S. pimpinellifolium. The position on the tomato map of genes affected by moderate and high impact mutations was also compared with that of known markers/QTLs (Quantitative Trait Loci) associated with reproductive and yield-related traits. The candidate genes/QTLs regulating heat tolerance in the selected landrace E42 could be further investigated to better understand the genetic mechanisms controlling traits for high and stable yield trait under high temperatures.The selection of tolerant varieties is a powerful strategy to ensure highly stable yield under elevated temperatures. In this paper, we report the phenotypic and genotypic characterization of 10 tomato landraces to identify the best performing under high temperatures. The phenotyping of five yield-related traits allowed us to select one genotype that exhibits highly stable yield performances in different environmental conditions. Moreover, a Genotyping-by-Sequencing approach allowed us to explore the genetic variability of the tested genotypes. The high and stable yielding landrace E42 was the most polymorphic one, with ~49% and ~47% private SNPs and InDels, respectively. The effect of 26,113 mutations on proteins’ structure was investigated and it was discovered that 37 had a high impact on the structure of 34 proteins of which some are putatively involved in responses to high temperatures. Additionally, 129 polymorphic sequences aligned against tomato wild species genomes revealed the presence in the genotype E42 of several introgressed regions deriving from  The rising rate in global temperatures predicted for the next few years ,2 can lestresses ,10. Therstresses . These tstresses ,13. If tstresses . Anotherstresses .1 hybrids and to better understand the genetic mechanisms underlying heat tolerance in tomatoes.The aim of the present work was to phenotype 10 tomato landraces grown under elevated temperatures for yield-related traits by comparing different environmental conditions and different years. These landraces were also genotyped in order to exploit their genetic variability. Herein, the mutations (SNPs and InDels) identified were annotated and a prediction of their effect on protein functions and structures was carried out. The phenotypic and genotypic data recorded in the present work were used for selecting stable heat-tolerant tomato landraces and for identifying candidate genes associated with thermotolerance. In the future, these data could be used as a starting point in breeding programs to constitute Fhttp://dx.doi.org/10.6070/H4TT4NXN). The two hybrids DOCET and JAG8810 have been used as control genotypes since they perform good yields under high temperatures . The 10 tomato genotypes coded E were selected from a wide tomato germplasm collection previously characterized for fruit morphology and nutritional traits and evaluated under elevated temperature conditions [−1) was applied to the soil before transplanting. Tillage treatments included plowing that was followed by one/two millings. Weeding and ridging were also carried out. Recommended levels of N (190 kg ha−1) and K (20 kg ha−1) were applied through irrigation. During cultivation, plants were irrigated as required. During the whole growing season, climatic data were recorded using the weather station VantagePro2 (Davis Instrument Corporation).During the summer of 2016 and 2017, 10 tomato landraces (coded E) and two hybrid genotypes, available at the University of Naples, Department of Agricultural Sciences, were grown in two regions of Southern Italy (Campania and Puglia) usually characterized by elevated temperatures during the summer season. Details on the genotypes are shown in nditions . Plants Five phenotypic traits associated to yield performances were evaluated: number of flowers per inflorescence (NFL), fruit set (FS), total number of fruits per plant (TNF), fruit weight (FW), and yield per plant (YP). NFL was evaluated counting the number of flowers per inflorescence from the second to the fifth truss on three plants per replicate, whereas FS was evaluated by counting the number of flowers setting fruit out of all flowers of each inflorescence from the second to the fifth truss on the same three plants per replicate. TNF was calculated by summing all fruits collected from all plants of the three replicates. The final value was divided for the number of total plants. FW was measured by weighting at least 50 fruits collected from all the plants and dividing the value for the number of total fruits used. Lastly, YP was evaluated using the total weight of all collected fruits and dividing it for the number of total plants of the three replicates. These three last parameters were determined at the red ripe fruit stage.iv value (coefficient of regression) was calculated for each genotype, considering the average yield of each field on the y-axis and an environment index (EI), estimated as Equation (1), on the x-axis.ijY is the yield of the thi genotype  at the thj location . The coefficient of regression (biv) describes the linear correlation between the yield of the single genotype and the different environments. In this linear regression model, a biv = 1 was considered the optimum value describing the phenotypic stability of a trait. The standard deviation of biv coefficients was calculated on the whole population to establish threshold borders of confidence for selecting stable-yielding genotypes. The average deviation from linearity (ADL) (Equation (2)) was estimated as:Qµ is the predicted yield mean in a best-fit non-linear model (quadratic model) of the thi genotype at the thj location, Lµ is the predicted mean in a linear model of the thi genotype at the thj location, and thi genotype in different locations. The ADL describes the average of deviation from linearity. When the ADL parameter calculated for each biv resulted in a significantly different value from zero, the coefficient of regression was considered inconsistent for selecting high-yielding and stable-yielding genotypes. Lastly, the coefficient of determination (2r) indicated the accuracy of the statistical model. In this way, 2r > 0.50 is a condition for the accuracy of the regression model, essential to identify high—and stable—yielding genotypes. The genotypes showing a YP mean higher than the grand mean, biv ≤ 1, 2r > 0.50 and ADL is not significantly different from 0. These were regarded as high—and stable—yielding genotypes. Multiple statistical test analyses were carried out using the software SPSS version 23 .In order to better understand the genotype by location by year interactions for all phenotypic traits, an ANOVA analysis was carried out, where the genotype, location, and year were considered fixed factors. The stability performance of each genotype in terms of yield per plant was evaluated using a linear regression model . In thisSolanum lycopersicum  was performed using BWA-MEM  [http://vcftools.sourceforge.net). Filtering parameters were set as follows: maximum missing value = 0.50 and minimum mean of Depth of Coverage (min-mean DP) = 5. All the mutations that were not polymorphic among the 12 analyzed genotypes were manually eliminated since they showed the same alternative allele with respect to the reference genome of the cv. Heinz. The visual display of sequencing data was performed using CircosVCF webtool [http://212.150.245.226/~tools/CircosVCF/). Then, in order to estimate the genetic distance among the genotypes, pairwise comparisons were performed by calculating the Identity-by-State (IBS) allele-sharing values using the software PLINK v.1.90b5.2 [http://snpeff.sourceforge.net/), using the SL3.0 version of the tomato genome to define the predicted SNPs’ effect and the annotation version iTAG3.2 to identify the affected protein. Four tags were used to estimate the putative impact of SNPs and InDels on genes’ expression and proteins’ translation. This is high for the variants assumed to have a disruptive impact on the protein function, moderate for non-disruptive variants that could change the protein effectiveness, low for the synonymous variants that should not affect the protein behavior, and, lastly, modifier effect for variants located in non-coding regions. Lastly, in order to investigate the origin of the genetic variability detected in the genotype E42, starting from the dataset of filtered InDels, an alignment was performed against genomic sequences of wild species publicly available . A global alignment was performed using the wild tomato species databases (available at ftp://ftp.solgenomics.net/genomes/) and the software Blast Genome Workbench v. 3.1.0 (https://www.ncbi.nlm.nih.gov/tools/gbench/). The screening of InDel loci was carried out by selecting only the polymorphisms showing an insertion or deletion of at least five nucleotides. The SSR-InDels were selected based on differences in the number of repeats (at least one repeat). Lastly, a sequence of about 100 bp, including the InDel mutation, was used as a query for the analysis. We considered putative wild species-derived loci that showed 100% identities on the InDel region and more than 90% identities in the whole 100-base pair sequence.Total genomic DNA was extracted from 100 mg of young leaf tissue from all the genotypes using the DNeasy plant mini kit . The DNA concentration was determined using the Qubit fluorometer  while the 260/280 and 260/230 ratios were measured using the Nanodrop spectrophotometer . For DNA sequencing, samples (1 µg in 30 µL of sterile milliQ water) were used to prepare libraries for the ddRAD sequencing, as described in Peterson et al.  with minMatches)  with defThe 10 tomato genotypes analyzed in this work were previously characterized for fruit quality and nutritional traits and were selected for their good performances under high temperature conditions ,17,18. It-test was performed to estimate statistical differences between the landraces and the heat tolerant control hybrids (DOCET and JAG8810) for all the evaluated traits in each field. As reported in r-value ranging from −0.63 to −0.45. TNF was positively correlated with YP in Campania in both years  and in Puglia in 2017 (r = 0.51). When considering the significant correlation indexes between YP and the four traits NFL, FS, TNF, and FW detected for each genotype (p < 0.001). The landraces’ performances evaluated in all the fields were compared to those of two tomato hybrids (DOCET and JAG8810) that are highly productive under high temperature conditions. A high variability of phenotypic data was observed for almost all the traits in the different growing locations and in the different years . For somgenotype , highly iv) value  value . This va and E42 . Three oiv = 0.96, YP = 2.93 kg/pt, ADL = 0, 2r = 0.72). In fact, notwithstanding the other two genotypes (E36 and E107), a YP pooled mean was higher than the grand mean. In addition, their coefficient of regression biv was higher than 1, which suggested that these genotypes achieved high yield only in favorable environments. The YP grand mean of the group in different locations was calculated (2.39 kg/pt) to establish which genotypes showed a YP pooled mean  higher than the group grand mean . In this way, ~42% of genotypes (five out of 12) showed a YP pooled mean higher than the grand mean. Among these genotypes, the landrace E42 was identified as a stable yielding genotype  in both the reference (83.0%) and alternative allele (10.1%). Low values were recorded for SNPs/InDels in a heterozygous condition (6.1%) and only 0.5% of missing