to withstand the harsh natural environment. This tolerance is largely associated with their metabolic and immune states. Because the hematological characteristics of mammals reflect their immunity and disease resistance [Camelus bactrianus. Camel species include the two-humped Bactrian camel (edarius) ,4. Chinaedarius) ,6 prevenountries ,8. Camelsistance ,10, decisistance ,13,14,15A GWAS is a powerful approach for identifying genomic regions that control important zoological traits in non-structured germplasms ,17. A suThe procedures and protocols for animal use were carried out according to the national codes of practice for the care and handling of farm animals and were approved by the animal care committee of the Camel Protection Association of Inner Mongolia.Camelus bactrianus breeds in East Asia, including four Chinese domestic breeds  and three Mongolian domestic breeds  was used to analyze the separated serum using the manufacturer’s reagents. The analyzer calculated the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (CHO), and glutamyl transpeptidase (GGT) (This study included 366 domestic Bactrian camels. The sample population represented the seven main domestic amel see ). Whole se (GGT) .The sequence data were generated using genotyping-by-sequencing (GBS) ,21. The The raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) in the GenBank database under accession number PRJNA565837.Camelus ferus genome [ftp://ftp.ncbi.nih.gov/genomes/Camelus_ferus/CHR_Un/. Population single-nucleotide polymorphism (SNPs) were detected using the SAMtools software [High-quality sequencing data were aligned to the s genome  using ths genome  (parametsoftware . Bayesiasoftware .http://treesoft.sourceforge.net/treebest.shtml) was used to calculate the distance matrix, which was the basis for constructing the phylogenetic tree using the neighbor-joining method. The distance between two bodies i and j was calculated using the following formula.L is the length of the SNP region, and the allele at position 1 is A/C. If the genotypes of the two individuals are AA and AA, then ijd = 0. If they are AA and AC or AC and AC, then ijd = 0.5. If they are AA and CC, then ijd = 1.A phylogenetic tree and principal component analysis (PCA) were used to investigate the population structure. The TreeBeST software (http://cnsgenomics.com/software/gcta/pca.html) was used to calculate the feature vectors and eigenvalues. The R software was used to draw the PCA distribution map. The SNP at position k in individual i is represented by ikd. If individual i is homozygous with the reference allele, then ikd = 0. If it is heterozygous, then ikd = 1. If individual i is homozygous with the non-reference allele, then ikd = 2. M is a matrix of n × S containing the standard genotype.The GCTA software (http://pngu.mgh.harvard.edu/~purcell/plink/), a Ped file, and then used the Frappe software to construct the population genetic structure and group lineage information.The SNP data of the 366 camels were analyzed using the Frappe software to establish the population structure. First, we created an input file for software tool, PLINK, (−6 (0.05/256616).The Bonferroni calibrated multiple test was used to determine the significance threshold, with a genomic significant level threshold of 0.05 per effective SNP locus and a chromosomal significant horizontal threshold of 1 per effective SNP locus. For the purpose of this study, the corresponding thresholds were set as 6.71 × 10http://www.xzlab.org/software.html), and the population structure and individual kinship were corrected using the mixed linear model (MLM).y is the phenotypic trait, X is the indicator matrix of the fixed effect, α is the estimated parameter of the fixed effect, Z is the indicator matrix of the SNP, β is the effect of the SNP, W is the indicator matrix of the random effect, μ is the predicted random individual, and e is a random residual and obeys e~.GWAS analysis was performed using the GEMMA software  was above 6.46% .At the genomic level, DNA sequence variations can be caused by an SNP, including single-base conversions and transversions. A total of 2,433,732 SNPs were identified using the SAMtools software, and the obtained SNPs were filtered . Lastly, 256,616 SNPs were obtained for subsequent analysis. The results of the ANNOVAR software analysis for SNP annotations are shown in The GWAS data were corrected for the presence of a population structure using three methods to identify relationships among the analyzed genotypes. The results of the phylogenetic tree showed that the 366 genotypes could be divided into seven groups, which is consistent with the number of breeds in the analysis . PCA resA structural analysis was performed on the 366 samples. Each column in p-value of 10−6, the analysis identified 1,635 trait–SNP associations as the top quantitative trait loci (QTLs)  . Howeveri (QTLs) .p-value < 10−6). WBC traits and RBC traits , platelet traits , GGT, and CHO were identified as associated with 5, 226, 41, 50, and 342 genes, respectively. To further evaluate the SNPs associated with the chosen traits, we aligned the tag sequence in which the SNP is located with all known sequences to infer its location and identify potential candidate genes that carry true etiological polymorphisms. The upstream and downstream 20 kb of 1635 SNP sites, which are known to be candidate genes in the camel genome, were searched randomly. Lastly, we identified 664 candidate genes associated with 13 blood traits (In this study, we performed a GWAS for camel hematological traits using the genotypes of 366 domestic Bactrian camels from China and Mongolia. To the best of our knowledge, this is the first GWAS for camel hematological traits using GBS.Population stratification affects the proportion of false positives in genome-wide association analysis. Therefore, corrections are needed to ensure the reliability of the statistical analysis ,26,27. VZNF772, MTX2, ESRRG, MEI4, IL11, FRMPD4, GABPA, NTF4, CRYBG3, ENPP5, COL16A1, and CD207 (for their full names, see The identification of markers that are tightly linked to phenotypic characteristics is the ultimate goal of genetic mapping ,33. The ZNF772 is likely to be involved in the regulation of early white blood cell differentiation [CRYBG3 and ENPP5, as important protein structure regulators, are likely to be involved in the regulation of platelet volume and size.WBCs are involved in the phagocytosis of bacteria and disease prevention. RBCs are essential for oxygen transport within the organism and the removal of the byproducts of respiration. The platelets play an important role in blood coagulation, wound healing, inflammatory response, and organ transplant rejection . The whintiation . Red blontiation . Gene ESntiation  and mighAlthough we identified 12 candidate genes, their mechanism of action remains unclear and requires further study and confirmation. These candidate genes lay the foundation for further exploration of the characteristics of blood traits in Bactrian camels to reveal their tolerance mechanisms.Camelus bactrianus. As a result, we identified 12 candidate genes and associated genetic markers, which provide a reference for further research on the camel’s special immune metabolism and disease resistance. Therefore, this study contributes to the understanding of the molecular regulation of blood traits in domestic animals.In conclusion, because blood traits are effective indicators of the camel’s immune and metabolic status, we attempted to identify genomic regions that control hematological traits in


