ROH) and homozygosity (FHOM). Our result shows that a total of 7,248 ROH exceeding 1Mb were detected in 262 pigs. In addition, Sus scrofa chromosome (SSC) 8 and SSC10, respectively, has the highest and lowest chromosome coverage by ROH. These results suggest that inbreeding estimation based on total ROH may be a useful method, especially for crossbreed or composite populations. We also calculated an inbreeding coefficient of 0.077 from the total ROH. Eight ROH islands were found in this study. These ROH islands harbored genes associated with fat deposition, muscular development, reproduction, ear shape, and adaptation, such as TRAF7, IGFBP7, XPO1, SLC26A8, PPARD, and OR1F1. These findings may help to understand the effects of environmental and artificial selection on the genome structure of composite pigs. Our results provide a basis for subsequent genomic selection (GS), and provides a reference for the hybrid utilization of other pig breeds.The primary purpose of the current study was to assess the genetic diversity, runs of homozygosity (ROH) and ROH islands in a Chinese composite pig and explore hotspot regions for traces of selection. First, we estimated the length, number, and frequency of ROH in 262 Xidu black pigs using the Porcine SNP50 BeadChip and compared the estimates of inbreeding coefficients, which were calculated based on ROHs (F Crossbreeding is a common strategy to improve livestock production because it can explore complementarities of additive genetic effects as well as heterosis caused by non-additive genetic effects . The EnsGenerally, crossbred offspring can be mated among themselves in each generation, and selection for specific traits and genetic improvement can be applied during this process. Therefore, it is essential to manage genetic diversity by avoiding high inbreeding rates in composite breed, which will retain high levels of heterozygosity and heterosis . It is dRuns of homozygosity are long continuous homozygous segments in the genome that are formed in an individual by the combination of two identical haplotypes from a common ancestor . As an iThe LD analysis is also an efficient approach for determining the level of genetic diversity within a studied population. Generally, LD can be defined as the non-random genetic relationship between two loci in a population . Thus, eThe main objectives of this study were: (1) to investigate the characteristics of ROH on the genome of the Xidu black pig, and also identify the genomic regions with high ROH frequency; and (2) to estimate genetic diversity parameters, such as inbreeding rates and LD in this composite pig population.All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Hubei Academy of Agriculture Sciences, and all methods involved pigs were in accordance with the agreement of Institutional Animal Care and Use Committee of the Hubei Academy of Agriculture Sciences (Permit number: 36/2016).Sus scrofa 11.1 was used in this study.The animal genomics dataset used in the current study were gotten from pigs raised in a composite swine breeding farm located in Enshi, Hubei, China. In order to make the sample representative, we chose individuals from the core group to avoid full siblings. At last, there were 262 individuals consisting of approximately three generations. The 262 animals were genotyped using the Porcine Single nucleotide polymorphism SNP50 BeadChip , consisting of 51,315 Single nucleotide polymorphism (SNPs) evenly distributed along the pig genome. Genotype quality control was carried out using PLINK v1.90  softwareWe identified ROH in individuals using PLINK v1.90 software, which uses a sliding window approach to detect autozygous segments. The algorithm is as follows: take a window of X SNPs and slide them across the genome. Determine at each window position whether the window looks sufficiently “homogeneous” (yes/no). Then, for each SNP, calculate the proportion of the “homozygous” window that overlaps that position. Call segmentation based on this metric, such as a threshold based on the average value. To define a ROH, the criterion and thresholds were as follows: (1) a minimum ROH length of 1Mb; (2) at least 50 homozygous SNPs included in a ROH; (3) a minimum density of a SNP in 100Kb; (4) a sliding window of 50 SNPs across the genome that moves one SNP at a time; and (5) up to one heterozygous SNP and five missing SNPs were allowed in a sliding window. Detected ROHs were later classified into three different classes based on their length: 1–5, 5–10, and >10Mb. The total number and length of ROHs were counted for all individuals.We identified the genomic regions that were mostly associated with ROHs, by calculating the proportion of SNPs in ROH. This was done by counting the number of times the SNP was detected in those ROH across individuals. Afterward, we selected the top 1% of SNPs that were commonly observed in ROHs. Adjacent SNPs that were above this threshold were finally merged into genomic regions which are called ROH islands, which is characterized by being shared by a majority of individuals in the population . We usedROHF) and the other based on excess of homozygosity (HOMF). Genomic inbreeding coefficients (ROHF) were computed for all individuals by the following formula, as proposed by ROHΣL is the length of ROHs, and autoL is the total length of the genome covered by the SNPs included in this chip. ROHF was also calculated based on three length classes: 1–5, 5–10, >10, and total (>1) Mb. Genomic inbreeding coefficients (HOMF) were calculated