data were observed. As expected, the two hybrids DOCET and JAG8810 showed the highest percentage of heterozygous SNPs/InDels . By a pairwise comparison of all the genotypes , based oIn order to exploit the genetic variability exhibited by the different landraces, the number of private SNPs and InDels was also calculated for each genotype . In mostIn order to understand the effects of the mutations on gene expression and protein translation, the annotations of the mutated genes and the prediction of the mutation effect were investigated . Out of Solyc02g087680 and Solyc02g087690 coding for a FACT complex subunit SSRP1 and Solyc12g044645 coding for the transcription factor AP2/B3 family protein. Interestingly, 14 different variants (7 SNPs and 7 InDels) resulted private for the genotype E42. Moreover, concerning the variants with predicted moderate effects on protein structure . For this purpose, 129 InDels were chosen and, for each of them, a 100-base pair sequence including the InDel was selected for the BLAST analysis. Among these, 27 InDels consisting of SSR (Simple Sequence Repeats) markers  and 22 InDels (61%) were identified as putatively derived from S. pimpinellifolium. The other two InDels-SSR and 14 InDels loci showed high identity percentages when aligned against the genomes of S. chilense and S. galapagense.Lastly, in order to investigate the origin of the high genetic variability of the genotype E42, starting from the dataset of 3,519 filtered InDels, an alignment of the selected InDels was performed against genomic sequences of wild species publicly available (1 hybrids used as control (DOCET and JAG8810), even showing a higher stability compared to them. The level of YP observed in all the genotypes, across the four experimental fields, showed that this trait was highly correlated with the TNF value, and the latter was mainly correlated with the NFL value. This occurred independently of the different FW values recorded herein, which ranged from the low values recorded in E42, characterized by very small fruits, to the high values recorded in E17, characterized by big-sized fruits. These results are in contrast with data reported by Ayenan et al. [Facing damage caused by abiotic stresses, such as drought and heat stress, represents a great challenge for agricultural science in the new century. Exploiting genetic resources is one tool to achieve this objective, in combination with optimized growing and management practices. In the present work, 10 tomato landraces have been tested for two years in two different open fields under high temperature conditions. As expected, the climatic parameters were highly variable in the four experimental conditions tested, including not only diurnal and nocturnal temperatures, but also relative humidity, wind, and rain. Other environmental factors, such as soil composition and texture, might have influenced the performances of the tested genotypes in the four trials. However, the phenotypic analyses performed on the 10 landraces allowed us to identify those that exhibited the best stable performances in terms of yield under a wide range of conditions, including high temperatures. Usually, heat tolerance has been evaluated under fixed climatic conditions in greenhouses or growth chambers ,26,27. Hn et al. , who fouS. pimpinellifolium, were mapped on chromosomes 1 and 7. The presence of these and/or other wild regions has potentially contributed to confer the observed thermotolerance to E42. It has been reported that the wild tomato species usually show high pollen viability and pollen number even under heat stress conditions [S. pimpinellifolium exhibit a good level of thermotolerance [The genotyping analyses performed in this scenario demonstrated that E42 has a high genetic variability compared to the other genotypes investigated. This could be due to the breeding history of this genotype, which likely included crossing events with tomato wild species. A group of selected InDel mutations aligned against the genome of five wild species revealed that in the genotype E42 putative introgressed regions, mainly deriving from the species nditions . Moreoveolerance ,39.Following the high-throughput genotyping of the landraces analysed, we demonstrated that 37 and 283 mutations were mapped within coding regions and exhibited a high and moderate impact on the corresponding proteins, respectively . TherefoS. pimpinellifolium. Since the good yield performances of E42 under high temperatures were mostly attributed to the high NFL and to the high TNF observed, we hypothesized that one or more genes influencing the number of flowers could exhibit informative mutations in E42. It has been reported that two QTLs carrying a S. pimpinellifolium allele and mapping on chromosomes 2 and 4 affect the number of flowers in tomatoes [S. lycopersicum and S. pimpinellifolium [The distribution of high and moderate mutations on the 12 tomato chromosomes is reported in tomatoes . Moreovelifolium . In the Solyc02g087680 and Solyc02g087690) and both coded for members of the FACT subunit complex SSRP1 protein family. It has been reported that, in Arabidopsis, this protein is an epigenetic regulator of seed dormancy and it is also involved in anthocyanin biosynthesis, and in high-light and UV stresses responses [Solyc07g042660, which codes for an RNA helicase, known to be involved in the responses to different abiotic stresses. Moreover, the expression levels of several DEAD-box helicases are regulated in response to environmental stresses, including salt, light, or temperature stresses [Solyc12g044645 coding for an AP2/B3 transcription factor family protein. This gene is homologous to At1g51120.1 identified in A. thaliana, which codes for a member of the RAV superfamily involved in seed germination, plant development [Solyc01g017220, coding for an ATP synthase epsilon chain, is reported to be involved in drought stress responses [Solyc02g070280, which codes for a protein involved in the transport of amino acids during the pedicel abscission [Solyc07g053640, which codes for an arabinogalactan-protein that could be involved in abscission events [Solyc08g079260, coding for a Hsp70 with a tetratricopeptide domain. Lastly, Solyc12g043090 codes for a trihelix transcription factor, which is involved in different stress responses. It also showed variations with moderate effects [Solyc07g053640 and in Solyc12g043090 were private for E42.Lastly, searching for genes potentially involved in the response to high temperatures, we could find mutations in four genes with a disrupting impact on the corresponding proteins with three of them showing SNPs/InDels private for the genotype E42. Two genes mapped close to each other on chromosome 2 (stresses ,44. Lastesponses . The genscission , and Soln events , might i effects . The mutS. pimpinellifolium. Considering that heat tolerance is a polygenic trait that involves many genes, the identification of candidate genes, obtained by combining the in-depth phenotypic and genotyping analyses carried out in this work, might help dissect this complex trait. In particular, the private SNPs and InDels detected in two QTLs mapping on chromosome 2 could be the starting point to design molecular markers targeting NFL, which is a trait that may contribute to induce a good response to heat. Additional candidate genes identified in this study will be further investigated to detect those involved in a stable response to high temperatures. Once functionally validated, these candidate genes could be useful to design molecular markers and/or apply genome editing techniques with the aim to obtain new tomato genotypes improved for heat tolerance.We evaluated 10 landraces under high temperatures in multi-environmental conditions to select the best genotypes in terms of stable yield. At the end of this work, we selected the genotype E42 as the best performer due to the high NFL, and, consequently, the high TNF values showed. Following a high-throughput genotyping, E42 also resulted in the most polymorphic genotype, showing some introgressed regions from the wild species


Lactobacillus plantarum (L. plantarum) fermented Astragalus in the diet of laying hens, with a final addition of 3‰. Its effects have been assessed on laying performance, egg quality, antioxidant and immunological status, and intestinal microbiota, and are compared to the control group, to the Astragalus group containing 3‰ unfermented Astragalus, and to the L. plantarum group containing 2% L. plantarum [5 × 108 colony-forming unit (CFU) per milliliter (mL)]. During the second half of the experimental period (15 to 28 days), the egg production rate was considerably higher in the fermented Astragalus group than that in the other groups, with the fermented Astragalus group having the lowest feed conversion ratio. No significant difference (P > 0.05) was noted among treatments on egg quality. Fermented Astragalus-treated hens exhibited significantly increased catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum, and reduced malondialdehyde (MDA) in serum. Furthermore, fermented Astragalus supplementation resulted in a significant increase in ileal microbiota abundance relative to control. In conclusion, feeding laying hens with L. plantarum fermented Astragalus has beneficial effects on production, antioxidant potential, immunity, and ileal microbiota. L. plantarum fermented Astragalus is expected to be a novel feed additive used in poultry production.In the era of increased antibiotic resistance and ever-stricter control on antibiotic use, it is urgent to develop green, safe, and non-residue alternatives to antibiotics applied to the poultry industry. To this end, we supplied the potential  Eggs are one of the most crucial sources of animal protein and nutritional content in human diets. Due to the widespread use of antibiotics in poultry, drug residues in eggs have been gaining worldwide concern over the past few years  was purchased from the China General Microbiological Culture Collection Center (CGMCC) . Te dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus was obtained from Gansu Huisen Pharmaceutical Development Co., Ltd.  and verified by Dr. JingYu Zhang . The purchased Astragalus was crushed into powder and filtered with a 100-mesh filter for further studies. The fermentation of Astragalus was performed following the method reported in our previous publications with slight modification  was inoculated with L. plantarum (1 × 106 CFU per gram) with a water content of 45% and Astragalus–L. plantarum mixtures were divided equally into 35 × 45-mm plastic film bags. The bags were sealed for fermentation at 37 °C for 30 days and then dried out at room temperature for future use.Astragalus group, Astragalus group, L. plantarum group, and control group), each containing five replicates, with 12 hens per replicate. The control group was fed with the basal diet; the L. plantarum group was fed with the basal diet supplemented with 2% Lactobacillus solution (5 × 108 CFU/mL) through uniform spraying; the Astragalus group was fed with the basal diet supplemented with 3‰ Astragalus, and fermented Astragalus group was fed with the basal diet supplemented with 3‰ fermented Astragalus . The trial lasted for 35 days . The hens were housed in a clean environment with good ventilation and artificial lighting allowed 16 h of lighting per day, and with water and food ad libitum. The basal diet of all groups was the same and prepared according to the NRC (1994) laying hen nutrition requirement standard. The composition and nutrient levels of the basal diet were shown in Table Two hundred and forty healthy Hy-Line Gray hens  were acclimated with the basal diets for 7 days. Then, hens were randomly divided into four groups  (feed intake/egg weight gain) during day 1 to day 14 and day 15 to day 28 were calculated to assess the laying performance. On day 14 and day 28, five eggs from each replicate were randomly sampled and measured egg quality parameters of egg shape index (ESI), eggshell strength (ESS), eggshell thickness (EST), albumen height (AH), Haugh unit (HU), yolk color (YC) and yolk weight (YW).On day 14 and day 28, one hen from each replicate was randomly selected. Following blood collection from the heart, the serum was isolated and stored at − 20 °C until use. The CAT assay kit , GSH-Px assay kit , SOD assay kit , T-AOC assay kit  and MDA assay kit  were purchased from Shanghai yuanye Bio-Technology Co., Ltd .