Vigna radiata L. Wilczek) is an annual grain legume crop affected by low availability of phosphorus. Phosphorus deficiency mainly affects the growth and development of plants along with changes in root morphology and increase in root-to-shoot ratio. Deciphering the genetic basis of phosphorus use efficiency (PUE) traits can benefit our understanding of mungbean tolerance to low-phosphorus condition. To address this issue, 144 diverse mungbean genotypes were evaluated for 12 PUE traits under hydroponics with optimum- and low-phosphorus levels. The broad sense heritability of traits ranged from 0.63 to 0.92 and 0.58 to 0.92 under optimum- and low-phosphorus conditions, respectively. This study, reports for the first time such a large number of genome wide Single nucleotide polymorphisms (SNPs)  in mungbean. Further, genome wide association study was conducted using 55,634 SNPs obtained by genotyping-by-sequencing method. The results indicated that total 136 SNPs shared by both GLM and MLM models were associated with tested PUE traits under different phosphorus regimes. We have identified SNPs with highest p value (–log10(p)) for some traits like, TLA and RDW with p value (–log10(p)) of more than 6.0 at LP/OP and OP condition. We have identified nine SNPs (three for TLA and six for RDW trait) which was found to be present in chromosomes 8, 4, and 7. One SNP present in Vradi07g06230 gene contains zinc finger CCCH domain. In total, 71 protein coding genes were identified, of which 13 genes were found to be putative candidate genes controlling PUE by regulating nutrient uptake and root architectural development pathways in mungbean. Moreover, we identified three potential candidate genes VRADI11G08340, VRADI01G05520, and VRADI04G10750 with missense SNPs in coding sequence region, which results in significant variation in protein structure at tertiary level. The identified SNPs and candidate genes provide the essential information for genetic studies and marker-assisted breeding program for improving low-phosphorus tolerance in mungbean.Mungbean ( Vigna includes about 100 species dispersed in subtropical and tropical region globally (Vigna mungo), cowpea (Vigna unguiculata), adzuki bean (Vigna angularis), moth bean (Vigna aconitifolia), creole bean (Vigna glabrescens), and tuber cowpea (Vigna vexillata) have been domesticated (Vigna radiata var. sublobata (Roxb.) Verdc., occurring in wild state in Western Ghats and Sub-Himalayan tract. From India, mungbean spread to Southeast Asia and through silk road  in South Asia and as sprouts, noodles, and paste in East Asia.The genus globally . Among tsticated . The wilsticated . Mungbeailk road . Mungbeailk road . The croilk road . MungbeaPhosphorus (P) is required for photosynthesis, redox reactions, energy production, and phosphorylation/dephosphorylation. The uptake of inorganic P by plants ranges from 10 to 25% . DespitePup1-specific protein kinase gene and phosphorus-starvation tolerance 1 is reported to be enhancer of early root growth which leads to more acquisition of P in plants  measures ability of plant for phosphorus uptake and its use for production of biomass or yield . Under Pn plants .The quantitative trait loci (QTLs) for PUE traits in response to P starvation have been reported in crop plants such as wheat , rice L, corn Z, common The association mapping (AM) utilizes panel of diverse genotypes for genome-wide association studies for identification of trait-linked molecular tags. QTLs are identified based on association of marker with trait elucidated by LD between makers and polymorphisms across the AM panel. AM has advantage of the populations undergoing many generations of recombination. Thus, AM provides much finer resolution than genetic mapping. Genome-wide association study (GWAS) have evolved over the last few years into a powerful tool which exploits hundreds of markers across all chromosomes to find genetic variations associated with particular trait . GWAS anSingle nucleotide polymorphism (SNP) markers are highly desirable for marker-assisted selection (MAS) due to their abundance, locus specificity, codominant inheritance, and high-throughput genotyping . Signifiw/v) HgCl2 for 3 min and washed thrice with distilled water to remove detergent. The seeds were kept for germination in germination paper. After emergence of cotyledonary leaves, uniformly germinated seedlings were transferred to modified Hoagland solution with two levels of P. Two P levels were balanced using KH2PO4 as optimum P (250 μM) and low P (3 μM). The composition comprised CaCl2⋅2H2O (0.75 mM), K2SO4 (0.92 mM), Fe-EDTA (0.04 mM), urea (5 mM), MgSO4 (1 mM), and micronutrients  , and 62 exotic germplasm lines . The exp0.6 μM)] . NutrienTwenty-one-day-old seedlings grown under optimum- and low-P conditions were carefully separated into leaf, stem, and root to record observations on PUE traits. Isolated complete root system of each plant was placed on a tray with no overlapping of roots for recording observations on root traits. Root traits recorded include total root volume (TRV), total root length (TRL), root average diameter (RAD), total root surface area (TSA), total root tips (TRT), and root forks (RF). These were measured using a root scanner (EPSON XL 1000) with WinRhizo software . Primary root length (PRL) was determined manually using meter scale. Chlorophyll concentration (CHL) was measured by using MC-100 chlorophyll concentration meter . Total leaf area (TLA) was measured using leaf area meter . Separated shoots and roots were dried at 60°C until constant mass of root dry weight (RDW) and shoot dry weight (SDW) was obtained and then root-to-shoot ration (RSR) was calculated. All the traits have been measured in three replications. The PRL and TRL have been measured in centimeters per plant. TSA, TRV, and RAD have been measured in square centimeters per plant, cubic centimeters per plant, and centimeters, respectively. CHL and LA have been measured in micromoles per square meter and square meters per plant, respectively. RDW and SDW have been measured in grams per plant . Descrip1 according to de novo-based GBS approach of STACKS. Subsequently, the putative SNPs were discovered among genotypes following The genomic DNA was isolated from the AM panel genotypes. The isolated genotypes were used to constitute two 144-plex GBS library and sequenced (2 × 150-bp single end) using Illumina HiSeq 4000 NGS platform according to The reference genome GBS-based SNPs identified in genic and intergenic component of genomes were annotated according to mungbean genome annotation .r2) and LD decay  had been determined. The LD had been measured as frequency correlation among pair of alleles across a pair of SNP loci.The genotyping data among mungbean genotypes were analyzed with PowerMarker v3.51  and MEGA10(p) value for each associated SNP. The adjusted p value threshold of significance was corrected for multiple comparisons according to false-discovery rate (FDR cut-off ≤ 0.05) (R2 (degree of associated SNP) and lowest FDR adjusted p values (threshold p < 1 × 10–5) were identified by integrating all the model-based outputs of TASSELv5.0.The genome-wide SNP genotyping information of mungbean AM panel was integrated with PUE trait phenotyping data, ancestry coefficient (Q matrix), and relative kinship matrix (K) data using general linear model (GLM) (Q model) and mixed linear model (MLM) approach. The quantile–quantile plot had been generated to determine relative distribution of observed and expected log ≤ 0.05) . The SNPArabidopsis ortholog of candidate gene from mungbean genome for analysis. The gene expression pattern has been searched using Expression Angler, an online search tools  for all traits except PRL, TRV, and TSA, while RDW showed general significance (p < 0.05)  . The mea < 0.05) . The coeV. radiata reference genome. In total, 76,160 high-quality SNPs  differentiating 144 samples were discovered using mungbean reference genome-based GBS assay. The genome-wide GBS-based SNP genotyping overall identified 55,634 chromosome-based SNPs showing polymorphism among mungbean genotypes of AM panel. The distribution of all SNPs was found to be in different components of genome including intergenic (27%), intragenic (23%), regulatory (31%), and scaffold (19%) variants  across 144 genotypes of AM panel. Notably, on an average, 75% of these reads were mapped on variants . A totalvariants . A maximvariants . In totavariants . The funvariants . The strThe identified 55,634 genome-wide GBS-based SNPs revealed polymorphism among AM panel mungbean genotypes with higher nucleotide diversity  potential. Any significant difference for nucleotide diversity value have not been found within the different set of genotype group such as, advanced varieties, landrace, etc. However, we have compared the diversity (θπ) value among the related species, like soybean and chickpea. It has been found that the diversity in soybean was θπ: 0.33 which was comparable to mungbean . In chickpea, θπ was found to be 0.99, which is higher as compared with mungbean. The unrooted neighbor-joining phylogenetic tree construction using 55,634 genome-wide SNPs depicted a distinct differentiation among the AM panel of mungbean . These gr2) and extent of LD decay were determined using 55,634 SNPs mapped on 11 mungbean chromosomes. We determined the LD decay of 55,634 SNP pairs by pooling the r2 estimates across 11 chromosomes of mungbean and plotting their average r2 against the 50-kb equal intervals of physical distance . The determination of LD patterns in a population of AM panel genotypes using 55,634 SNPs  showed a higher LD estimate  and less-extensive LD decay  at 50–100 kb physical distance in mungbean chromosomes (r2 ≥ 0.3) was observed with increase in the physical distance (cM) of SNP markers mapped on the 11 chromosomes. The population genetic structure among AM panel genotypes was determined using SNPs (mapped on 11 mungbean chromosomes) employing STRUCTUREv2.3.4 software. The admixture model-based STRUCTURE simulations were performed at different levels of possible population