as O is the number of observed homozygous genotypes, E is the number of expected homozygous genotypes by chance, and L is the total number of genotyped autosomal SNPs. Pearson’s correlation was used to compare the inbreeding coefficients estimated by these methods using R.In this study, two types of genomic inbreeding coefficients were calculated, one based on ROH  were calculated using the parameters “--ld-window 99,999 --ld-window-kb 1,000 --ld-window-r2 0” in PLINK v1.90. To visualize the decline of LD, the physical distances between SNPs were divided into 100-Kb intervals, and the average of r2 in each group was then estimated.The LD was measured using the oH) and expected (eH) heterozygosity estimates were 0.37 and 0.35, respectively, and the average MAF was 0.26. We observed that the oH was somewhat higher than the eH.After quality control, 262 pigs and 38,275 SNPs were retained. The average observed (Sus scrofa chromosome 8 (SSC8) was 57.84Mb . In this study, a total of 7,248 ROHs were identified in the 262 animals with an average of 27.66 ROH per animal. The average ROH length was 6.32Mb, and the longest fragment in The relationship between the total ROH number and the total length of the genome covered by ROH in each individual is shown in For chromosomes, the distribution of total number of ROHs in each chromosome and percentage coverage per chromosome are presented in ROHF and HOMF) using the genotype data of 262 individuals. The mean value of the inbreeding coefficient based on total observed ROHs  was 0.077 and ranged from 0.002 to 0.344. The estimated FHOM inbreeding coefficients was −0.054 with a range from −0.199 to 0.251 in this population . Traditionally, the inbreeding coefficient was estimated based on pedigree data. However, when it comes to cross-bred individuals, things get complicated because the genealogical relationships between parental breeds cannot be established. Moreover, in reality, pedigree information might be incorrect and incomplete, and does not usually take into account the various stochastic events of recombination, which might have occurred during meiosis  were the main source of FROH_total. The inbreeding coefficients obtained by different categories of ROHs with FHOM ranged from 0.63 to 0.83, with the highest correlation found between FROH_total and FHOM. These results are in line with previous research in other pig populations , a composite breed, which is likely due to the admixture present in RDC. A similar result was obtained for a crossbred cattle in Vrindavani  in Xidu black pigs was about 25Kb. This value was less than 79.54Kb (r2=0.3) for Meishan pigs  obtained by the same method  extended up to 150–200Kb. Assuming that the total length of the pig genome is 2.5Gb, this suggests that at least 12,500–16,667SNPs would be required for effective GWAS in this breed. This result could be particularly useful when designing breed-specific SNP array panels in future genomic study and selection programs for this composite pig breed.A total of 38,275 SNPs were used to calculate the average LD between all adjacent SNPs, with a distance less than 100Kb. The average and SD of estimated ult that  estimateult that . When the method . The eff mapping . Howeverue GEBV; . In thisIn summary, in this study, we investigated the patterns of ROH, inbreeding coefficients and LD in the Xidu black pigs. To our knowledge, this is the first study about ROH patterns and autozygosity islands in a composite pig breed. The results of this study suggest that inbreeding based on total ROH may be a useful method, especially for crossbred or composite populations. The detected ROH patterns in this population suggests recent inbreeding events, agreeing with the newest developments in this composite pig breed. Besides, the reported genes within the identified ROH islands point to phenotypic characteristics related to reproduction, fat deposition, ear shape, and environmental adaptation. We believe that these findings will further assist in genome-wide association studies, GS, as well as the design and implementation of breed improvement and conservation programs.https://figshare.com/articles/dataset/Genome-wide_assessment_of_runs_of_homozygosity_and_estimates_of_genomic_inbreeding_in_a_Chinese_composite_pig_breed/14904567.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Hubei Academy of Agriculture Sciences, and all methods involved pigs were in accordance with the agreement of Institutional Animal Care and Use Committee of the Hubei Academy of Agriculture Sciences (Permit number: 36/2016).SM and XP: data curation and supervision. SM: funding acquisition. JW: project administration. ZX: writing – original draft. SM, XP, and FO: writing – review and editing. JZ, YZ, MQ, HS, ZL, LL, and BD: data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Camellia accessions uncovered independent evolutionary histories and parallel domestication in two widely cultivated varieties, var. sinensis and var. assamica. It also revealed extensive intra- and interspecific introgressions contributing to genetic diversity in modern cultivars. Strong signatures of selection were associated with biosynthetic and metabolic pathways that contribute to flavor characteristics as well as genes likely involved in the Green Revolution in the tea industry. Our results offer genetic and molecular insights into the evolutionary history of Camellia sinensis and provide genomic resources to further facilitate gene editing to enhance desirable traits in tea crops.Tea