−ΔΔCt method.After blood samples collection, liver, spleen, ileum, and cecum samples were harvested for interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) mRNA expression evaluation. Total RNA was extracted from these tissues using RNAiso Plus  and reverse transcribed into cDNA with PrimeScript™ RT reagent Kit with gDNA Eraser   according to the manufacturer’s protocol. The primers used in the study were synthesized by Sangon Biotech (Shanghai) Co., Ltd. , and primer sequences are summarized in Table Astragalus group (14IB), 14-d ileum L. plantarum group (14IC), 14-d ileum fermented Astragalus group (14ID), 14-d cecum control group (14CA), 14-d cecum Astragalus group (14CB), 14-d cecum L. plantarum group (14CC), 14-d cecum fermented Astragalus group (14CD), 28-d ileum control group (28IA), 28-d ileum Astragalus group (28IB), 28-d ileum L. plantarum group (28IC), 28-d ileum fermented Astragalus group (28ID), 28-d cecum control group (28CA), 28-d cecum Astragalus group (28CB), 28-d cecum L. plantarum group (28CC), and 28-d cecum fermented Astragalus group (28CD). All collected samples were immediately stored at – 20 °C until extraction. DNA extraction was performed with a commercial DNA extraction kit  and quantified by a Qubit 2.0 fluorometer . The extracted DNA was qualitatively assessed by 0.8% agarose gel electrophoresis and spectrophotometry  and stored at − 20 °C until further analysis.On day 14 and day 28, a total of 48 hens were randomly selected (12 hens per group) and euthanized via an intravenous injection of pentobarbital sodium (150 mg/kg) to collect ileal and cecal contents  under the Accession number SRA: PRJNA533918.For amplicon library generation, the V4 region of the 16S rRNA gene of all DNA samples was amplified with gene-specific primers . PCR amplifications were performed using Q5 high-fidelity PCR DNA polymerase  and completed under the following conditions: a pre-denaturation at 98 °C for 30 s; 27 cycles of 98 °C for 15 s, 50 °C for 30 s, and 72 °C for 30 s; a final extension at 72 °C for 5 min. Amplicons were purified using the Axygen AxyPrep DNA Gel Extraction Kit . DNA libraries were validated and quantified using the TruSeq Nano DNA LT Library Preparation Kit  and Quant-iT™ PicoGreen™ dsDNA Assay Kit . After quantification, the barcoded V4 amplicons were pooled to a final concentration of 2 nmol/L and sequenced using an Illumina MiSeq platform to generate 300 bp paired-end reads. Raw reads were quality-filtered to remove any reads less than 150 bp using Quantitative Insights into Microbial Ecology (QIIME) version 1.8 , and significance levels are indicated as *P > 0.05), with hens fed with fermented Astragalus had the highest laying rate. During day 15 to day 28, hens fed with fermented Astragalus had the highest laying rate, 7.14% higher than that of the control group (P < 0.05). Although the differences were not statistically significant (P > 0.05), the laying rate of the Astragalus group and L. plantarum group were also increased by 3.25% and 2.99%, respectively in comparison with the control group. Furthermore, the FCR of the fermented Astragalus group was reduced by 6.6% compared with that of the control group (P < 0.05), while the FCR of the Astragalus group and L. plantarum group displayed no significant differences as compared with the controls (P > 0.05). In addition, no significant differences in the phenotype of the egg quality including ESI, ESS, EST, AH, YC, HU, and YW were observed among dietary treatments, suggesting that dietary supplements have no significant effects on egg quality in this study. Therefore, we identified that dietary supplementation of fermented Astragalus can markedly improve egg production and decrease FCR, and the effect is substantially superior to that of Astragalus and L. plantarum.The effects of different dietary supplements on the laying hen production performance and egg quality are listed in Tables P > 0.05). However, serum CAT, GSH-Px, SOD, and T-AOC concentrations were increased by 61.5%, 62.4%, 68.0%, and 52.6% (P < 0.05) at the end of experimentation in the fermented Astragalus group as compared with the controls. No statistically significant differences were observed for CAT, GSH-Px, and SOD among the control, Astragalus, and L. plantarum groups (P > 0.05). Among the effects of different dietary supplements on MDA activity in serum of laying hens, hens fed with fermented Astragalus, Astragalus diet were significantly decreased by 54.7% and 43.0% than that of the control treatment (P < 0.05); treatment with L. plantarum diet did not dramatically differ from the control treatment (P > 0.05). The results presented above show that dietary supplementation of fermented Astragalus can markedly improve laying hen antioxidant status, and the effect is significantly superior to that of Astragalus and L. plantarum.The effects of different dietary supplements on the laying hen antioxidant status are listed in Table Astragalus to the diets respectively increased the mRNA content on day 14 of IFN-γ and TNF-α in the ileum by 1.7-fold (P < 0.01) and 3.1-fold (P < 0.001), and the mRNA content on day 14 of IFN-γ in the cecum by 2.1-fold (P < 0.01). Interestingly, we found that the highest amount of IFN-γ and TNF-α mRNA in the liver, spleen, ileum, and cecum were present in the fermented Astragalus group at 28 days.The expression levels of IFN-γ and TNF-α mRNA in the liver, spleen, ileum, and cecum were assessed at 14 and 28 days. As shown in Figs. A total of 48 intestinal content samples were analyzed by 16S rRNA gene sequencing and produced a total of 2,006,223 high-quality sequences with an average of 41,796 reads. After OUT clustering at 97% sequence identity, a total of 216,116 OTUs were classified into 49,235 phyla, 48,677 classes, 48,634 orders, 40,101 families, 24,072 genera and 4995 species  of the bacterial community in the ileum. PLS-DA was performed to evaluate the similarity (β-diversity) of microbial community structure among groups  of cecal bacterial phyla of hens fed with different dietary supplements was presented in approximately the same amount on days 14 and 28. On day 28, fermented Astragalus led to a reduced abundance of ileal Firmicutes, with an increased abundance of ileal Bacteroidetes and Proteobacteria. Genus level analysis showed that the Lactobacillus and Bacteroides accounted for the largest proportion of the intestinal microbiota as shown in Fig. Lactobacillus showed high abundance in the ileum and the extremely low abundance in the cecum. In contrast, Bacteroides showed high abundance in the cecum and the extremely low abundance in the ileum. On day 14, fermented Astragalus addition increased the abundance of cecal Bacteroides by 5.06% as compared with the control, with no significant influence on the abundance of ileal Lactobacillus. On day 28, fermented Astragalus addition significantly decreased the abundance of ileal Lactobacillus by 48.51% as compared with the control, with no significant influence on the abundance of cecal Bacteroides.A total of 20 phyla were identified within the intestinal microbiota among 48 samples as shown in Fig. L. plantarum fermented Astragalus supplementation on the performance, egg quality, antioxidant status of serum, and gut microbiota in laying hens. When taking out the feeding trial, we observed that diets supplemented with fermented Astragalus increased egg production rate (P < 0.05) and decreased feed conversion rate (P < 0.05), which may likely be attributed to the improvement of laying hen health status. In vivo, free radicals are harmful by-products generated during normal cellular metabolism and are prone to attack unsaturated fatty acid on the biological membrane, triggering lipid oxidation and lipid peroxide accumulation that result in impairment of organism health  on day 28. Our findings are consistent with our previous studies on broilers (Qiao et al. Astragalus fermented by L. plantarum can enhance the antioxidant ability of both broilers and laying hens. In our previous research, we compared the differences between fermented Astragalus and Astragalus. During the fermentation, the pH was markedly reduced, as a consequence of the increase of organic acids content, which could inhibit the growth of miscellaneous bacteria (Qiao et al. Astragalus polysaccharide, total saponins, and total flavonoids in Astragalus, and fermented Astragalus possess more abundant microflora (Qiao et al. Astragalus active components by fermentation may be responsible for the enhancement of the performance, egg quality, and antioxidant status of serum in laying hens fed with the basal diet supplemented with 3‰ fermented Astragalus.The present study was undertaken to investigate the effects of Astragalus polysaccharide has attracted rising interests in its anti-cancer effects. A previous study has observed that Astragalus polysaccharide can significantly enhance the proliferation of spleen lymphocytes and increase phagocytosis of peritoneal macrophages in mice and is capable of up-regulating the expression of IL-2, TNF-α, and IFN-γ in peripheral blood (Li et al. L. plantarum merely increased the mRNA expression of ileal TNF-α, Astragalus increased the mRNA expression of splenic and cecal IFN-γ and that of hepatic, splenic and cecal TNF-α. Interestingly, fermented Astragalus significantly increased the mRNA expression of both IFN-γ and TNF-α in all the liver, spleen, ileum, and cecum. However, there are comparatively few findings to date regarding the impact of L. plantarum fermented Astragalus on host immune responses. We speculate that the increased content of Astragalus polysaccharide in the Astragalus after fermentation leads to enhance the body’s immune function by increasing the expression of cytokines. Certainly, further investigations will be obliged to fully illustrate that whether there are any endophytic bacteria of Astragalus also responsible for immune activation.In recent, Astragalus on intestinal microbiota of laying hens. Our results showed that fermented Astragalus addition increases the diversity of the ileal bacterial community with the increase of feeding time. Furthermore, at the phylum level, Firmicutes, Bacteroidetes, and Proteobacteria were the most dominant phyla in the intestinal microbiota of hens, which are consistent with previous studies (Danzeisen et al. Astragalus addition led to a reduced abundance of ileal Firmicutes, with an increased abundance of ileal Bacteroidetes and Proteobacteria. We speculate that the increased diversity of the ileal bacterial community might be explained by the fact that the abundance of ileal Firmicutes was reduced to enhance the abundance of other phyla. At the genus level, Lactobacillus as the largest proportion of ileal microbiota of hens is generally highly relevant to feed digestibility (Yan et al. Astragalus addition significantly decreased the abundance of ileal Lactobacillus by 48.51% as compared with the control at 28 days. These results were totally different from our previous report on the effect of fermented Astragalus on the broiler chicken fecal microbiota, which found that the count of Lactobacillus was increased in chickens fed fermented Astragalus as compared with those in the control group. Those factors responsible for the differences should be further studied.Intestinal microbiota plays a major role in maintaining host health, immunity, and production performance, it has become a research hotspot in recent years (Yeoman et al. L. plantarum fermented Astragalus as an efficient dietary additive could significantly promote the production performance, antioxidant capacity, and ileal microbiota diversity of laying hens during the late laying period. A higher expression level of IFN-γ and TNF-α in the liver, spleen, ileum, and cecum of laying hens supplemented with fermented Astragalus indicates a particular role of fermented Astragalus on the innate immune system, and this needs a comprehensive investigation in the future to fully illustrate the exact mechanism.In conclusion, this study suggested that