numbers (K = 1 to 10) with 20 replications. The population numbers (K) were determined using the second-order statistics of STRUCTUREv2.3.4 by delta K estimation. The results showed a maximum value of delta K at 3  = 4.0) was used as a cutoff to identify the significant marker trait associations. A total of 1,171 and 139 significant SNPs were identified by GLM and MLM, respectively. Of these, 136 SNPs were identified by both models  explained by the SNPs ranged from 13.28 to 17.25%.Under optimum-P condition (OP), a total of 597 SNPs identified by GLM were found to be associated with all tested traits in the study. The GLM detected 39 and 18 significant SNPs for RAD and CHL, respectively; for which no significant SNP was detected by MLM. The MLM-detected maximum number of SNP loci (13) was found to be associated with TRT with 13.93% contribution of phenotypic variation. The MLM detected 3, 4, 7, 9, 6, 10, 10, 6, and 1 significant SNPs for PRL, TRL, TSA, TRV, RF, TLA, RDW, SDW, and RSR, respectively . The pheUnder low-P (LP) condition, a total of 317 significant SNPs associated with tested traits were detected by GLM. GLM detected 35, 45, 18, and 8 SNPs for TRL, TSA, TRV, and RAD, respectively, for which no significant SNP was detected by MLM. MLM detected 1, 2, 7, 4, 5, 10, 2, and 1 significant SNPs for PRL, TRT, RF, CHL, TLA, RDW, SDW, and RSR, respectively. The phenotypic variation explained by SNPs ranged from 12.88 to 14.59%. At LP/OP condition (trait ratios), a total of 257 and 38 significant SNPs associated with tested traits were identified by GLM and MLM, respectively. MLM detected 2, 2, 6, 2, 1, 18, 5, 1, and 1 significant SNPs for TRL, RAD, TRT, RF, CHL, TLA, RDW, SDW, and RSR, respectively. No significant SNPs were identified by both models for trait ratios of TSA and TRV. The phenotypic variation explained by SNPs for trait ratios ranged from 12.06 to 15.02%.At low-P and optimum-P conditions, MLM detected a maximum number of SNP loci (10) associated with RDW trait with around 14.59 and 17.25% of average phenotypic variation, respectively. Whereas, at LP/OP condition, a number of five SNP loci were found to be associated with RDW trait. At LP/OP conditions, MLM detected a maximum number of SNP loci (18) found to be associated with TLA trait and contributed around 15% of phenotypic variation. Whereas, at OP and LP conditions, a number of 10 and 5 SNP loci were found to be associated with TLA traits, respectively. The association results were further confirmed by analyzing quantile–quantile plot . In tota2 search against Arabidopsis genome database . The study identified a total of 13 genes with highest similarity and identity (≥80%) to the Arabidopsis genes. These genes were well characterized and found to be involved in different nutrient uptake and root architecture development pathway  (At1G32080), At5PTASE9 (At2G01900), Ca2+-sensor protein kinases-NIN-like protein 7 (CPK-NLP7) (At2G37590), cellulose synthase-like D gene (CSLD3) (At5G16910), K+ channel Arabidopsis thaliana (KAT1) (At4G11480), ubiquitin-conjugating enzyme E2 13 (UBC13) (At3G46460), fibroblast growth factor receptor substrate 3 (FRS3) (At2G27110), GTPase root hair defective 3 (RHD3) (At4G39250), iron-regulated transport er1 (IRT1) (At5G58440), PHO80 (At5G64260), K+ uptake 7 (KUP7) (At5G09400), speechless (SPCH) (At5G53210), and KAN protein (KANADI) (At4G25440). We found that three genes VRADI11G08340, VRADI01G05520, and VRADI04G10750 containing missense SNP at CDS region. These three genes may regulate PUE traits by changing at protein structure level. Therefore, we determined the protein structure by I-TASSER3 for these three genes in order to decipher the functional complexity at protein structure level. However, we were able to predict the protein structure for two genes   . The str effects . For exaAT1g32080, AT2g01900, AT2g27110, ATg37590, AT3g46460, AT4g11480, AT5g16910, AT5g53210, AT5g58440, and AT5g64260 gene  had the highest expression in lateral root cap, pericycle, lateral root cap, whole root, seed root cell and hairs, whole root, root hair, pollen, seed, and shoot apical meristem, respectively  reported in our study can be utilized for genotyping and QTL mapping for several traits as well as comparative genome mapping involving mungbean and legumes. Additionally, 55,634 chromosome-based SNPs can be used for marker-based applications of mungbean breeding.The discovered SNPs were annotated in different components of protein-coding genes of mungbean genomes. The structural and functional annotation revealed that a total of 29,923 SNPs present in intergenic region and 25,663 SNPs in intragenic region. Among the intragenic variants, 10,423 and 13,030 SNPs were found to be present in intron and CDS regions, respectively. Further, the coding variants represented 5,643 and 7,387 missense and synonymous variants. A large number of SNPs were found to be present in regulatory region. The upstream and downstream regulatory region consisted of likely equal number of SNPs . The structural and functional annotation of genes will accelerate the pace of AM to identify a number of genes/QTLs governing qualitative and quantitative agronomic traits in mungbean. Tajima’s D is a statistical test which is performed to test the null hypothesis of mutation-drift equilibrium and constant population size. A significantly higher Tajima’s D (3.21848) value were observed in our used AM panel. The results indicate that mungbean may undergo for a population reduction , populatIn our study, high-resolution LD pattern were determined in a structured population of 144 mungbean accessions. In soybean and chickpea, several studies have been carried out using GBS and WGRS (Whole genome re-sequence) approach to find the LD pattern and dissect complex trait . In a str2, 0.62) and less extensive LD decay with approximately 50–100 kb physical distance in mungbean chromosomes. In a previous study, the LD decay was found to be extensive with approximately 60–100 kb physical distance in a panel of cultivated and wild mungbean accessions , inositol polyphosphate 5-phosphatase (At5PTASE9), and CPK-NLP7. The PLGG1 encodes a chloroplast protein which regulates lateral root development in response to ABA (Arabidopsis. Interestingly, the AT5PTASE9, inositol polyphosphate 5-phosphatase is previously reported to be involved in phosphate homeostasis (AT5PTASE9 is a member of the AT5PTASE family, which regulates several responses, such as the calcium influx, ROS production, bulk endocytosis, and induction of stress-responsive genes (CPK-NLP7 is found to be involved in a transcriptional regulatory network that regulates the root and shoot architecture in response to changes in nitrogen availability (VRADI11G08340, VRADI01G05520, and VRADI04G10750 between native and mutated sequences suggests that the respective protein differ in domains and structure for post-transcriptional modification (Among these 13 putative candidate genes, we identified missense SNPs in CDS region of three genes, e to ABA . Trull eeostasis . The AT5ve genes . The CPKlability . The misfication . The diffication . TherefoWe performed GWAS for PUE traits in a population containing 144 diverse mungbean genotypes with 55,634 SNP markers. The genetic diversity analysis, LD analysis, and population structure of this study will accelerate the pace of mungbean genomics research which can be directed toward genetic improvement of mungbean. The identified SNPs and candidate genes associated with PUE traits can be used in mungbean breeding program to enhance phosphorus deficiency tolerance. These results demonstrated the efficacy and reliability of implementing high-resolution AM in mungbean which has a narrow genetic base and least availability of genomic resources. However, these genes need to be validated further to shed light on their functional role in regulation of the PUE traits in mungbean. Overall, the large number of genomic resources developed in our study will lead to enrich mungbean genomics. These resources can also be utilized to advance genomic-assisted breeding in mungbean.https://www.ncbi. nlm.nih.gov/sra/PRJNA609409.The original contributions presented in the study are publicly available. This data can be found here: HD, GM, RP, MS, and VR planned and designed the research. VR, MA, SM, and AS performed the hydroponics experiment. VR, P, TB, KT, and PG helped in data recording. VR, MA, and GM conducted genotyping of the association mapping panel. VR and SD conducted the STRUCTURE, TASSEL, and Bioinformatic analysis. HD, VR, and SD prepared the manuscript. RN, TS, and SK edited the manuscript for publication. All authors have read and approved the final manuscript.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. The reviewer MKP declared a past co-authorship with one of the authors GM to the handling editor.