is an important global beverage crop and is largely clonally propagated. Despite previous studies on the species, its genetic and evolutionary history deserves further research. Here, we present a haplotype-resolved assembly of an Oolong tea cultivar, Tieguanyin. Analysis of allele-specific expression suggests a potential mechanism in response to mutation load during long-term clonal propagation. Population genomic analysis using 190  Camellia accessions provide insights into the evolutionary history of the tea plant Camellia sinensis.Haplotype-resolved genome assembly of an Oolong tea cultivar Tieguanyin and population genomic analyses of 190  However, this method has some disadvantages, including greater vulnerability to crop loss through shared disease susceptibility. Clonal crops can be prone to accumulating deleterious mutations, leading to high mutation load in plants that reproduce asexually due to ‘Muller’s ratchet’2. High levels of deleterious mutations in individuals can ultimately reduce relative fitness, associated with reduction of agronomic performance1. Diplontic selection can purge deleterious mutations and involves selecting specific cells bearing favorable alleles from a mixture of other cell lineages3. However, evolutionary consequences of mutation load in clonally propagated crops remain unclear.Many agronomically important crops are clonally propagated, including potato, cassava and tea. Such clonal propagation can be effective to maintain valuable genotypes that may segregate or be lost through sexual recombinationC. sinensis, is a widely consumed beverage that contains multiple polyphenolic compounds considered beneficial to human health4. Although the origin of tea drinking is unclear5, archeological evidence from the Mausoleum of Han Yangling indicates that tea drinking was popular by the 2nd century BCE during the Western Han dynasty6. With more than two billion cups consumed every day, tea is an extremely important crop economically and globally, yielding an annual global harvest of ~5 million tons, worth about US $5.7 billion (ref. 7). Tea is classified into two varieties, C. sinensis var. sinensis (CSS) and var. assamica (CSA) with a number of distinct features, such as leaf size8. Both varieties are flavorful, carry health-promoting bioactive compounds and have been domesticated for commercial tea production.Tea, produced from 11; however, the mosaic assemblies likely missed allelic variations underlying important selected traits. One of the studies of the tea genome generated a phased assembly based on construction of a genetic map. This strategy required a large effort to perform resequencing and variant calling of 135 sperm cells12, hindering application to other crops. Population structure and genetic diversity in tea plants have been extensively discussed recently11, which substantially contributed to the study of tea genomics. Nevertheless, the complex evolutionary history and uncertain phylogeny, especially the reticulate evolutionary pattern with wild close relatives, remain to be examined.Recent studies have provided reference genomes for the two varieties13. This leads to a high level of heterozygosity in the genome, providing a model to investigate allelic variations that may play important roles during evolution. Hybridization among variable tea cultivars is known to produce offspring with desirable traits superior to both parents, indicating the importance of heterosis in tea breeding14. Abundant germplasm resources and the well-documented pedigree of cultivars make this species an attractive model system for studying the mechanism underlying heterosis. Here we show a chromosome-scale genome for the Chinese Oolong tea variety Tieguanyin , with two haplotypes fully represented. We also resequenced several leading tea accessions and close relatives to explore genetic diversity among geographically distinct tea populations. Our results provide insight into the mechanism of heterosis and the evolutionary history of the tea plant and uncover important signatures of selection.Tea plants exhibit allogamy and self-incompatibility15) based on k-mer counting  completeness for the monoploid genome (Table 16 and linked into 15 pseudo-chromosomes that anchored 3.03 Gb (98.96%) of the monoploid genome  of PacBio long reads was 5.41 Gb  of the annotated genes with two defined alleles Table , and a v.07 Fig. . We furt.07 Fig. . These dP < 0.05 and false discovery rate <0.05; Fig. CsSRC2 gene showed a consistent expression pattern across the six tested tissues. The ortholog in Arabidopsisthaliana encodes an activator of a calcium-dependent pathway that mediates reactive oxygen species production in response to cold stress20. We observed two 3-bp insertions and one 78-bp deletion in the second exon of haplotype B, introducing two additional amino acids (lysine and asparagine) while removing 26 amino acids from the deduced protein sequences , without resequencing the parental genomes. We found that 30.1% of genes  showed significant ASE in tea leaves  was mixed, with a substantial contribution of genetic material originated from or similar to Huangdan (purple) and TGY (red and purple). In addition, the Admixture analysis is consistent with the documented breeding event that Fuyun 6 is a typical descendant of Fudingdabai11, showing a mixture of Fudingdabai (cyan) and one unknown CSA accession (pink) in Fuyun 6  within the CCSA subgroup (6.44 × 10−4) than that within the four CCSS populations  to investigate population