OARX_76354330.1 and s64890.1 could be identified as functional SNPs for the growth traits of Hu sheep. CAPN6, as a candidate gene, was significantly different in the biceps femoris and longissimus dorsi muscles of weaning (60-day) and 6-month sheep, which facilitated the discovery of causal variants for BW and contributed to the marker-assisted selection breeding of Hu sheep.Body weight (BW) is a critical economic trait for meat production in sheep. The current study aimed to perform a genome-wide association study (GWAS) to detect significant single-nucleotide polymorphisms (SNPs) that are associated with BW in Hu sheep. The comparison and analysis of the G1 and G2 generations of a nucleus meat Hu sheep breeding herd revealed four SNPs identified by GWAS. The subsequent verification of the significant SNP loci in the Hu sheep G3 generation nucleus herd also detected nine SNPs in significant SNP regions. Two SNPs were significantly associated with the BW of Hu sheep ( Genome-1) . The GWA1) . A previ1) . In addi weaning , and 10  weaning . In the  weaning . FOS likon sheep . Moreoveri sheep . Birth wri sheep . BW measThere is no GWAS report on Hu sheep. Hence, the present study aimed to (1) perform a GWAS to detect significant SNPs that are associated with the birth weight, weaning weight, 6-month weight, and yearling weight, using the data from 240 Hu sheep genotyped with the Illumina Ovine SNP50 BeadChip; (2) verify significant SNPs in the progeny of Hu sheep; (3) study the expression differences of candidate genes during sheep growth.Since 2006, the Herbivore Livestock Research Group of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, has established two Hu sheep nucleus herds in Huzhou and Hangzhou. The 240 Hu sheep selected for the study were from the parent (G1) and progeny (G2) of the Hu sheep nucleus herd Culture Department in Huzhou Taihu Lake Culture Cooperative. A total of 202 females of the G3 generation nucleus herd in Hangzhou were used to verify significant SNPs. The feeding management of two Hu sheep nucleus herds was based on the same method applied to meat sheep.BW measurements, including birth weight, weaning weight, 6-month weight, and yearling weight, were taken in a quiet, relaxed state in the two nucleus herds of Hu sheep. To reduce manmade errors in BW measurements, each sheep was measured at least twice by the same person, and the average was the final measurement. Blood samples were collected from the posterior venous plexus of Hu sheep to extract genomic DNA using the standard phenol/chloroform method .Genotyping of individual SNPs was performed using the Ovine SNP50BeadChip . Subsequently, the data were entered into Bead Studio software for related analysis. Chips with call rate <0.99 were removed from further analysis. After the genotypes of the software output were sorted and proofread, statistical analysis was performed, and the inaccurate SNP loci were eliminated or corrected by the software to obtain the characteristics of the genome-wide data. The nucleic acids were typed using a genome-wide genotyping system, and the data were visualized into genotyping results using the Genome Studio software, saved in.txt format, and exported.r2 threshold of 0.2. Thereafter, genotypic data were processed for quality control. The mixed linear model (MLM) of TASSEL 5.0 software2 was used for GWAS analysis of SNPs, and the SNPs related to the phenotype of the Hu sheep core traits were mined. The MLM model calibrates three confounding factors: gender, group structure, and kinship. The specific model isPopulation structure analysis was performed using admixture v1.3. For kinship plot, a heat map of the values in the kinship matrix was created. Kinship matrix was analyzed using the TASSEL5.0. PLINK 1.90p was used to pre-filter the SNP data. Biallelic sites with the maximum genotype-missing rate of 0.2, and minimum minor allele frequency (MAF) of 0.05 were retained. Missing genotypes were imput using beagle 5.1 (version: 24Mar20.5f5). The imputed data were then screened using the LD-based variant pruner and haplotype block estimator implemented in PLINK 1.90p with a windows size of 50 kb, step size of 10, and a pairwise Y: phenotypic value vector of the weight trait of the core group of Hu sheep; β: a fixed effect vector other than the SNP and the population structure; α: SNP effect vector; v: group structure effect vector; u: multi-gene background effect vector; e: residual effect vector; X, S, Q, and Z: correlation matrices of β, α, v, and u, respectively.where p value. The F and p values were calculated by MLM and then tested. The specific formula is as follows:While performing the correlation analysis of the traits of the nucleus herd of Hu sheep, if there are errors in the multiple hypothesis tests, it is necessary to analyze and correct the N: number of independent SNPs used in the analysis. After genome-wide association analysis to obtain significant SNPs, the 500 bp sequence upstream and downstream of the SNP locus was downloaded and aligned against the sequences from databases, such as NCBI and Ovis aries_v4.0 (UCSC)3 to determine SNP locations and the adjacent genetic information for designing primers for PCR and direct sequencing , 0.2 μL of 10 μM forward primer, and 0.2 μL of 10 μM reverse primer. The amplification reaction was pre-denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 25 s, annealing at 55°C for 25 s, extension at 72°C for 20 s; extension at 72°C for 10 min, followed by storage at 4°C. Mutation Surveyor 5.02  was used to discover the SNPs of each individual by Sanger sequencing. PopGen32 software was employed to calculate the gene frequency, genotype frequency, polymorphism information content (PIC), Shannon information content (SIC), and site heterozygosity (H) of each SNP. The GLM procedure in SPSS 19.0 software  was performed to evaluate the SNP-phenotype association of G3 generation nucleus herd.The PCR reaction was performed in a volume of 20 μL containing 1 μL of genomic DNA, 10 μL of 2 × TiosbioCandidate functional genes associated with BW of sheep were selected. RNA from biceps femoris muscle and longissimus dorsi muscle in weaning (60-day) and 6-month sheep was extracted using an animal tissue total RNA kit , and reverse transcription was performed using a Polestar first cDNA synthesis kit + gDNA removal . Quantitative PCR was conducted using the KOD SYBR qPCR Mix  for the detection and quantification of the expression differences of candidate genes in the skeletal muscle of weaning and 6-month sheep.The corresponding primer pairs were listed in The genomic DNA was detected by the whole genome-based detection platform, and the quality control management was performed on 240 individual samples and 54241 SNP sites using Plink1.90 software. We excluded the following: (1) 3577 SNPs with call rate <90%; (2) 20 SNPs not in accordance with the Hardy–Weinberg equilibrium; (3) 4435 SNPs with a minor allele frequency <0.05. Also, 14 individuals were excluded because of the call rate <99%. After quality control, 226 sheep and 35,363 independent SNPs were used for the final analysis.–6 and 2.28 × 10–5 were regarded as genome-wide and suggestive significant values, respectively. The results show that four SNPs were significantly correlated with birth weight. s37755.1 (p = 3.01 × 10–7) was located on chromosome 1, s39389.1 (p = 9.31 × 10–6) on chromosome 7, and OARX_76354330.1 (p = 1.45 × 10–5) on chromosome 27. One SNP was significantly correlated with the 6-month weight. The most significant SNP was OARX_76354330.1 (p = 1.56 × 10–5) located on chromosome 27. In addition, one SNP was correlated with the yearling weight: s64890.1 (p = 2.31 × 10–5) located on chromosome 12; it was close to the threshold value, so it was also included in genome-wide suggestive SNPs, and done with subsequent population verification  and 6-month sheep (p < 0.05), but the difference in the expression of ITGA11 or SCMH1 was not significant , collagen type 1 (COL-1), osteocalcin (OCN), osteopontin (OPN), and dwarf-related transcription factors 2 (RUNX2), in the mouse osteoblast MC3T3-E1 cells, highlighting its regulatory role in bone formation (s37755.1 is located within the SCMH1 (GeneID: 101103519). Scmh1 is a mammalian homolog of the Drosophila Sex comb on midleg (Scm) gene . Scmh1 mormation .ITGA11 (GeneID: 101105162). ITGA11 belongs to the integrin family. Integrin α11 was overexpressed in the stroma of most head and neck squamous cell carcinomas (HNSCC) and correlated positively with alpha smooth muscle actin expression (s39389.1 is located within the pression . ITGA11 pression .CAPN6 (Gene ID: 101110122). Calpain 6 (CAPN6) is a calcium-dependent intracellular lysosomal protease. CAPN6 is a non-proteolytic protease with microtubule binding and stabilizing activities that promotes cytoskeletal structure and microtubule stability in osteoclasts  and 6-month sheep, which proved that during the growth process of Hu sheep, CAPN6 was involved in the development of skeletal muscles. Recent studies have also identified SNP sites in CAPN that significantly affect sheep BW  and 6-month sheep. This phenomenon would facilitate the discovery of causal variants for BW and contribute toward the marker-assisted selection breeding of Hu sheep.In summary, four SNPs identified by GWAS were associated with the BW of Hu sheep, and GSE152717.Data supporting this study has been deposited in GEO – accession number The animal study was reviewed and approved by All procedures performed in studies involving animals were conducted according to the Guidelines for the Care and Use of Animals of Zhejiang Academy of Agricultural Sciences, 2017. The study was approved by the Ethical Committee of Zhejiang Academy of Agricultural Sciences, Hangzhou, China.FS, XS, and YJ conceived the project. YC performed the study and wrote the manuscript. HS, JJ, PX, and JW participated in the analysis of data. YJ is the leader of the project. All authors 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.