Pseudotsuga menziesii). Results suggest a complex genomic architecture of cold adaptation, in which traits are either highly polygenic or controlled by both large and small effect genes. Newly discovered associations for cold adaptation in Douglas-fir included 130 genes involved in many important biological functions such as primary and secondary metabolism, growth and reproductive development, transcription regulation, stress and signaling, and DNA processes. These genes were related to growth, phenology and cold hardiness and strongly depend on variation in environmental variables such degree days below 0c, precipitation, elevation and distance from the coast. This study is a step forward in our understanding of the complex interconnection between environment and genomics and their role in cold-associated trait variation in boreal tree species, providing a baseline for the species’ predictions under climate change.Understanding the genomic and environmental basis of cold adaptation is key to understand how plants survive and adapt to different environmental conditions across their natural range. Univariate and multivariate genome-wide association (GWAS) and genotype-environment association (GEA) analyses were used to test associations among genome-wide SNPs obtained from whole-genome resequencing, measures of growth, phenology, emergence, cold hardiness, and range-wide environmental variation in coastal Douglas-fir ( Understanding the fitness consequences of naturally occurring genetic variation is of great biological interest. In natural populations of widely distributed species, the challenge is to identify genes underlying traits that increase the species’ ability to survive, thrive and reproduce ,2. SpeciIn the Northern hemisphere, the interactions between growth, phenology and cold hardiness are main determinants of survival in tree populations. Cold adaptation in forest trees involves significant physiological, cellular, genetic and morphological changes, and is highly synchronized with environmental cues such as photoperiod, day length and temperature ,10. TempThe identification of genes underlying variation in growth, phenology and cold hardiness has been particularly difficult due to the highly polygenic nature of the traits. Univariate genome-wide association studies (GWAS) successfully identify genes of major effect (in which a new advantageous mutation is rapidly driven to fixation) but have very low power to detect weakly selected loci, characteristic of polygenic adaptation ,13,14. TP. menziesii) is an economically and ecologically important species in western North America. Considered one of the most important sources of lumber worldwide, it is grown extensively in plantations throughout Europe [menziesii and glauca, have been identified in coastal  and intermountain regions ; although phylogeographic studies using mitochondrial and chloroplast DNA suggest the presence of three, instead of two, distinct varieties  [Douglas-fir  . Common Mexican) , adaptatMexican) ,22. ColdMexican) , QTL mapMexican) ,25, and Mexican) . Genome-Mexican) . SequencMexican) . In thisSeeds from ten individuals spanning most of the coastal Douglas-fir’s natural distribution  were collected for whole-genome re-sequencing analysis. After collection, seeds were soaked in water at room temperature for four days, then haploid megagametophytes were dissected from each seed. DNA was extracted with a Qiagen DNeasy mini-prep Plant kit, and DNA quality and concentration were evaluated using nanodrop and picogreen on a Qubit 2.0 Fluorometer, respectively. Sequencing libraries were constructed using Illumina’s TruSeq Nano DNA Library Prep Kit . Prior tRaw reads from whole-genome re-sequencing data of ten Douglas-fir individuals were aligned to the 16 Gbp Douglas-fir reference genome version Psme v1.0 ,28; usinSeeds were obtained from 288 open-pollinated parent trees from throughout the range of Douglas-fir in western Washington and Oregon. Progeny from the parent trees were measured in a common garden study for adaptive traits including growth, phenology, emergence, and cold hardiness . The same-value < 1 × 10−10), and by using the Douglas-fir’s genome annotations [Seeds were soaked in a solution of 70% water and 30% of 3% hydrogen peroxide for 12 h. After that, megagametophyte haploid tissues from ten half-sib individuals for each family were pooled together to infer the maternal genotype. Megagametophytes DNA was extracted using the Qiagen DNeasy mini-prep Plant kit and an Eppendorf automated pipetting workstation. The extraction protocol included one day of tissue lysis and incubation at 96 °C, followed by several steps of precipitation and filtering. DNA quality and concentration were assessed using nanopore and picogreen on a Qubit 2.0 Fluorometer, respectively. Samples were genotyped using a custom-based multi-species Illumina Infinium SNP array comprising 80 k SNP markers from which 20 k were designed for Douglas-fir and 60 k for sugar pine. Ilumina’s Genome Studio Genotyping Module v2.0.4 was used to call genotypes, filter, and generate genotyping statistics for all samples and SNPs. Filtering was applied to Douglas-fir and sugar pine SNPs separately and the pass-filter SNP data were subsequently merged. All SNPs with a call frequency ≤ 0.65 and all individuals with a call rate ≤ 0.8 were not included. Further, markers were filtered out based on minor allele frequency (>0.01) to remove all monomorphic and low-quality SNPs. SNP functional annotations were obtained from aligning against the full NCBI non-redundant protein sequences database (nr) using BLASTP  implemented in the “adegenet” R package ,37. PopuTo rule out the possibility that any patterns observed were due to isolation by distance (IBD), pairwise genetic and physical distances were calculated in the R package adegenet ,37. A maPhenotypic data were measured as part of a large genecology experiment containing 1338 families from 1048 locations planted in a randomized plot design, seedlings were grown for two years in a common garden in Corvallis, Oregon . A subseA multivariate GWAS was performed by fitting a Bayesian sparse linear mixed model (BSLMM) in GEMMA v0.98 . BSLMM u using β . Analysi using β ,47.2), and the dominance and additive effects were also calculated with TASSEL v.5.In addition, a univariate GWAS using mixed linear model (MLM) was performed in TASSEL v.5. The first principal components of a PCA were used as co-variates to control for population structure, and a kinship matrix to account for relatedness . BreedinElevation data were obtained from GIS coverages using a digital elevation model (DEM). Climate data were obtained using ClimateNA . Climatep-value = 0.012) for each of the first two axes.A redundancy analysis (RDA) as implemented in the vegan R package  was perfIn addition to the multivariate GEA analysis, two univariate GEAs were performed using the programs Bayenv v.2  and TASSPcadapt R package  was usedOf the 458M SNPs called in the whole genome re-sequenced individuals, a total of 1.2M SNPs were retained after filtering following the SNP calling process. All SNPs were scored by Illumina using in silico design scores to maximize the number of markers for genotyping that will provide high conversion rates. As a result, 20 k best-scoring SNPs were included in the Illumina Infinium array for SNP genotyping. From this, genotypes were obtained from 16,146 SNPs. Posterior genotype clustering, and removal of monomorphic and low-quality SNPs led to 20,397 SNPs, from which 5815 were originally designed for sugar pine. SNPs selected for posterior analyses were distributed along 5892 scaffolds in the Douglas-fir genome and matched 6833 coding regions (4301 genes plus 2532 transcripts from unknown genes). The distribution of SNPs minor allele frequencies can be seen in A K value of 2 was shown to be most optimal using the choose K script included in fast STRUCTURE. Distruct  used theAll correlations among phenotypic traits and environmental variables are represented in heatmaps in PCAdapt identified 582 outlier loci distributed across 290 scaffolds in the Douglas-fir genome . From thr = 0.09, p-value = 0.001). There was no significant relationship between genetic distance and geographic distance when individuals were grouped into populations . See When individuals were not grouped into populations, mantel tests revealed very low levels of isolation by distance  correction for multiple-testing . A totalIn contrast, GEMMA identified 7536 significant associations among 6034 SNPs and all 23 traits . SignifiIndividual SNP markers explained from 6 to 24%  of the phenotypic variation for all traits, whereas combined SNP markers explained up to 93% . Cold haTASSEL univariate association analyses