divergence, which showed that the population divergence among four CSS subgroups (average = 3.67 × 10−2) was much smaller than that between the two CSA subgroups  and one outgroup, 21 individual plants from 14 Camellia species were resequenced at the whole-genome level  within the species from section Thea with C. sinensis as P3. The f3 analysis showed significant adjusted negative Z scores (adjusted Z score < −1.96) in most tested triplets , including transporting ATPase activity and metalloexopeptidase activity  showed that C. sinensis underwent two demographic bottlenecks, both coinciding with known periods of environmental change . However, the second Ne drop was restricted to CSS and occurred during the extremely low temperatures25 of the Last Glacial Maximum , followed by a rapid demographic expansion  gene -abscisic acid old Fig. . In addiene Fig. , leadingR)-limonene, (E)-β-ocimene, pinene, myrcene and α-farnesene  and CsCYP90B1 (CsDWF4)), associated with photomorphogenesis, were also under artificial selection in the early domestication of CSA and the improvement process of CSS, respectively  and identify allelic imbalance. ASEGs were classified into two major patterns: consistent ASEGs and inconsistent ASEGs . Consistent ASEGs had an allele with biased expression across all the tested tissues of tea plants, supporting a dominance effect on heterosis. Genes with expression biased toward one parental allele in some samples but shifted to another allele in other samples  indicate an overdominance effect28. In contrast to hybrid rice28, we observed considerably more consistent ASEGs than inconsistent ASEGs  in C. sinensis, suggesting that the dominance effect played a major role in the highly heterozygous tea genome. The large number of consistent ASEGs is likely caused by accumulation of somatic mutations due to the long period of clonal propagation in tea plants. Study of the mechanism of widespread ASE possibly due to epigenetic modifications32 is a further work that deserves much effort. Basing on our results, we propose that the dominance effect likely provides a potential mechanism to overcome mutation load in clonally propagated tea plants.TGY is a world-renowned Oolong tea cultivar, which was selected during the reign of Yongzheng Emperor in the Qing Dynasty . A ~300-year clonal propagation has led to accumulation of substantial somatic mutations in the genome, allowing us to separate the two haplotypes using our newly developed algorithms , even in the same cultivar.The two ancient bottlenecks in CSS, both coinciding with a dramatic temperature decline, should lead to a substantial reduction in population diversity and smaller CsCYP734A1 (CsBAS1) and CsCYP90B1 (CsDWF4)) associated with photomorphogenesis were under artificial selection in CSS and CSA cultivars, respectively. These two genes are involved in brassinosteroid biosynthesis. Loss of function in the Arabidopsis dwf4 mutant results in dwarfism due to abnormal cell elongation34, while the double mutant in BAS1 along with its functionally redundant paralog (SOB7) displays elongated hypocotyl and decreased sensitivity to light35. Similar to wheat Rht genes and the rice sd1 gene36, the two genes CsBAS1 and CsDWF4 likely contributed to the Green Revolution in tea industry as they may have introduced dwarfing traits into this crop. In conclusion, this study provides important insights into genome evolution, allelic imbalance, population genetics and further directions for crop breeding of tea plants. Our newly developed genomic resources can advance molecular biology research and ultimately offer tools and knowledge for shortening the 20–25-year breeding cycle through gene-targeted improvement of the tea crop.Our efforts to detect signatures of artificial selection provided evidence of parallel domestication in CSA and CSS. The two varieties possess distinct features, such as various aromatic chemicals, different plant heights and cold tolerance, which were likely targets of artificial selection over the domestication history. Our results uncovered that several protein-coding genes associated with these economically important traits underwent domestication. Key genes related to biosynthetic metabolism of alkaloid and aromatic chemicals, including caffeine and catechins, contributed to the feature of interest in tea plants. In contrast to ACSA, CCSA and CCSS have reduced plant height, with CSA being small trees or semi-shrubs and CSS being shrubs. The morphological modification (plant height) in CCSA and CCSS is likely associated with domestication, as two cytochrome P450 genes  in Beijing. Genomic DNA from each sample was isolated using the DNeasy Plant Mini kit (Qiagen) following the manufacturer’s instructions. For PacBio long-read sequencing, we first applied the BluePippin system for size selection. SMRTbell libraries (30–50 kb) were then constructed according to the protocol released from PacBio. A total of three single-molecule real-time cells were sequenced on a PacBio Sequel II platform, generating 359 Gb of subreads. DNA samples that were used for whole-genome resequencing were sequenced using the Illumina NovaSeq platform with a read length of 150 bp and an insert size of 300–500 bp. In addition, the 10x Genomics library was constructed using high-molecular-weight DNA (>50 kb) according to the manufacturer’s protocol (https://support.10xgenomics.com/de-novo-assembly/library-prepr/doc/user-guide-chromium-genome-reagent-kit-v1-chemistry). Reads of approximately 300 Gb were sequenced