Runs of homozygosity (ROH) are commonly used to estimate inbreeding coefficients and identify selection signatures in livestock population. The present study determined ROH patterns, estimated the inbreeding levels, and identified the genome regions with high ROH frequency (ROH hotspots) in Chinese Merino. Our results showed that the genome of Chinese Merino harbored lower ROH abundance. Moreover, the inbreeding levels were relatively low. Thirteen ROH hotspots consisting of 190 genes were identified. The ROH hotspots overlapped the selective signatures might be associated with body size, horn traits, immune traits and environment adaption. These findings could contribute to an optimum breeding program by identifying the candidate genes related to economically traits in Chinese Merino.Ovies aries chromosome (OAR) 21 and OAR3 exhibited the highest and lowest coverage of chromosomes by ROH, respectively. OAR1 displayed the highest number of ROH, while the lowest number of ROH was found on OAR24. An inbreeding coefficient of 0.023 was calculated from ROH greater than 1 Mb. Thirteen regions on chromosomes 1, 2, 3, 5, 6, 10, 11, and 16 were found to contain ROH hotspots. Within the genome regions of OAR6 and OAR11, NCAPG/LCORL, FGF11 and TP53 were identified as the candidate genes related to body size, while the genome region of OAR10 harbored RXFP2 gene responsible for the horn trait. These findings indicate the adaptive to directional trait selection in Chinese Merino.In this study, we estimated the number, length, and frequency of runs of homozygosity (ROH) in 635 Chinese Merino and identified genomic regions with high ROH frequency using the OvineSNP50 whole-genome genotyping array. A total of 6039 ROH exceeding 1 Mb were detected in 634 animals. The average number of ROH in each animal was 9.23 and the average length was 5.87 Mb. Most of the ROH were less than 10 Mb, accounting for 88.77% of the total number of detected ROH. In addition,  PED) is largely dependent on the accuracy, completeness and depth of pedigree information, and pedigree errors are common in many livestock populations. Moreover, FPED tends to ignore the history of selective breeding and underestimates the level of inbreeding, possibly due to the pedigree records [Runs of homozygosity (ROH) are defined as contiguous homozygous genotype segments that arise from the transmission of identical haplotypes from parents to their offspring . The dis records .ROH) is an optimal approach to reflect the present-day and ancient inbreeding levels [At present, several alternative methods have been proposed to estimate inbreeding coefficient based on the whole-genome single nucleotide polymorphism (SNP)-derived metrics of autozygosity. For instance, the ROH-based inbreeding coefficient  is the most well-known sheep breed with fine wool in China. The breeding goals for a sheep breed include fine and soft wool, polled females, and large body size. However, there is limited information about the distribution of ROH across the genome in Chinese Merino. Therefore, this study aimed to investigate the characteristics of ROH in Chinese Merino using the Illumina Ovine 50 K SNP BeadChip, as well as to identify the genomic regions with high ROH frequency, namely ROH islands or ROH hotpots, which could have occurred during the selective breeding for production-related traits.Ovis aries. The eligible SNPs were then filtered according to the following criteria: (1) Call frequency ⩾ 0.95, (2) minor allele frequency (MAF ⩾ 0.01), (3) Hardy–Weinberg equilibrium (HWE > 0.000001), and (4) individual call rate >0.9. The samples that did not satisfy these criteria were excluded.Ear tissue samples were collected from 635 female Chinese Merino (Xinjiang type) sheep in Bohu farm and Gongnaisi farm . All samples were genotyped for 54241 SNPs using the OvineSNP50 Genotyping BeadChip . The quality control of SNP data was performed with PLINK v1.90 software. SNPs were removed from the analysis if they did not align to the autosome of The runs of homozygosity (ROH) in each sample were evaluated using PLINK v1.90 software. The following criteria were used for ROH estimation: (1) The minimum length of ROH was set to 1 Mb, (2) five missing SNPs and up to one possible heterozygous genotype were permitted for each ROH window, (3) the minimum number of SNPs was set to 40, (4) a minimum density of one SNP every 100 kb, and (5) the maximum gap between two consecutive SNPs was set to 1 Mb.Ovies aries chromosome (OAR) and then dividing by the number of animals that had ROH on that OAR. Subsequently, the mean ROH length was divided by the length (in Mb) of OAR.The mean number, average length, and total number of ROH in each animal were estimated. The percentage of chromosomes covered by ROH was also determined. First, the mean ROH length was calculated by summing all ROH (Mb) on the ROH) of each animal was calculated as follows: FROH = LROH / LAUT, where LROH is the total length of all ROH in an animal genome, and LAUT refers to the length of autosomal genome covered by the SNPs included in the array.Based on ROH data, the genomic inbreeding coefficient (FROH (FROHOAR) were estimated as follows: FROHOAR = LROHOAR / LOAR, where LROHOAR is the total length of ROH in an OAR, and LOAR is the length of an OAR covered by the corresponding SNPs.In addition, the values of chromosomal FTo identify the most commonly ROH-associated genomic regions, the percentage of SNP occurrence in ROH was calculated by counting the number of times each SNP within the detected ROH and then dividing the number of animals. The obtained data were plotted against the chromosomal positions of SNPs. The genomic regions with top 1% SNPs were considered as a potential ROH ROH hotspot in the genome ,22.To verify whether recombination can affect ROH hotspots, ROH were mapped based on the position of SNP  . The aveThe identified genomic hotspots were used for gene annotation using Biomart according to the sheep reference genome (Oar 3.1). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were then performed using Metascape with default parameters .In total, 6039 ROH with varying length were identified in all 635 samples, and the average length of ROH was 5.87 Mb. Notably, the longest ROH (up to 60.19 Mb) containing 1247 SNPs was found on chromosome 3, while the shortest ROH was only 1.54 Mb. Among the studied samples, 634 of them had at least one ROH. The average number of ROH in each animal was 9.23 (ranging from 1 to 29 ROH), while the average total length of ROH was 55.78 Mb. Three Merino sheep had the highest homozygosity levels of 392.74 Mb, 356.76 Mb and 327.21 Mb, which were relatively close to 15% of the genome. On the contrary, the Merino sheep with lowest homozygosity had only one ROH of 2.66 Mb. The frequencies of ROH in Chinese Merino under different ROH length categories are presented in n = 686), while the lowest number of ROH (n = 69) was observed on OAR24. The highest genome coverage by ROH was found on OAR21 (14.26%), while the lowest coverage was on OAR3 (3.76%). In overall, the number of ROH tended to reduce with decreasing chromosome length, and the percentages of genome coverage by ROH exhibited significantly different patterns.The number of ROH in each chromosome and the percentage of chromosomes covered by ROH are demonstrated in Furthermore, the genetic position of each SNP was used to map the ROH hotspots, and the abundance levels of ROH in different length categories were used to assess the historical demographic patterns of the breed. The time to the most recent common ancestor (TMRCA) in the four different categories of the breed was evaluated . The powROH) under different length categories are presented in ROH gradually increased as a function of ROH length, ranging from 0.008 to 0.022. In addition, the coefficient of variation was also increased with increasing ROH length. As shown in ROH of Chinese Merino (0.023) was relatively low, with the highest FROH of up to 0.16.Descriptive statistics for ROH-based inbreeding coefficients (FROH in each chromosome (FROHOAR) is illustrated in ROHOAR were comparatively different in the genome of Chinese Merino. The highest value of FROHOAR was found on OAR10, while the lowest value was observed on OAR24.The distribution of FBy determining the abundance of SNPs in ROH, several genome regions were identified as potential ROH hotspots. As shown in 10P > 2). The results indicated that the top 20 clusters were related to the regulation of cyclin-dependent protein serine/threonine kinase activity, positive regulation of immunoglobulin production, oocyte development, regulation of neuroblast proliferation, and regulation of growth and embryo implantation  was greater than 0.1, suggesting a relatively high level of relatedness among the individuals in this breed. However, these results consistently overlapped with the recent decrease in effective population, which might be attributed to the broad use of artificial insemination and decreased number of Chinese Merino raised in farm due to low wool prices in recent years.In Chinese Merino, the mean values of Fis breed . Similarp breeds ,22. The p breeds . The FRObreeding . AccordiH values , the eviROHOAR value was found on Oar10, and these findings were not consistent with those of the Valle del Belice breed [In addition, ROH was used to measure the chromosomal inbreeding coefficients of Chinese Merino. Notably, the highest Fce breed . PossiblThe maximum frequency of SNPs in ROH in Chinese Merino was around 6.15%–9.46%, Similar SNP frequency in ROH was found in Italian sheep breeds and Spanish sheep breeds ,22. On tThe existence of ROH hotspots might be partially attributed to genetic recombination rate . The recNCAPG/LCORL gene. Similar findings were also reported in Italian sheep breeds [NCAPG/LCORL region were found in many sheep breeds [TP53 is the most plausible functional candidate gene for body size in sheep [The ROH hotspot with highest SNP occurrence was located on OAR6 35.07–38.67 Mb), which consisted of the p breeds , Spanishp breeds , Swiss sp breeds ,31. Morep breeds ,32,33, ip breeds . A previp breeds ,37, chic.67 Mb, wp breeds ,40,41. Fp breeds  and Signp breeds . A guiltin sheep . Taken aRXFP2) gene was harbored in this genomic region. The ROH hotspot region also overlapped the selective signatures in RXFP2 across many sheep breeds [RXFP2 region was associated with horn phenotype in sheep [RXFP2 was also identified in Merino [The ROH on OAR10 27.31–29.84 Mb) found in this study overlapped a common ROH island in Swiss sheep breeds  and Spanp breeds ,46,47,48in sheep . The sel–29.84 Mbn Merino ,51. AddiMELK and GNE) [IL4, IL13, IL5, and IRF1) within the genome region, and this selection signature has been detected in sheep breeds [Besides, the ROH hotspots on OAR2 (51.10–52.41 Mb) could be overlapped with a region under climate-associated selection that harbors two genes  . The ROHp breeds .In this study, the distribution patterns of ROH in Chinese Merino were analyzed based on the Ovine SNP50 BeadChip data. Our results demonstrated that the genome of Chinese Merino harbored a lower abundance of ROH, and the levels of ROH abundance were consistent with the effective population size. Moreover, both short and long ROH were detected, indicating that the ancient and recent inbreeding may have an impact on the genome of Merino sheep breed. It was also found that the ROH hotspots in sheep were formed through recombination and selection. In addition, several candidate genes were detected in ROH hotspots, revealing that the selection process for body size, horn, and immune traits in Chinese Merino has left distinctive signatures and formed ROH hotspots. The findings of this study can contribute to the identification of candidate genes associated with economically important traits in Merino sheep.