identified 74 SNPs significantly associated with 17 environmental variables . Most SNThe multivariate (RDA) GEA method identified a total of 528 significant SNPs for continentality (255 SNPs), precipitation as snow (219 SNPs), and summer heat moisture index (54 SNPs) . Of thesPinus yunnanensis [In agreement with previous studies of natural populations of highly-outcrossing, widely distributed boreal and temperate conifer species ,9,26,33,nanensis . In contnanensis ,59, and nanensis . FurtherPinus taeda [Pinus lambertiana [Populus balsamifera [Our results highlight the role of both coding and non-coding regions in cold adaptation of the species. We believe these regions are widely distributed across the genome, however, in the absence of a high-density linkage map or a chromosome-scale reference genome, our study could only identify genomic locations at the scaffold-level. We identified multiple SNPs or genes within the same scaffolds. Due to the highly fragmented nature of the Douglas-fir reference genome, we assume genes located in the same scaffold have a high chance of being linked. A widespread genomic location of adaptive genes was also found in recent GWAS and GEA analyses in us taeda ,61, Pinubertiana  and Popusamifera .Zea mays [Populus trichocarpa [Pinus contorta [Pinus sylvestris [Both GWAS and GEA analyses suggested a polygenic basis of cold adaptation, with many genes associated with cold-related traits and climate adaptation. This is coincident with recent genome-wide analyses in widespread, outcrossing plant species with large population sizes such as Zea mays , Populuschocarpa , Pinus ccontorta , and Pinlvestris . Trait aPinus contorta [Pinus taeda [Aspects of climate are highly correlated with each other, complicating the interpretation of genome-wide environmental associations. Our GEA analyses identified several hundreds of SNPs associated with a large number of temperature and precipitation related variables , suggestcontorta  and Pinuus taeda . MultivaPseudotsuga menziessii [Picea sitchensis [Populus trichocarpa [Pinus sylvestris [Abies sachalinensis [Acer rubrum, Betula alleghaniensis, Quercus rubra and Juglans nigra [Trees living in cold environments must balance the timing of growth initiation with the risk of frost . If a trnziessii ,26, Picetchensis , Populuschocarpa , Pinus slvestris ,72,73, Alinensis , Acer runs nigra .Our results showed the complex relationships between genotypes, growth , emergence , phenology , cold hardiness, and environmental variables such as degree days below 0c, precipitation as snow, and continentality (TD) . The impCold adaptation in forest trees involves significant physiological, cellular, genetic and morphological changes ,10. In oPlant secondary metabolites play an important role in adaptation to changes in the environment . In our Arabidopsis, overexpression of HOS1 (RING-type E3 ligase) and AtCHIP (U-box type E3 ligase) led to reduced cold hardiness [Plants employ complex signaling pathways to regulate the expression of defense and stress genes and other mechanisms that allow resistance to environmental stress . The ubiardiness . In our P. sitchensis [Members of the thaumatin family (“antifreeze proteins”) are upregulated during cold stress and are potentially regulated by photoperiod in tchensis . In our tchensis ,82. Membtchensis ,84 and ptchensis , growth tchensis . In our Picea mariana [Due to the rapid nature of climate change and the slow nature of plant migrations, proactive strategies should be employed by forest managers to mitigate potential damages resulting from the increased frequency of extreme cold and drought events . As glob mariana . Moreove mariana . Our stu


In Ethiopia, iodine deficiency disorder (IDD) is a major public health problem. The most visible effect of IDD is the appearance of goiters, and 28 million people are affected by goiter. Therefore, this study aimed to assess the prevalence and associated factors of goiter among high school adolescent girls at Tach Armachiho district, Northwest Ethiopia. p value of less than 0.05 in the multivariable analysis were considered as significant factors for goiter.  An institution-based cross-sectional study was conducted from October to November 2018. A total of 620 high school adolescent girls were selected using the simple random sampling technique, and structured questionnaires having observational check-list were used for data collection. The presence of goiter was estimated using criteria set by World Health Organization. Iodine content of the salt was estimated by using spot testing kits. Both bivariable and multivariable logistic regressions were used to identify associated factors. The degree of association between independent and dependent variables was assessed by using odds ratio with 95% confidence interval. Those variables having  A total of 614 adolescent girls gave a complete response. The prevalence of goiter among adolescent girls was 24.1%. In the multivariable logistic regression analysis, age of adolescent girls (adjusted odds ratio (AOR) = 1.57, 95% CI: 1.01–2.46), residence , family history , low dietary diversity , and medium dietary diversity  were significantly associated with adolescent girls goiter.  Goiter among high school adolescent girls in this study was high. Age of adolescent, residence, family history of goiter, and dietary diversity were statistically significant factors for goiter. Therefore, more emphasis will be given for late adolescent age, having family history of goiter, low dietary diversity, and rural residence to improve the burden of adolescent goiter. Goiter, which refers to an abnormal enlargement of the thyroid gland, is one of the most common endocrine problems for children and adolescents . Each ofThe consequences of iodine deficiency are goiter, hypothyroidism, physical and neurophysiologic defects, mental retardation, and brain damage . The eduDifferent studies show that the prevalence and associated factors of goiter among adolescent girls varies from country to country. For instance, in Africa, the burden of goiter ranges from 34.5% to 37% on an average; in Ethiopia, about 30% of the adolescent girls develop goiter . The magAlthough the Government of Ethiopia has increased its efforts to prevent goiter, its magnitude among adolescent girls is still high, and factors vary from place to place. Therefore, this study aimed to assess the magnitude of goiter and associated factors among adolescent girls in Tach Armachiho district.An institution-based cross-sectional study was conducted from October to November 2018 among adolescent high school girls at Tach Armachiho district, Northwest Ethiopia, located 854 km from Addis Ababa, the capital of Ethiopia. The district has 38 kebeles . Its altitude is 1050 to 1800 ms above sea level; 42% of its area is mountainous, with a temperature of 25–42 degree Celsius. According to the 2018 report, the district had four high schools with 1,972 adolescents. One district hospital and nine health centers provide health services.The single population proportion formula was used to determine the sample size by considering 37.6% previous prevalence of goiter , 95% conAdolescent girls who were learning in high school during data collection were included in the study, and adolescent girls who suffered from serious illness during the period of data collection were excluded from the study.For the general data collection, eight nurses, two general practitioners, and two public health experts participated as supervisors, after they were given two-day training by the principal investigator. In order to maintain data quality, a pretest was administered on 5% of the sample in Tsegede district out of the study area. Onsite supervision was performed, and each copy of the questionnaire was checked for completeness and accuracy before data entry.Physical examination of a thyroid gland was performed by two skilled general practitioners (GPs), using WHO/ICCIDD/UNICEF clinical criteria. Accordingly, goiter was defined as grade 0 if no palpable mass in the neck was detected, grade 1 if there was a mass in the neck consistent with palpable enlarged thyroid, but not visible when the neck was in the normal position, whereas grade 2 if there was a swelling in the neck that was visible when the neck was in a normal position and is consistent with an enlarged thyroid when the neck is palpated . Lastly, the child was deemed as having goiter when he/she had goiter of grade 1 or 2 .Iodine level of salt was determined by a rapid test kit (ARCHIV MANNAR no. 107397). The kit was used in houses of all participants; the interviewer asked households to provide a teaspoon of salt used for cooking and added two drops of test solution to determine the iodine content in the