on the Illumina NovaSeq platform with the 150-bp paired-end sequencing model.The TGY plant used for PacBio sequencing and de novo genome assembly was maintained by the Tea Research Institute, Fujian Academy of Agricultural Sciences. Leaves were collected from a single TGY individual, planted in the county of Anxi located in Fujian Province, China . In addition, we constructed a comprehensive dataset by incorporating our resequenced data (129 samples) as well as recently published 61 non-redundant resequenced accessions17 (version 1.9) with optimized parameters designed for polyploid genomes to assemble heterozygous genome sequences as far as possible . To further correct systematic errors of PacBio sequencing, we generated ~183 Gb (58× coverage) of Illumina short reads from the same TGY individual. These short reads were mapped against the Canu initial genome assembly using BWA37 MEM with default parameters, and variants that were considered to result from sequencing errors were polished using Pilon38 with parameters ‘--mindepth 4 --threads 6 --tracks --changes --fix bases --verbose’.We assembled the TGY genome by incorporating Illumina short-read sequences and PacBio single-molecule real-time long-read sequences as well as sequences from high-throughput chromatin conformation capture (Hi-C) technologies. A total of 359 Gb (~114× coverage) of subreads generated from the PacBio Sequel II platform were subjected to self-correction, trimming and assembly. All three steps were accomplished using Canu39. Chimeric DNA fragments that represented sequences from proximal regions were sequenced on the Illumina NovaSeq platform with the paired-end model. The resulting non-redundant contigs were subjected to ALLHiC scaffolding with a diploid model18 and then partitioned into 15 groups, representing 15 pseudo-chromosomes. The chromosome number and orientation were renamed according to the chromosome-scale assembly of CSS-SCZ published previously9 for comparison. For the haplotype-resolved genome assembly, we first detected misassembled contigs that displayed abnormal long-range contact patterns from paired-end read alignments against the Canu initial assembly using Juicer tools40 and the 3D-DNA pipeline16, and only the first round of Hi-C corrected contigs were retained for haplotype phasing. We further applied a read-depth strategy to identify and duplicate collapsed contigs in the Canu initial assembly   were fully resolved at the chromosomal level.We provided two levels of chromosome-scale assemblies, including a monoploid genome and a haplotype-resolved assembly. For the monoploid genome, we first used our newly developed program Khaper to select primary contigs and filter redundant sequences . Significance of enrichment was determined using Fisher’s exact test, with P values adjusted using the Benjamini–Hochberg multiple-hypothesis-testing correction.To annotate protein-coding genes, we applied the same method as described previously for the sugarcane genome19), relying on a ‘true’ phased SNP dataset, which can be generated by incorporating PacBio long reads and 10x Genomics linked reads. The concept of the ‘true’ phased SNP dataset is to find consistently phased SNPs in PacBio read phasing and 10x read phasing. To achieve this, we first constructed an accurate variant-calling file (VCF) based on Illumina WGS short reads following the GATK45 best practices workflow suggested on the official website. Subsequently, approximately 80 Gb of PacBio long reads with length >10 kb were randomly selected and mapped against the reference genome using minimap2 (ref. 46) with the parameter ‘--secondary=no’, which means that only the best alignment was reported for each long read. The resulting BAM file along with the Illumina VCF was subjected to WhatsHap (version 1.1) phasing47 with default parameters, and the phased SNPs with the ‘PS’ label were extracted for further comparison. For phasing of 10x Genomics linked reads, we used proc10xG Python scripts (https://github.com/ucdavis-bioinformatics/proc10xG) to extract and trim reads of gem barcode information and primer sequences, respectively. This pipeline used BWA MEM for 10x linked reads mapping, and the resulting BAM file was also subjected to WhatsHap SNP phasing. Consistently phased SNPs in the two datasets were considered as ‘true’ phased SNPs, which were further used for assessment of ALLHiC phasing.A switch error indicates that a single base that is supposed to be present in one haplotype is incorrectly anchored onto another. This kind of assembly error is likely prevalent in the haplotype-resolved genome assembly. To detect switch errors in our phased chromosome-scale TGY genome assembly, we developed a new pipeline .We next aligned two haplotypes in our ALLHiC assembly using the Nucmer program41. Because haplotype-resolved genome assembly is available for the TGY genome, each allele can be annotated from DNA sequences. The allele definition can be achieved using a synteny-based strategy and a coordinate-based method. Synteny blocks between two haplotypes were identified using MCScanX49, and paired genes within each synteny block with high similarity were considered as alleles A and B. Gene models with exactly the same coding sequences were considered as a single allele. In addition, gene models that were not present in syntenic blocks were mapped against the monoploid assembly using GMAP50. Potential alleles were considered if two genes had more than 50% overlap on coordinates.We used the same method as we did