Massively parallel sequencing (MPS) has rapidly become a promising method for forensic DNA typing, due to its ability to detect a large number of markers and samples simultaneously in a single reaction, and sequence information can be obtained directly. In the present study, two kinds of forensic genetic markers, short tandem repeat (STR) and identity-informative single nucleotide polymorphism (iiSNP) were analyzed simultaneously using ForenSeq DNA Signature Prep Kit, a commercially available kit on MPS platform. A total of 152 DNA markers, including 27 autosomal STR (A-STR) loci, 24 Y chromosomal STR (Y-STR) loci, 7 X chromosomal STR (X-STR) loci and 94 iiSNP loci were genotyped for 107 Tibetan individuals . Compared with length-based STR typing methods, 112 more A-STR alleles, 41 more Y-STR alleles, and 24 more X-STR alleles were observed at 17 A-STRs, 9 Y-STRs, and 5 X-STRs using sequence-based approaches. Thirty-nine novel sequence variations were observed at 20 STR loci. When the flanking regions were also analyzed in addition to target SNPs at the 94 iiSNPs, 38 more alleles were identified. Our study provided an adequate genotype and frequencies data of the two types of genetic markers for forensic practice. Moreover, we also proved that this panel is highly polymorphic and informative in Tibetan population, and should be efficient in forensic kinship testing and personal identification cases. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) are commonly used genetic markers in present forensic cases3. Allele calling can thus be inferred from fragment length by comparison with a locus specific allelic ladder that has been previously sequenced, where the number of repeat units is distinct2. Thus, each allele is regarded as a length-based (LB) allele using this approach. With the advancement of sequencing technologies over the last decade, the existence of sequence structure variations in alleles with the same length has been uncovered4.STRs, usually 2–6 bp in length, are commonly typed with the amplified fragment length polymorphism (Amp-FLP) strategy combining fluorescently labelled multiplex PCR and capillary electrophoresis (CE)5. Several autosomal SNP marker sets and detection methods, such as single-base extension, chip-based microarrays, and allele-specific hybridization arrays, have been developed to compensate for the relatively weaker discrimination power of single loci caused by the bi-allelic nature of the human genome7. However, these methods are not widely used in forensic practice due to the requirement of higher DNA inputs or the limited ability to detect a vast number of SNP loci in a single reaction8.SNPs, which could be amplified with smaller amplicons, are bi-allelic genetic markers with lower mutation rates compared with STRs10. Thus, more alleles can be identified based on the analysis of full sequences of SNPs.Different from detection methods mentioned above, massively parallel sequencing (MPS), also known as next-generation sequencing (NGS), provides new technology for forensic genetic marker typing. Numerous markers and samples can be investigated simultaneously with MPS, and there is no need to consider the problem of the size overlapping of amplified fragments or the availability of fluorescent labels as CE method does. For STRs, both length and sequence data can be achieved; thus, allele calling may be more informative and the allele’s sequence characteristics are identified, resulting in sequence-based (SB) alleles. For SNPs, not only the target SNPs but also the variations in the flanking regions can be identified simultaneously, and form the potential microhaplotype11, STRait Razor13, STRinNGS14, SEQ Mapper15, FDStools16 et al. Sequencer manufacturers also carried out supplementary analysis packages to fit for the data produced by their sequencers, such as ForenSeq Universal Analysis Software17  and Ion Torrent Suite Software Plugins18 .This new technology puts forward new challenges to researchers. First of all, the immense variable and complex data produced by MPS platforms is hard to be analysed manually. Meanwhile, the software packages developed for LB datasets are not efficient anymore. New bioinformatic methods are required to process and interpret these extensive data. An optimal package for MPS data analysis needs to be accurate, time-saving and easy to operate. Several packages has been published to make this process convenient for forensic uses, such as TSSV4. Some researchers have tried to answer these questions20 but a perfect nomenclature is still under development. A unified minimal nomenclature of the complex sequences obtained by MPS technologies was recommended by the International Society for Forensic Genetics (ISFG) in 201622 to facilitate communication between laboratories and to make this data backward compatible with LB data produced on CE platform. In early 2019, the STRAND Working Group was formalized to discuss the expanding and advancing topics of STR sequence nomenclature23. Quality control of string sequences and alleles has also been suggested by ISFG24.Moreover, the LB nomenclature of CE method for STR is not suitable for the complex sequence variations detected by MPS platforms. It is urgent to know how the MPS data should be analysed and reported, what connections do these data have with LB alleles, and how to record and search such datasets in a database28. The ForenSeq DNA Signature Prep Kit  is one of the library preparation kits that simultaneously targets the sequences of Amelogenin, 27 autosomal STRs (A-STRs), 24 Y-STRs, 7 X-STRs, and 94 identity informative SNPs (iiSNPs) in Primer Mix A (DPMA), with the option to include an additional 56 ancestry informative SNPs (aiSNPs) and 22 phenotype informative SNPs (piSNPs) in Primer Mix B (DPMB). A pair-ended sequencing will be performed after the library preparation and then the raw data will be imported to UAS to analyse automatically. Validations using the ForenSeq Signature system have demonstrated its advantages in forensic practice relative to other library preparation kits33, but the knowledge of alleles and genotype frequencies of these 58 STRs is still inadequate for accurate lineage analysis39 and is not sufficient for population genetic studies, which limits its utility in forensic casework.Aiming to facilitate MPS in forensic genetics practice, several commercial and custom STR typing systems have been developed based on different MPS platforms for different purposes40 and can be further classified as Wei Tibetan, Kangba Tibetan and Amdo Tibetan41 by linguistics. The diversities of LB STR alleles using CE methods have been reported in some studies43, but the polymorphisms of SB STR and SNP alleles on MPS platforms using the Forenseq system have not been researched in this ethnic group.The Tibetan ethnic group is one of the oldest ethnic groups in China and in South Asia, and the culture of ancient Tibet was thrived from the tenth to the sixteenth century. The Tibetan ethnic group includes a population of 6.3 million people according to the 2010 Chinese census and they have resided mainly on the highest plateau in the world, the Qinghai-Tibetan Plateau , for hundreds of generations. They have a distinctive language, clothing, customs, religious characteristics from other Chinese or South Asian ethnic groupsIn this study, a Tibetan population from Lhasa (Wei Tibetan), the capital of the Tibetan Autonomous Region, was analysed. Sequence variations in 58 STRs and 94 iiSNPs, and population data from these two kinds of markers were reported. Parameters of evidence weight were also performed.2, while the average total number of reads was 96,239 and 110,292 for each sample for the two runs (Supplementary Table The average cluster density was 740 k/mmhttps://strider.online/) with the assigned accession number STR00014922.A-STRs dataset were verified and accepted by STRidER , flanking region variants only (FRVO) and repeat region plus flanking region variants (RRFR) 44 Table . As showFR) Fig. .Table 1Nhttps://genome.ucsc.edu/). The highest increased allele number due to flanking region variations were observed at D7S820, whose alleles with SNPs accounted for 80% of the total kinds of SB alleles. In addition, in the 214 alleles detected in D7S820, 94.39% were observed with flanking region SNPs. D13S317 presented the second highest increase with an FRV ratio of 62.50% and 55.14% of the alleles being observed with flanking SNPs.Twenty-five SNPs and two InDels were detected in the flanking region of 21 STRs  (Supplementary Table obs were D3S1358 (17.15%), D2S441 (13.90%) and D5S818 (10.39%) successively.Loci with increased number of alleles in SB data compared with LB data also showed gains in observed heterozygosities (Hduo) and trios (CPEtrio) using SB data were higher than those using LB data  ranged from 0.2046 (DYS391) to 0.8672 (DYS481) and 0.2046 (DYS391) to 0.9296 (DYF387S1) when using LB data and SB data, respectively. The increased percentage of GD values of SB data compared with that of LB data ranged from 0 to 77.08% (DYS437). A total of 50 haplotypes were observed in both LB and SB data, with a haplotype diversity (HD) of 0.9971 and 48 haplotypes were unique (0.96)  after Bonferroni correction  , no sequence variation (neither repeat nor flanking region) was observed at the D22S1045 locus. Overall, the sequence variations observed herein were consisted with the observations reported in previous literature50.The Tibetan population described in this study exhibited many sequence variations in repeat regions and flanking regions based on MPS data. A total of 33 STRs showed a higher diversity of alleles when considering sequence variations rather than considering length-based alleles only, while 25 loci showed no increase in allele number by the SB method. Thirty-nine novel alleles were detected, although only 107 samples were studied. Twenty-five SNPs and two InDels were detected in the flanking regions of 21 STRs. InDels existing in the flanking regions of sequences may influence the length call definitions of alleles. Variants with a substantially differences in frequency distributions between different populations is an indicator of the ancestry-informative value of the locus52, it was not surprising to observe the LD phenomenon for iiSNPs pairs. These loci were considered as independent when calculating forensic parameters since these iiSNP pairs were located on different chromosomes52. We suppose it was the special population structure of the aimed population group that caused the disequilibrium. Meanwhile, considering the small sample size of this study, failure of LD testing may also result from random effect.Five pairs and three pairs of loci showed LD after Bonferroni correction when data of target SNPs and full sequences of iiSNPs were considered, respectively. Referring to previous similar studiesAiming to correctly interpret the complex data produced by MPS platforms, a convenient and sophisticated software package for data analysis may promote the use of MPS platforms for this type of forensic genetic study. The two software packages we used here for concordance analysis have their own characteristics. As an offline software with customized web browser interface for forensic use, UAS v1.2.16173 can report the LB allele callings for STRs and genotypes of target SNPs, and can calculate RMP and TDP for specific populations. Although this version of UAS doesn’t support flanking region analysis, it has been improved and allowed the analysis in the upgraded version (UAS v1.3 or later). STRait Razor v2s can analyse more than 300 loci , and focus on both repeat regions and flanking regions of the investigated loci, and can report a SB allele in concordance with the minimal nomenclature requirements recommended by ISFG. Moreover, a much more informative form of SNPs  can be obtained using an MPS platform rather than alleles based on target SNPs only, which may facilitate mixture