salt. It was filled in a small cup and spreaded flat, two drops of test solution were added on the surface of salt by piercing the white ampule, the color of salt was compared with a color chart within one minute, and the iodine concentration was determined (intense color). If no color change appeared on the salt after one minute, up to five drops of recheck solution were added in red ampule on a fresh sample, two drops of test solution were added on the same spot, and the color was compared with the color chart. The iodine content was determined, if it is 0 parts per million (no iodine), <15 ppm (light blue), ≥15 ppm (deep blue) , 20.Household's wealth index, adopted from EDHS 2011, was determined using principal component analysis (PCA) by considering the household assets, such as quantity of cereal products, type of house, livestock, and agricultural land ownership. First, variables were coded between 0 and 1 and then entered and analyzed using PCA. Those variables having a communality value of greater than 0.5 were used to produce factor scores. Finally, the factor scores were summed and ranked into tertiles as poor, medium, and rich .The collected data were entered by using Epi Info 7 and transferred to SPSS version 20 for further analysis. Data cleaning was performed to check for accuracy, consistency, and missed values. Frequencies, proportions, and summary statistics were used to describe the variables.p values in the bivariable analysis were entered into the multivariable analysis to control the possible effects of confounders. Variables with p value <0.05 in the multivariable analysis were considered as statistically significant for adolescent goiter.The result was presented using tables, and statistical associations were assessed by using bivariable and multivariable logistic regressions to obtain crude and adjusted odds ratios with a 95% confidence interval. Variables with less than or equal to 0.2 A total of 614 adolescent girls participated with a response rate of 99.03%. The mean age of the participants was 16.78 years with SD ± 1.66. A majority of the participants, 380 (61.9%), were in the age group of 16–19 years; 588 (95.8%) were Orthodox Christian; 290 (47.2%) were grade nine completed; 435 (70.8%) were rural dwellers, and 214 (34.9%) had low economic status; 510 (83.1%) of the fathers were farmers; 517 (84.2%) of the mothers/care givers were housewives, and 466 (75.9%) were unable to read and write .In this study, 448 (73%) households used adequate iodized salt (≥15 ppm). Of the participants, 580 (84.4%) used iodized salt during food preparation, 259 (50%) added it at the end of cooking, and 422 (68.7%) households stored salt without exposing it to direct sunlight. One hundred and sixty (26%) of the participants had a family history of goiter, and 448 (71.3%) had adequate dietary diversity score in the last 24 hours .The overall prevalence of goiter among adolescent girls was 24.1% ; 13.2% and 10.9% of the goiters were grade 1 and grade 2, respectively.The result of the multivariable analysis revealed that age, residence, family history of goiter, and DDS were significantly associated with goiter. Accordingly, the odds of goiter were 1.57 times more likely among late than early adolescent girls . Likewise, the odds of getting goiter were 2 times higher among participants who lived in rural areas than in urban settings . Adolescent girls who had a family history of goiter were more likely to develop the problem than their counterparts . Similarly, higher odds of goiter were noted among adolescent girls who consumed inadequately diversified diets than their counterparts  .The prevalence of goiter among adolescent girls school at 24.1% (20.0–29.9%) posed a moderate public health challenge to the study area . This fiLate adolescent (16–19 years) girls were more likely to be affected by goiter than early age groups. This finding was consistent with the results of studies done in Hawassa, Goba, and Robe, Ethiopia , 15, 17,In our study, rural dweller adolescent girls were more likely to have goiter compared with urban residents. The result was consistent with the findings of Gamogofa  and Lay Adolescent girls who had a family history of goiter were more likely to develop goiter than their counterparts. This finding was supported by the result obtained in Lay Armachiho and Gamogofa, Ethiopia , 29, BraDietary diversity of adolescent girls was significantly associated with goiter. Poor DDS among adolescent girls was almost 8 times more likely to develop goiter compared to adolescent girls without the problem. The result was supported by studies done in the Amhara regional state, Adama city and Bale, Ethiopia –35, respThe overall prevalence of goiter among adolescent girls was a moderate public health problem. Age, residence, family history, and DDS were significantly associated with goiter. Therefore, due emphases should be given to late adolescents by counseling about diversified diets and the causes and prevention of goiter.


Staphylococcus aureus in Africa are scanty, partly due to the high costs and long turnaround times imposed by conventional genotyping methods such as spa and multilocus sequence typing (MLST), which means there is a need for alternative typing approaches. This study evaluated the discriminatory power, cost of and time required for genotyping Kenyan staphylococcal isolates using iPlex MassARRAY compared to conventional methods. Data on the clonal distribution of S. aureus isolates from three counties were characterized using iPlex MassARRAY, spa and MLST typing methods. Ten single-nucleotide polymorphisms (SNPs) from the S. aureus MLST loci were assessed by MassARRAY. Fifty-four clinical pa typing and MLST revealed 22 spa types and 21 sequence types (STs) that displayed unique regional distribution. spa type t355 (ST152) was the dominant type overall while t037/t2029 (ST 241) dominated among the methicillin-resistant S. aureus (MRSA) isolates. MassARRAY showed 83 % and 82 % accuracy against spa typing and MLST, respectively, in isolate classification. Moreover, MassARRAY identified all MRSA strains and a novel spa type. MassARRAY had a reduced turnaround time (<12 h) compared to spa typing (4 days) and MLST (20 days). The MassARRAY reagent and consumable costs per isolate were approximately $18 USD compared to spa typing ($30 USD) and MLST ($126 USD). The MassARRAY assay identified 14 unique SNP genotypes, while sS. aureus isolates to identify clinically significant genotypes, MRSA strains and emerging strain types. This study demonstrated that iPlex MassARRAY can be adapted as a useful surveillance tool to provide a faster, more affordable and fairly accurate method for genotyping African  Staphylococcus aureus (MRSA) is a recognized global threat in the clinical management of infections caused by the pathogen  were sub-cultured by streaking a loopful of inoculum on Muller–Hinton agar plates  and incubating it for 18–24 h at 37 °C. The ZR/Fungal/Bacterial DNA MiniPrep kit  was used for extraction following the manufacturer’s instructions, with two modifications: the processing time for the cell disruption step was set at 10 min and DNA was eluted with 60 µl of elution buffer. DNA was quantified using a Qubit DsDNA quantification kit . For the iPlex MassARRAY assays, DNA concentrations were normalized to 10 ng µl−1. DNA samples were stored at −20 °C.All of the clinical isolates and four reference isolates .The combination of nine SNPs for each isolate was used to generate SNP genotypes. To identify the smallest number of SNPs with the highest resolution, different SNP combinations were created by sequentially and progressively reducing the number of SNPs while comparing the resultant classifications with those of the full nine-member SNPs. First, individual SNPs were excluded to create eight-member SNP classifications. Four SNPs  proved to be individually discriminative, while the remaining five  contributed to the resolution by acting in combination . Next, different SNP pairs from the five SNPs that had combinatorial resolution were excluded to create seven-member SNP classifications. Six different combinations of seven SNPs had a similar resolution to that of the nine-member classification. Lastly, different sets of three SNPs from the five SNPs with combinatorial resolution were excluded to create six-member SNP groupings. In the end, a minimum of six SNPs was required to achieve the resolution of the nine-member SNP profiles. Two unique six-member SNP combinations were identified . In this paper, classifications based on the former combination are presented.spa typing and MLST.The assay grouped 44/54 isolates into 14 different SNP genotypes. spa and MLST STs. The iPlex MassARRAY showed 83 % (34/41) accuracy