for an autopolyploid sugarcane genome project to identify alleles51 for pairwise comparison of allelic genes with default parameters. The edit distance between two alleles was counted if any base substitution or indel was detected using the Text Levenshtein distance model, implemented in PERL. The similarity score was calculated as the number of unsubstituted bases divided by the length of the alignment block.We used the MAFFT program52), using the ‘NEAR’ seeding scheme, which favors short and strong similarities that are assumed to occur between closely related sequences. Haplotype A for each chromosome was used as input ‘as is’, with no external repeat masking except for simple repeats using tantan53 (lastdb parameters ‘-P0 -uNEAR -R01’). LAST alignments were then performed with lastal parameters ‘-E0.05 -C2’, followed by splitting alignments into one-to-one matches using last-split54. LAST alignments resulted in one MAF file that contained all high-scoring segment pairs per pairwise chromosome comparison. These resulting high-scoring segment pairs form the basis for calculating sequence identities in each pairwise comparison. Identities between haplotypes were calculated based on 10-Mb non-overlapping windows at the most stringent level with no indels or gaps within an alignment block. To identify different types of genetic variations between haplotypes, the Nucmer48 program was used to map HB to HA genomic sequences, and SNPs were identified from the alignment file with ambiguous best matches. Furthermore, we applied Assemblytics55 to identify short indels (1–10 bp) and large structural variants on the basis of the alignments above.Pairwise comparison between haplotypes was performed using LAST version 959  were generated using three biological replicates. RNA-seq reads were trimmed using the Trimmomaticwww.omicshare.com/tools). All functional enrichment analyses were calculated against a background gene set , and background genes were submitted to the Mendeley database (10.17632/9nr63jfhtd.1) along with a functional annotation.GO enrichment and KEGG pathway analysis were performed using OmicShare tools (Q < 30) were trimmed from raw reads using Trimmomatic56, and the resulting clean reads were aligned against the monoploid reference genome of TGY using BWA37 with default parameters. To analyze population genetics, we focused on SNPs and small indels (1–10 bp). These variants were identified using the GATK45 pipeline following the best practices workflow suggested on the official website. To remove erroneous mismatches around small indels, IndelRealigner was applied to process the alignment BAM files. HaplotypeCaller and GenotypeCaller were used to call variants from all samples. SNPs were subjected to quality control and removed if they met the following criteria: (1) SNPs only present in one of the two datasets , (2) SNPs in repeat regions, (3) SNPs with read depth >1,000 or <5, (4) SNPs with missing rate >40%, (5) SNPs with <5-bp distance from nearby variant sites, (6) non-biallelic SNPs. The SnpEff60 program was used to annotate SNPs and large-effect SNPs with modification of start or stop codon, and alternative splice sites were extracted for further analysis. SNP accuracy was assessed based on manual checking of 100 randomly selected SNPs in JBrowse61, showing an accuracy of 95%.We sequenced a total of 7.2 Tb of paired-end reads on the Illumina NovoSeq platform. This resulted in an average coverage of 12.75× per accession. To avoid potential DNA contamination, such as index swapping, we constructed dual-indexed libraries with unique indices for each sample. Double indices contain a total of 16 bases and were inserted in the flanking regions of the target DNA fragments. This allowed us to unambiguously separate DNA sequences pooled from different libraries and avoided potential index hopping. In addition, raw reads that had any mismatch with index sequences were clustered as undetermined sequences and finally removed from our analysis. Adaptors and low-quality bases  were considered single-copy genes. A total of 11,334 single-copy genes were identified based on our method. Nuclear SNPs were further extracted from genomic regions located in single-copy genic regions. For heterozygous SNP sites, the major alleles were determined and retained for further analysis if they had more Illumina reads supported than the secondary alleles. The resulting SNPs were converted to aligned FASTA format. Maximum-likelihood trees were constructed using two popular programs: IQ-TREE22 software was used to infer the ancestral population among the resequenced tea accessions with different k values (from 1 to 10) tested. To avoid parameter standard errors, we allowed testing with 2,000 bootstraps. The optimal ancestral population structure was determined based on cross-validation error, with k = 7 showing the smallest cross-validation error and thus considered to be the best population size.Admixture64 version 0.1.16 were used to perform PCA and other population divergency statistics, including nucleotide diversity and genetic differentiation (FST). Linkage disequilibrium decay was calculated using PopLDdecay  with default parameters, and the decay distance of linkage disequilibrium indicates the Pearson’s correlation efficient (r2) decreased to half of the maximum.PLINK1.9 and VCFtools65. BAM files generated from each accession were filtered with parameters ‘-only_proper_pairs 1 -uniqueOnly 1 -remove_bads 1 -minQ 20 -minMap 30’. After that, we used the ‘-doSaf’ parameter to calculate the site allele-frequency likelihood based on individual genotype likelihoods, assuming HWE, and then used the realSFS with expectation–maximization algorithm to obtain a maximum-likelihood estimate of the folded SFS. The stairway plot66 was used for estimating the population demography history. Stairway plot was performed with 200 bootstraps, a generation time of 3 years and a mutation rate per generation per site of 6.5 × 10−9.We first calculated site-frequency spectrum (SFS) using ANGSD67 and Tajima’s D-test as well as population fixation statistics (FST). To avoid false positive signals, we first filtered 26,318,206 of 35,725,355 (73.7%) SNPs located at TE regions, 12,030 of 35,725,355 (0.03%) SNPs at NUMT regions and 4,496 of 35,725,355 (0.01%) SNPs at NUPT regions before sweep finding. Subsequently, we applied the XP-EHH approach to identify positive selection sites by measuring cross-population extended haplotype homozygosity, which was implemented in the selscan program (https://github.com/szpiech/selscan). The XP-EHH score for each chromosome was calculated individually, and the top 5% sites with positive XP-EHH values were considered as signals for candidate selective sweeps. These candidate selective sweeps were further validated using Tajima’s D statistic and FST analysis. Tajima’s D statistic was calculated in sliding windows with a 10-kb window size and a 5-kb step size using the ANGSD program65, and the empirical lowest 5% windows were retained for validation of the candidate selective sweeps identified by XP-EHH. Similarly, FST values were calculated in VCFtools using the same sliding window size, and the top 5% regions were retained. XP-EHH candidate regions either supported by Tajima’s D statistic or the FST value between two tested populations were considered as the final set of selective sweeps.Patterns of selective sweeps associated with artificial selection were investigated based on three genetic differentiation metrics, including XP-EHHf3 values using the program ADMIXTOOLS22; Z scores were adjusted based on a Benjamini–Hochberg false discovery-rate correction method.To detect introgression between cultivated tea plants and close relatives, we calculated D statistic using the program doAbbababa2, implemented in ANGSD62. Patterson’s D statistic is widely used to examine site patterns  in genome alignments for a specified four-taxon tree. Given four taxa with the relationship ‘, P3), O’, a D statistic significantly different from zero indicates introgression between populations P1 and P3  or between P2 and P3 69.To detect introgression between cultivated tea plants and close relatives, we calculated the Patterson’s fd statistics22, which is a modified version of a statistic originally developed to evaluate admixture at a genome-wide level. C. oleifera was used as an outgroup to infer phylogeny of the tested triplets , with a combination of any of the four cultivated tea populations (P2) and close relatives from Camellia section Thea (P1 or P3). Modified fd statistics were calculated for each 100-kb non-overlapping window with the high-quality of SNP data identified above as input using a set of Python scripts . Windows with a negative Patterson’s D statistic and fd > 1 were ignored as suggested24. Within each cultivated tea population, we used a threshold of the 95th percentile to detect outliers of the fd distribution that could be considered as introgressed loci from close relatives.Introgressed loci were identified based on the modified four-taxon Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41588-021-00895-y.Supplementary InformationSupplementary Notes 1 and 2, Figs. 1–13 and Tables 1–13 and 15–17Reporting SummarySupplementary Table 14Information and statistics of resequenced tea accessions.


In order to understand the genetic basis of starch pasting viscosity characteristics of Chinese spring wheat, we assessed the genetic variation of RVA parameters determined by the Rapid Visco Analyser in a panel of 192 Chinese spring wheat accessions grown in Er’shi, Shihezi and Zhaosu during 2012 and 2013 cropping seasons. A genome-wide association study with 47,362 single nucleotide polymorphism (SNP) markers was conducted to detect marker-trait associations using mixed linear model. Phenotypic variations of RVA parameters ranged from 1.6 to 30.7% and broad-sense heritabilities ranged from 0.62 to 0.91. Forty-one SNP markers at 25 loci were significantly associated with seven RVA traits in at least two environments; among these, 20 SNPs were located in coding sequences (CDS) of 18 annotation genes, which can lead to discovering novel genes underpinning starch gelatinization in spring wheat. Haplotype analysis revealed one block for breakdown (BD) on chromosome 3B and two blocks for pasting temperature (T) on chromosome 7B. Cultivars with superior haplotypes at these loci showed better starch pasting viscosity than the average of all cultivars surveyed. The identified loci and associated markers provide valuable sources for future functional characterization and genetic improvement of starch quality in wheat. Triticum aestivum L.) is one of the most important staple food crops worldwide. With the improvement of living standards, people pay more attention to the quality of end-use products of wheat. Improvement of quality traits has become a major objective in wheat breeding . Each genotype was sown in ten rows, with a row length of 3 m, a row-to-row distance of 25 cm and plant-to-plant distance of 10 