deconvolution in the future.21 contains repeat number information based on allele length. Similar to the principles of the CE method, the ‘CE callings’ for SB alleles were determined by comparing the length of the fragment with the same structure length relative to a reference sequence. It is important to note that the CE callings may not represent the actual numbers of repeat units of an allele, especially in alleles with flanking region variations. The annotation of the flanking variants in the nomenclature can indicate the true status of a sequence, which is important for researchers to quickly determine the diversity of a given sequence. An InDel that exists in a flanking region may not be identified but can influence the length of a fragment. In this study, an InDel at D7S820 could explain the influence of InDels on nomenclature . Hence, the SB allele name of the string sequence was “D7S820 [CE10.1]-Chr7-GRCh38 84,160,226–84,160,277 [TATC]10 84,160,204-A; 84,160,204.1A”, regardless of if the actual number of repeat units was 10. Moreover, the exact position of the A insertion in the above example was ambiguous. The insertion may exist at any position from 84,160,204 to 84,160,212. Consistent with this observation, the possible InDels have not been defined in the reference template yet53. Similar allele callings were observed in 11 loci . This type of inconsistency between alleles and sequences was reported by Novroski et al.34In order to adapt to the backward compatibility of the CE typing system, the nomenclature recommended by ISFGure Fig. . Through21, while others used custom-defined nomenclature37. Discordant nomenclatures can lead to inconsistent allele calling between laboratories, further confusing precise allele and InDel calling between populations. An optimised consolidation of allele nomenclature and reference genome coordinates for SB alleles should be addressed for a convenient method for data communication between laboratories is urgently needed.Moreover, discrepancies in SB allele nomenclature could be observed when using different coordinates of sequence guides and analysis tools. In previous studies of SB alleles, some researchers followed the nomenclature recommended by ISFGLastly, a high system efficiency of the selected 58 STRs and 94 iiSNPs was demonstrated in this Tibetan population. MPS methods make the combined detection of STRs and SNPs more convenient, thereby improving the system efficiency dramatically. Cases involving personal identification and parentage testing of duos and trios can thus be solved with reliable results. The combination of detection of STRs and SNPs may help to solve problems in complex kinship analysis more efficiently. A lofty goal of this field would be reducing the number of markers (or removing loci with low diversity) when conducting duo and trio testings at an acceptable performance level. Furthermore, exploring customized marker subsets for different identification purposes is also an area of future interest.This study investigated sequence polymorphisms of 58 STRs and 94 iiSNPs in a Tibetan population using massively parallel sequencing, and provided an accurate sequence-based allele frequencies dataset for these loci. Distinct sequence variations were observed in both repeat and flanking regions of these loci, which indicated that the ForenSeq DNA Signature system is highly polymorphic and informative in the studied population. Our study also demonstrated a potential capability for this system to be applied in kinship testing and personal identification cases.Peripheral blood samples were collected using FTA cards from 107 unrelated individuals  from Wei Tibetan population in the Tibetan Autonomous Region of western China.54. Briefly, 1.2 mm diameters of FTA cards were punched directly as an input template without DNA extraction. Target amplification and tagging were performed under advised thermal cycling parameters. Index 1 (i7) and index 2 (i5) adapters were added for target enrichment purposes. Then the libraries were purified using Sample Purification Beads (SPB) and normalized using Library Normalization Beads 1 (LNB1). Finally, 5 μL of the normalized library from each sample was pooled into a single microcentrifuge tube. Seven microliters of pooled libraries were added into 591 μL Hybridization Buffer (HT1) and mixed with 2 μL of diluted Human Sequencing Control (HSC) mixture. Sequencing was performed on a MiSeq FGx instrument  using the MiSeq FGx Reagent Kit  following the manufacturer’s instructions. Two runs were performed to cover all samples in this study.DNA libraries were constructed using the ForenSeq DNA Signature Prep Kit according to the manufacturer’s recommendations17. The intralocus balance threshold was measured as the intensity (number of reads) of the minimum intensity typed allele divided by the intensity of the maximum intensity typed allele and was set as 0.60 (default setting) for all loci except for D22S1045 , which was suggested to be analysed with caution by the manufacture due to the extreme heterozygote imbalance. Alleles were reported using length calling and sequence calling, which contain the repeat region of the locus.The raw sequencing data of STRs was first analysed using ForenSeq UAS (v1.2.16173) with default analytical and interpretation thresholds  for allele calling12 software with the default analytical threshold (AT = 2 reads) and heterozygote threshold . In-house workbooks (written by VBA using Microsoft Excel) were developed to modify the format of the nomenclature produced by STRait Razor v2s so as to completely conform to the requirements recommended by ISFG21 and the revised Forensic STR Sequence Guide_v353. Manual corrections were also performed to verify the nomenclature of SB alleles identified as ‘novel’ by STRait Razor v2s. The 94 iiSNPs were genotyped using ForenSeq UAS with default settings , from which we obtained the alleles and genotypes according to the target SNPs. Comprehensive nomenclature following Parson et al.21 were obtained using STRait Razor v2s12, from which we obtained the alleles and genotypes considering the full sequences of SNPs.The actual ACR was determined for heterozygote loci by dividing the lower number of reads by the higher number of reads. Then, the FASTQ files were re-analysed by STRait Razor v2shttps://strbase.nist.gov/str). Novel alleles were newly discovered if they had not been previously reported in the STR Sequencing Project 44. The SNPs and InDels in flanking regions were compared to the UCSC Genome Browser  and were also verified in the NCBI database .The allele sequence variants of STRs were classified into three categories: repeat region variants only (RRVO), flanking region variants only (FRVO), and repeat region plus flanking region variants (RRFR) Fig. . Referenhttps://strider.online/) for quality control22.The genotyping data of A-STRs and corresponding string sequences were submitted to STRidER (All samples were genotyped using the Goldeneye DNA ID System 25A amplification system. The system contained the 20 expanded Combined DNA Index System (CODIS) core loci plus Penta E, Penta D, D6S1043, a Y indel and Amelogenin for sex identification. DNA amplification was performed according to the manufacturer’s instructions. The PCR products were detected using CE on an ABI 3500xL Genetic Analyzer . The results were analysed with GeneMapper ID-X Analysis Software . Concordance analysis was performed between the LB alleles produced by Miseq and the corresponding CE results. The comparison of genotyping results from UAS and STRait Razor v2s was also performed.55. The expected and observed heterozygosity , polymorphism information content (PIC), discrimination power (DP), random match probability (RMP), and power of exclusion (PE) for both duos and trios of the 27 A-STRs were calculated with Cervus 3.0.756. For X-STR loci, the differentiation of the X-STR allele frequency distribution of females and males was performed using Arlequin 3.5.2.255. HWE test and LD analysis were performed for X-STRs of females. The mean exclusion chance for father/daughter duos (MECduo) and father/mother/daughter trios (MECtrio) were calculated on the ChrX-STR.org.2.0 website (https://chrx-str.org/)57. Finally, an LD test for the 27 A-STRs combined the 7 X-STRs of females was performed using Arlequin 3.5.2.255. Relevant Y-STR parameters, which included genetic diversity (GD), haplotype diversity (HD), allele frequencies and haplotype frequencies, were calculated using an in-house workbook (written by VBA using Microsoft Excel). The formulas were as follows:i represents the allele frequency, N represents the number of haplotypes, and pj represents the haplotype frequency.A counting method was utilized to obtain the LB and SB allele frequencies. For A-STRs, a HWE test and pairwise LD analysis was performed using Arlequin 3.5.2.255. Cervus 3.0.756 was used to calculate allele/full sequence frequencies, Hexp, Hobs, PIC, DP, RMP, PEduo and PEtrio. The effective number of alleles (Ae) was defined as the reciprocal of the homozygosity:i represents the frequency of the ith allele according to Kidd46. We also evaluated the performance of combination of LB A-STRs and target iiSNPs, SB A-STRs and full sequence of iiSNPs with the same methods.HWE and LD for both target SNPs and full sequences of iiSNPs were performed using Arlequin 3.5.2.2All of the experimental process in this study were strictly followed the ethical research principles, and all methods following were performed in accordance with the relevant guidelines and regulations. All samples were anonymously collected after informed consent was obtained. This study was approved by the Ethics Committee of Sun Yat-sen University with the approval number of No. 11[2012].Supplementary Tables


African indigenous taurine cattle display unique adaptive traits shaped by husbandry management, regional climate and exposure to endemic pathogens. They are less productive with respect to milk and meat production which has been associated with amongst others, small size, traditional beliefs, husbandry practices, limited feed resources, disease burden and lack of sustained breeding for trait improvement. This resulted in the severe dwindling of their population size rendering them vulnerable to extinction.The Namchi taurine cattle breed is referred to as [Namchi (Doayo)] and shows resistance traits against trypanosome infection and exposure to tick infestation. Nonetheless, the historically later introduced Zebu cattle are the main cattle breeds in Africa today, even though they suffer more from locally prevailing pathogens.Bos indicus indicus) breeds: Ngaoundere Gudali, White Fulani and Red Fulani.By using a whole genome sequencing approach, we sequenced with high depth for the first time the genomes of five cattle breeds from Cameroon in order to provide a valuable genetic resource for future African cattle breeding: the Namchi, an endangered trypano-tolerant taurine breed, the Kapsiki, an indigenous trypano-susceptible taurine breed, and three Zebu  obtained from analysis using the genome ARS-UCD1.2 were compared with reference genomes of European Bos taurus breeds Kapsiki, N’Dama and Holstein together while the B. indicus breeds White and Red Fulani, Gudali and Brahman clustered separately. The deviating result for Namchi indicates a hybrid status of the selected animal with a recent introgression of Zebu genes into its genome.A total of ~ 100 million (M) SNPs were identified and 7.7 M of those were breed-specific. An approximately 11.1 M constituted of small insertions and deletions. By using only breed-specific non-synonymous variants we identified genes as genetic signatures and associated Gene Ontology (GO) terms that could explain certain cattle-breed specific phenotypes such as increased tolerance against trypanosome parasites in the Namchi breed and heat tolerance in the Kapsiki breed. Phylogenetic analysis grouped, except for Namchi, the The findings