against spa typing and 82 % (32/39) against MLST. The SNP genotype TTACAC corresponded to a novel spa type t17841. iPlex MassARRAY identified all MRSA isolates in this collection. One MRSA was of the ATACGT genotype, which corresponded to t355. t037, which was the spa type of three MRSAs, and t2029 (one MRSA) were represented by the ATACAT genotype. ACGTGT corresponded to t007 (one MRSA).The 14 SNP genotypes were compared with corresponding spa types t318/t021 (ST30) and t037/t2029 (ST241) were considered to be minor, as the isolates are closely related and indistinguishable by MLST (Three discrepancies in the iPlex MassARRAY assay were observed: SNP genotype TCACGC could not distinguish between t3772 and t13194 (ST 25 and ST80); SNP genotype TTGTGT could not distinguish between t314 and t272 (ST121 and 152); and TCACAT could not distinguish between t084 and t131 (ST 15 and ST1290). Discrepancies involving  by MLST .spa typing and MLST, which took approximately 4 and 20 days, respectively (spa typing and $126 USD/isolate for MLST for the collection of isolates analysed.The iPlex MassARRAY assay took approximately 1 day to complete compared to ectively  to complspa typing/MLST of a collection of Kenyan isolates. The isolate types ST152, ST5, ST8, ST15, ST30 and ST241 identified by MassARRAY have been reported in other African countries, such as Cameroon, Madagascar, Morocco, Niger, Senegal [Six MLST SNPs identified 14 unique genotypes and reflected the heterogeneity and distribution depicted by  Senegal  and Ghan Senegal , highliget al. noted an approximately 30 % reduction in costs compared to performing a full MLST analysis [MassARRAY demonstrated the clear advantages of reduced turnaround time and reagent and consumable costs per isolate based on the time and costs in the Kenyan context, which could vary in different settings. In one study, when the iPlex MassARRAY assay costs were compared to SYBR green real-time PCR, there was a 60 % reduction in reagent costs . Trembizanalysis . The cosNeisseria gonorrhea in Australia [As the MassARRAY technology is increasingly being adopted, studies utilizing its application in bacterial genotyping are being reported. In one study, a 14-member SNP assay on the MassARRAY platform was developed for genetic characterization of ustralia  and subsustralia . Even thustralia .spa typing, MLST or whole-genome sequencing on hundreds to thousands of isolates.While the increasing availability of whole-genome sequencing platforms and the concomitant reduction in sequencing costs is recognized, this assay remains superior in terms of the cost and speed of obtaining specific information, such as strain type and presence or absence of resistance or virulence genes, while avoiding the rigours of handling and analysing whole-genome sequencing data. Additionally, the assay would be most relevant for long-term surveillance studies, where it would be impractical to perform spa/MLST types, an observation noted by others [spa/MLST types did not belong to major circulating strains, as they were each represented by one isolate, none of which was an MRSA strain. Previously, it has been suggested that increasing the number of SNPs can resolve minor S. aureus clones [In some instances, the assay could not distinguish between two or more y others . However clones . While tThere were instances, for example with the pta294 SNP, where particular SNPs could not be called. A possible explanation for this could be sequence variation in the target locus for the primer in the primary PCR reactions, an observation that has been noted elsewhere . The quaEven though the SNPs analysed had generally high identification rates, the combinatorial nature of the method, which requires all SNP results for genotype assignment, meant that 10 isolates could not be classified despite having most of the alleles. This could be solved by further optimization of assay conditions and rescreening of the missing loci to complete the panel for classification.spa types [spa /STs from an international collection of isolates. With increased validation, it should be possible to have an online MLST-style platform where SNPs can be submitted for inter-laboratory comparability of data.The lack of a database for MLST SNPs that can be used for matching particular SNP profiles to known STs or pa types  is a majS. aureus in Kenya but also serve to validate the utility of iPlex MassARRAY for surveillance.Compelling epidemiological conclusions cannot be drawn from this study due to the modest collection of 54 isolates analysed. A larger collection of isolates from diverse regions and clinical syndromes would not only give a reliable reflection of the epidemiology of S. aureus isolates, including MRSA strains. The iPlex MassARRAY demonstrated the advantages of reduced turnaround time and assay costs compared to two conventional typing methods. With increased validation, the assay could serve as a complement to existing typing methods in S. aureus surveillance studies to establish the epidemiology of S. aureus strains and monitor for the presence or emergence of high-risk strains in a timely fashion. In conclusion, six SNPs derived from the MLST loci provided comparable discriminatory power for resolving a heterogeneous and regionally unique collection of Kenyan clinical Click here for additional data file.


Brassica rapa accessions. Cluster analysis separated these 34 accessions into three clusters based on heading types. The millions of SNP loci, a large set of resource for KASP markers, as well as the newly developed KASP markers in this study may facilitate further genetic and molecular breeding studies in Brassica rapa.Kompetitive allele-specific PCR (KASP) is a cost-effective single-step SNP genotyping technology, With an objective to enhance the marker repertoire and develop high efficient KASP-SNP markers in Chinese cabbage, we re-sequenced four Chinese cabbage doubled haploid (DH) lines, Y177-47, Y635-10, Y510-1 and Y510-9, and generated a total of more than 38.5 billion clean base pairs. A total of 827,720 SNP loci were identified with an estimated density of 3,217 SNPs/Mb. Further, a total of 387,354 SNPs with at least 30 bp to the next most adjacent SNPs on either side were selected as resource for KASP markers. From this resource, 258 (96.27%) of 268 SNP loci were successfully transformed into KASP-SNP markers using a Roche LightCycler 480-II instrument. Among these markers, 221 (85.66%) were co-dominant markers, 220 (85.27%) were non-synonymous SNPs, and 257 (99.6%) were newly developed markers. In addition, 53 markers were applied for genotyping of 34  Brassica rapa L. ssp. pekinensis), which contains an AA genome, is one of the most important vegetable crops worldwide and is a valuable plant for genome evolution study in Brassica species [B.rapa accessions showed that the Chinese cabbage group was positioned at a more distant point from the root than other groups, such as turnip, sarson, pak choi, caixin and zicaita [B.rapa [Chinese cabbage , 2 flowering Chinese cabbage (Brassica rapa L. ssp. chinensis var. parachinensis), 2 Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea), and 22 Chinese cabbage (Four Chinese cabbage doubled haploid (DH) lines, Y177-47, Y635-10, Y510-1 and Y510-9, were used for whole-genome re-sequencing with paired-end strategy on the Illumina platform. To validate the utility of these KASP markers, an additional panel of 29 accessions of  cabbage . All thoAll plants were grown in farm field. Young leaves of 14-day-old seedlings were collected for DNA extraction. Genomic DNA of all accessions was extracted from young leaves using a modified CTAB method .The sequencing libraries were constructed following the manufacturer’s instructions (Illumina Inc.). The Illumina HiSeq 2000 platform was used to generate 100-base paired-end reads for Y177-47 and Y635-10, while 150-base paired-end reads were generated for Y510-1 and Y510-9 on the Illumina HiSeq X Ten platform. The raw data were deposited in the Sequence Read Archive (SRA) in NCBI as SRP221414.B. rapa reference genome V1.5 with Burrows-Wheeler Aligner (BWA 0.7.12-r1039) using default parameters [http://brassicadb.org/brad/datasets/pub/Genomes/Brassica_rapa, which consists of 10 chromosomes and 40357 scaffolds. The SAMtools software package (version 1.3.1) was used to call SNPs across all samples simultaneously [After pre-processing of reads, where adapter sequences were removed and low quality reads were discarded, clean reads generated from the four accessions were mapped to the rameters . The refaneously . Only SNaneously .2O. The PCR started with an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 50 s, 56°C for 50 s, 72°C for 50 s, and a final extension at 72°C for 10 min. Sequencing