provide the first comprehensive set of genome-wide variant data of the most important Cameroonian cattle breeds. The genomic data shall constitute a foundation for breed amelioration whilst exploiting the heritable traits and support conservation efforts for the endangered local cattle breeds. More than 150 cattle breeds or distinct populations have been recorded in Africa . Their pBos primigenius primigenius and B. primigenius opisthonomus from two centers of domestication, namely the Middle East and North Africa, respectively [In Sub-Saharan Africa, trypanosomiasis (Nagana), dermatophilosis, tick-borne diseases and gastrointestinal helminthiasis are the major endemic diseases affecting cattle productivity , 5. Indiectively , 7.Bos taurus longifrons introduced already 10,000 years ago [Bos taurus brachyceros domesticated on the continent some 6500 years ago [Today Namchi and Kapsiki are geographically restricted to endemic areas of human and animal trypanosomiasis in Northern Cameroon. Whereas N’dama and Kuri cattle are grouped as residual longhorn ears ago , 8, Baouears ago .The Kapsiki cattle form a population of approximately 5000 animals that are found mainly in the Mayo Tsanaga (Rhumsiki) area of the Far North region . In contBos indicus Zebu cattle in Africa fall into two distinct groups, the West African Zebu (WAZ) and East African Zebu (EAZ). In Cameroon, 99% of the estimated population of six million cattle are WAZ breeds. They consist of two major sub-types of the Sokoto and Adamawa Gudali [a Gudali . In Centa Gudali . All Zeba Gudali , 14 and Better knowledge of unique adaptive traits against locally prevailing pathogens is needed not only for breed conservation, but also for future genetic amelioration of cattle breeds to mitigate food insecurity problems in Africa. Long-term selection pressure has operated on the genomic architecture and on regions that control traits for adaptive fitness . For exaBos indicus breeds. Furthermore, breed-specific non-synonymous variants were detected, which can be linked to important traits such as trypanotolerance in Namchi and heat tolerance in Kapsiki.In this study, we characterize for the first time the complete genomes of five cattle breeds in Cameroon, namely the endangered taurine trypanotolerant Namchi, the trypano-susceptible Kapsiki taurine, and the three Zebu breeds Gudali, White Fulani and Red Fulani, which are all trypano-susceptible. Using the genomic data, ~ 100 million (M) SNPs were identified in this study of which 7.7 M (~ 8%) were considered as novel variants. In general, lower genetic diversity was found in African taurine cattle breeds than in the Cameroonian Genomic DNA from the cattle breeds Gudali, White Fulani, Red Fulani, Namchi and Kapsiki were sequenced (150-bp paired-end reads) with the Illumina HiSeq4000 sequencing platform and libraries were sequenced using 150-bp paired-end reads. This generated a total of ~ 840 Gb of raw reads for all five breed samples analyzed together, averaging to ~ 167 Gb per sample which provides, to the best of our knowledge, the first comprehensive set of high depth, whole genome variant data of these breeds.9 reads per sample  SNPs were identified in this study of which 7.7 M (~ 8%) were not found in the 1000 Bulls Genomes Project and considered as novel variants Table ; Fig. 2Am et al.  on East m et al.  in AfricBos taurus Holstein, Asian Bos indicus Brahman and African trypanotolerant N’Dama breeds. A total of 1,649,795 SNPs were common across all breeds, and 302,546 SNPs were Zebu-specific, distributed between Brahman, Red Fulani, White Fulani and Gudali cattle breeds  we used the genome ARS-UCD1.2 but not UMD3.1 and compared it with reference genomes of European Bos indicus breeds  using all autosomal SNPs Fig. . The firl Africa , 27. Gudl Africa , 27. Thel Africa , 24, 25.The SNPs and InDels were annotated in order to identify the location of the variant in terms of genomic features using the tool snpEFF . In geneBreed-specific variants with high and moderate impact such as frameshift, missense, splice acceptor, splice donor, start lost and stop gained, that may putatively change amino-acids codons are located in and/or close to genes that may lead to functional changes were examined in each chromosome. Overall, 4349 genes with such mutations were identified: Most of them were from the breeds White Fulani and Kapsiki (both > 2000 genes) and the remaining genes were separated on the other breeds as follows: in Red Fulani 11 genes, in Gudali 8 genes, in Namchi 6 genes, and in the three control samples Brahman 27 genes, Holstein 11 genes, and in N’Dama 10 genes, respectively  and the surfactant protein SFTPD were identified carrying either missense variants or frameshift variants  enrichment analysis for each breed separately. Fifty-two significantly enriched GO terms were identified Fig.  with mosTaken together, the functional annotation and Gene Ontology analysis identified breed-specific high and moderate impact variants as genetic traits which could help explaining cattle-breed specific phenotypes, such as heat tolerance in Kapsiki and trypanotolerance in Namchi.Adaptation to local environment is multifactorial involving several genes –3. To coTrypanosoma congolense, T. vivax and T. brucei is actively driven by the innate immune response. IL-12, INF-γ and TNF-α are primarily produced by cells of the innate immune system and trigger phagocytic cell activation and inflammation, thus contributing to the control of parasite growth [Stress response, olfactory receptors and immune responses play a critical role in adaptation to the tropical environment and diseases , 25. Mame growth . Sialic e growth . In conte growth .OR9G1 gene and in the SIGLEC-1 gene in Kapsiki . All the candidate genes could hence constitute a valuable resource for development and genetic amelioration of tropical cattle breeds, particularly in Africa. Furthermore, the full high depth sequence data widens our knowledge on the value of native breeds as genetic resources for future cattle breeding, and the power of selection signature analyses.The whole genome of five indigenous Cameroonian cattle Namchi, Kapsiki, Gudali, White Fulani and Red Fulani was sequenced and analyzed for the first time, and variant calling results were compared to the reference genomes of European The data used for this paper was obtained from the project “Pathogen detection in African cattle Breeds” Abanda et al.  and PaguOne representative individual of each of the five different cattle breeds was selected Table .Table 3Setaria, trypanosomes and Borrelia spp.  using the Wizard Genomic DNA Purification Kit  according to the manufacturer’s instructions. DNA isolation and concentration was verified by fluorescent methods using Picogreen (Life Technologies). Libraries were generated from 2 μg of genomic DNA per specimen using the Illumina TruSeq DNA PCR-Free Library Prep Kit  following the manufacturer’s protocol. 2 × 150 bp paired-end libraries sequencing was conducted on the Illumina HiSeq4000 platform with the manufacturer’s proprietary TruSeq SBS Kit V3-HS.Blood samples of 5 ml volume per animal were collected in ethylene diamine tetra acetic acid (EDTA)-coated vacutainers during the routine examination. The blood was centrifuged at 3000 rpm for 15 min. Then, genomic DNA was extracted from the buffy coat . Adaptor read sequences were removed using SeqPurge from ngs-bits4 . For comparison with other cattle breeds, whole genome raw sequencing data from NCBI Sequence Read Archive SRA was extracted for the breeds Holstein (SRR934414), N’Dama (SRR3693376) and Brahman (SRR6649996). Paired-end reads from the five samples along with these three controls from the SRA archive were mapped against the reference Bos taurus Hereford breed genome UMD3.1 (NCBI refSeq accession GCA_000003055.5) and ARS-UCD1.2 (NCBI RefSeq accession GCF_002263795.1) respectively, using BWA-MEM version 0.7.10-r789 [The quality of the generated raw Illumina reads was determined using Fast QC software  or 1000 Bull Genomes Project (for mapping to ARS-UCD1.2) were classified as breed-specific novel variants. The average ratios of homozygous versus heterozygous SNPs were calculated for each breed. This ratio is expected to be 1:2 in a freely mating population; therefore, any departure from this condition such as the presence of admixture in the population will be manifested by an increase in the homozygous/heterozygous ratio .Reads that were not mapped to the reference genome UMD3.1 and ARS-UCD1.2, respectively, were extracted from alignment BAM files and sorted by name using Samtools. The sorted BAM files were given as input to AbySS (version 2.1.5) and assembled using the parameter “k = 25” indicating k-mer size = 25 in standard de Bruijn graph mode. Resulting contigs.fa files were subdivided into contigs with a length > 500 bp. Then the remaining contigs were searched against Blastn database using Nucleotide-Nucleotide BLAST (version 2.8.1+) with the parameters “-num_alignments 1”and “-num_descriptions 1” to show alignments and descriptions for the top 1 matching database match only. The BLAST output was then parsed using the R language (version 3.4.0) to determine for each sample the species of the BLAST hit, the percent identity, length of match and query, and BLAST e-value. Mean values of these statistics were calculated for each species in each sample.Bos taurus Holstein, Asian Bos indicus Brahman and African trypanotolerant N’Dama breeds. Breed-specific non-synonymous (bs-ns) SNPs, InDels with moderate and high impact in the genome and new variants not found in any publicly available database were extracted from WAS and WAZ using the data repositories Ensembl release 76, dbSNP138, Entrez Gene, NCBI and Uniprot. The variant carrying genes were functionally characterized based on different gene ontology (GO) terms using clusterProfiler (v3.12) R package(v3.5.2) [For downstream analysis of single nucleotide polymorphisms (SNPs) we used the genome ARS-UCD1.2 but not UMD3.1 and compared it with reference genomes of European (v3.5.2) .To understand the genetic relationships between indigenous cattle breeds and other subfamilies of Bovidae, a principal component analysis (PCA) was performed with EIGENSTRAT. For the phylogenetic tree reconstruction, the variant files were converted to FASTA format with Vcf-kit8 (https://vcf-kit.readthedocs.io/en/latest/). Multiple sequence alignment (MSA) was generated using Muscle with default options . ProttesAdditional file 1: Table S1. Pairwise alignment of contigs assembled from unmapped reads to the non-redundant nucleotide database.Additional file 2: Figure S2. Distribution of SNPs, InDels and breed-specific SNPs per chromosome and breed. Bar plot illustrates the number of SNPs found in at least two breeds (green), breed-specific SNPs (blue) and InDels (orange) across all the breeds for each chromosome.Additional file 3: Table S3. High impact Namchi breed specific SNP. Table S4. High impact Kapsiki breed specific SNP. Table S5. High impact Red Fulani breed specific SNP. Table S6. High impact White Fulani breed specific SNP. Table S7. High impact Gudali breed specific SNP. Table S8. High impact Brahman breed specific SNP. Table S9. High impact Holstein breed specific SNP. Table S10. High impact N’Dama breed specific SNP.Additional file 4: Figure S11. Distribution of SNPs per cattle breed of chromosome 23 between location 25,350,340 and 25,593,072 containing the BoLA gene. The X axis represents genomic location and y-axis represents ratio of non-reference base. Value 1 indicates that all reads carry the non-reference base at a given location whereas a value of 0.5 and 0 indicates half and none of the reads carry the non-reference base, respectively.Additional file 5: Table S12. Trypanosome, Onchocerca filarial and gastro-intestinal parasites detected from five animals of each cattle breed detected by microscopy and molecular diagnostics using ribosomal nuclear makers.