results were analyzed by DNASTAR v7.1.0 (http://www.dnastar.com/).To validate SNPs identified from whole genome re-sequencing, 142 of them were selected for PCR amplification and Sanger sequencing. Primers were designed using the Primer Premier 5.0 program  for at l5'-GAAGGTGACCAAG-TTCATGCT-3') and HEX (5'-GAAGGTC-GGAGTCAACGGATT-3') tails respectively at the 5' end.For each selected SNP, two allele-specific forward primers and one common reverse primer were designed using the Primer Premier 5.0 program  accordin2O, 30 μL common reverse primer (μM), and 12 μL of each tailed allele-specific forward primer (μM). KASP assays were carried out in 96-well formats using Roche LightCycler 480-II instrument , and set up as 8 μl reaction volume . PCR cycling was performed using the following protocol: hot start at 94°C for 15 min, followed by ten touchdown cycles , followed by 26 additional cycles of annealing  (https://biosearch-cdn.azureedge.net/assetsv6/running-KASP-on-Roche-LC480.pdf). Fluorescence was read by the LightCycler480II and analyzed using the LightCycler 480 Software (Version 1.5.1).The newly developed KASP-SNP markers were validated by examining polymorphisms among the four sequenced Chinese cabbage lines. Before PCR application, the primer assay mixture was prepared, which comprised 46 μL ddHB. rapa reference genome V1.5. Only the best-hit alignment  was retained. The SNP identifier was recorded using the format chr_position, for example, A02_1646719 indicates that there is a single nucleotide polymorphism in physical position 1,646,719 of chromosome A02. Similarly, the allele-specific forward primer sequence of KASP-SNP markers from the article reported by Su et al. [The 50 bp forward flanking sequences of RNA-based SNPs were downloaded from the article reported by Paritosh et al. . We blasBrassica rapa accessions were genotyped using 53 randomly selected KASP-SNP markers. Genotypes of these 29 Chinese cabbage accessions together with 4 DH lines used for re-sequencing and the reference material Chiifu-401-42 were used for diversity analysis. For each marker, minor allele frequency (MAF), the total number of alleles, genetic diversity, heterozygosity and the polymorphism information content (PIC) were calculated with PowerMarker software (version 3.25) [Brassica rapa accessions based on 53 markers was calculated using PowerMarker software V3.25. The dendrogram was drawn by MEGA (version 5.1) using neighbor-joining algorithm (http://www.megasoftware.net/download_form).Twenty nine on 3.25) . The CSCB. rapa reference genome (version 1.5) [Whole genome re-sequencing yielded approximately 62, 56, 99 and 94 million raw reads , which rion 1.5) . The mapBrassica rapa [Through comparisons with the reference genome of the inbred Chiifu-401-42 (V1.5), we detected a total of 827,720 SNPs distributed among all ten chromosomes , with anica rapa .in silico SNPs, a total of 41 primer pairs were designed for Sanger sequencing, and 30 primers yielded clear and single bands of the expected fragment size from the four original Chinese cabbage DH lines  SNPs were identified in expressed regions or splicing regions, and 56,142 (14.49%) were annotated in introns. In particular, 33,582 (8.67%) SNPs were classified as non-synonymous SNPs resulting in amino acid changes, and 443 (0.11%) SNPs gave rise to variants in stop codons or splicing regions, also altering the amino acid sequences of coding genes.In order to establish a KASP-SNPs resource, we filtered for SNP loci with at least 30 bp to the next most adjacent SNPs on either side. As a result, 387,354 SNPs were obtained, which could serve as resource for KASP-SNPs. All these SNPs and their flanking 30 bp sequence were supplied in To develop novel KASP-SNP markers, we selected 268 SNP loci to design markers from the above resource based on two criteria. First, SNPs are evenly distributed across the whole genome with a density of 1 marker/Mb. Second, the SNPs located in the exonic region with non-synonymous variation were preferred.Brassica rapa No.1), and the primer sequences and other information are listed in In total, 258 (96.27%) worked well and gave rise to polymorphisms, which categorized the four sequenced genotypes into two clusters, while the other ten primers produced no signal. All the validated KASP markers were named using the format KBr1  with a density of 3,217 SNPs/Mb. The number of SNP loci detected in our study is considerably larger than that detected by Sanger sequencing and transcriptome sequencing , 34–36, Ps/Mb in  and SangPs/Mb in . The larin silico SNPs (142 loci) was selected for validation using the Sanger sequencing method, which yielded a 100% SNP validation rate. This result means that almost all of the SNPs found in this study are authentic nucleotide variations in Chinese cabbage. The SNP validation rate is considerably higher than those reported in past studies [Brassica rapa.A subset of  studies , 17. TheNumerous methods have been developed for SNP genotyping, including allele-specific PCR (AS-PCR), cleaved amplified polymorphic sequences (CAPS) and high-resolution melting (HRM) curve assays. All these methods require gel electrophoresis or high-resolution melting to separate products, which are low throughput, high cost, and labor intensiveness . A numbeBrassica rapa, (b) the SNP loci identified in this study are mostly authentic, and (c) stringent criteria were used for selecting SNP loci.In this study, we established a KASP-SNPs resource which comprised 387,354 SNPs with at least 30 bp to the next most adjacent SNPs on either side. From this resource, we selected 268 SNP loci, designed primers and carried out KASP assays using Roche LightCycler 480-II instrument. In total, 258 (96.27%) were successfully converted into KASP markers, the marker conversion rate was considerably higher than SSR markers 28.44%) , CAPS ma% , CAPS 2 and backcross populations [Co-dominant SNP assays are preferred markers compared with dominant SNP assays due to the ability to differentiate heterozygotes from the homozygous genotype in segregant populations, such as Fulations . Of the Compared to non-coding genomic markers, SNPs developed from functional genes are potentially more closely related to phenotypic variations , 46. In Brassica rapa, and could be used for high-density genetic linkage map construction, gene/QTL mapping, cultivar identification, and so forth.Of all 258 KASP markers developed in this study, 257 are novel SNP markers, which extended the KASP marker resource in Brassica rapa accessions. The 53 KASP markers produced 106 allelic variations with 2 alleles per SNP. The PIC values ranged from 0.1676 to 0.3750 with an average of 0.3276, which is much higher than recently developed KASP markers for chickpea (0.12) and Grasspea (0.2457) [Brassica rapa (0.35) [Brassica rapa accessions into three distinct clusters, which matched with the Brassica rapa heading types. Materials in cluster I were all non-heading type. Three Pak-choi, two Zicaitai and two flowering Chinese cabbage were classified into three subgroups based on variety distinctness and were much closer to each other than to Chinese cabbage, which is consistent to previous studies [A total of 53 KASP markers were selected for KASP assay genotyping with 34 0.2457) , 22, and , 22, anB.rapa accessions genotyped with 53 KASP markers can be separated into three distinct clusters, which matched with the Brassica rapa heading types. Furthermore, three Pak-choi, two Zicaitai and two flowering Chinese cabbage were divided into three subgroups according to varieties, which demonstrated KASP markers mined here are not only useful in Chinese cabbage, but also for other Brassica rapa crops. The large set of SNPs discovered in this study and newly developed KASP markers will be useful for genetic improvement of Chinese cabbage through breeding.In summary, We re-sequenced 4 Chinese cabbage lines and obtained more than 38.5 billion high quality sequenced bases. Based on genome-wide SNP detection and validation through Sanger sequencing, we identified 827,720 SNP loci with a high accuracy rate. A total of 387,354 SNPs with at least 30 bp to the next most adjacent SNPs on either side were selected as a KASP-SNPs resource. Among which, 258 SNP loci were successfully transformed into KASP markers, the marker conversion rate was high to 96.27%. Additionally, 221 (85.66%) were co-dominant markers, 220 (85.27%) were non-synonymous SNPs, and 257 (99.6%) were newly developed markers. Thirty four S1 Appendix(FASTA)Click here for additional data file.S1 FigPrimer sequences (A) and KASP genotyping (C) of a dominant KASP marker, optimized primer sequences (B) and KASP genotyping (D) with co-dominant feature.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.