cm. There were differences in climate and soil conditions among Er’shi, Shihezi and Zhaosu, and different temperatures between the years 2012 and 2013. Planting and harvest dates and trial management varied according to the recommendations of each location. The accessions were harvested at maturity and cleaned prior to quality test.Flour milling was performed in a mill  to flour extraction rates of around 65%. Prior to milling, the hard, medium hard (mixtures of hard and soft wheat) and soft wheats were tempered overnight to moisture contents of around 16, 15, and 14%, respectively.Pasting properties of flour were determined with a Rapid Visco Analyser . The 3 g flour was suspended in 25 ml of distilled water before the solution was placed inside RVA instrument. The programs of temperature in the following order: held at 50°C for 60 s, heated from 50 to 95°C at a rate of 1°C/5 s and held at 95°C for 150 s, then cooled to 50°C at a rate of 1°C/5 s and held at 50°C for 120 s. RVA parameters including peak viscosity (PV), trough viscosity (TV), breakdown (BD), final viscosity (FV), setback (SB), peak time (PT) and pasting temperature (T) were determined.Fresh leaf samples were collected from 10-day old seedlings and sent to the CapitalBio Technology company4 cycles were used, followed by a simulation runs of 105 cycles with an admixture model. The K values of 1–10 and 3 independent runs were selected to attain reliable results. Web-based analysis “Structure Harvester v0.6.93”The Bayesian clustering technique was used with 3400 SNP markers to classify groups of genotypically same individuals using the statistical software STRUCTURE v.2.3.4 . Burn-inA principal component analysis (PCA) using filtered SNPs was performed with the TASSEL v.5.2.43 and the first three PCA values were plotted in three dimensions.p < 4.18E-4 . Linkage disequilibrium (LD) was calculated by TASSEL 5.0 with the markers whose positions were known. The LD decay plot was generated by intra-chromosomal r2 and base pair distance using R package ggplot2.Phenotypic data in different environments and best linear unbiased prediction (BLUP) values were analyzed, respectively, for association analysis to identify the marker-trait associations (MTAs) employing mixed linear model (MLM) in TASSEL v.5.2.43 . The MLMThe Chinese spring reference genome (IWGSC RefSeq v1.0) and gene annotations in GFF3 format were retrieved from the Ensemble database release 44p) above the threshold, and the phenotypic values of accessions with different alleles reached significant level (p < 0.05) in multi-environment, haplotype analysis was carried out by Haploview version 4.2 software  of RVA traits were calculated as h2 = σ2g/(σ2g + σ2gl/l +σ2gs/s + σ2gls/ls), where σ2g, σ2gl, σ2gs, σ2gls variances for genotypic, genotype by location interaction, genotype by season interaction and genotype by location by season interaction, respectively, whereas l and s were the numbers of locations and seasons, respectively. SNP density plots, Manhattan and quantile-quantile (Q-Q) plots were generated in R package CMplot while histograms were performed in Origin 8.0.ANOVA and correlation analysis were carried out using SPSS version 22.0. The coefficient of variation (CV) was calculated by dividing the standard deviation by the average of trait values. The BLUP values of all traits over 2 years across environments were calculated by the R package Lme4 . Broad-sh2 estimates were 0.91, 0.77, 0.86, 0.89, 0.82, 0.62 and 0.85, respectively , BD (r = 0.81**) and FV (r = 0.63**); TV was significantly and positively correlated with FV (r = 0.94**), SB (r = 0.72**) and PT (r = 0.63**); FV was significantly and positively correlated with SB (r = 0.89**) and PT (r = 0.48**); BD was significantly and negatively correlated with PT  on chromosomes 7A (1), 7B (1) and 7D (2) at 534, 489 and 463 Mb, respectively, were significantly associated with both PV and TV, while one SNP  on chromosome 7D at 13 Mb was significantly associated with both PV and BD. Thirty-four SNP markers associated with RVA parameters were mapped in the annotated genes; 20 SNPs among these were located in the CDS of 18 annotation genes, which were considered as candidate genes  revealed significant differences (p < 0.05) of the traits between two alleles in at least four environments  associated with BD showed that the 776–782 Mb interval on chromosome 3B had an 862-kb block, and six SNPs including BS00023017_51 were clustered in the block (BS00024883_51/BS00023017_51/IAAV8892/RFL_Contig2578_862/wsnp_Ex_c33879_42293206/wsnp_CAP11_c59_99263) haplotypes showed significantly higher BD (p < 0.05) than the average of all cultivars surveyed in four environments  associated with T, and two blocks in 442–454 Mb interval on chromosome 7B were detected. Two cultivars with CC/AA/CC , 17 cultivars with AA/TT/GG/GG (wsnp_Ex_c64815_63464750/RAC875_c4438_419/IAAV3414/TA001679-0356) haplotypes showed higher T than the average of all cultivars surveyed, exhibiting significant differences in some locations  encodes granule-bound starch synthase, generating a higher PV and BD  around there.Significant and stable MTAs for PV were mainly clustered to chromosomes 2B, 3A, 3B, 4A, 4B, 5A, 7A, 7B and 7D ; severalV and BD . Some stosome 4A . In the radation . In the GENE-4993_69 on chromosome 7B, Excalibur_c9183_1397 and Tdurum_contig69003_459 on chromosome 7D, BS00067650_51, Wx-B1 on chromosome 4A, where no locus associated with FV was detected in this research. Nine SNPs in seven loci on chromosomes 2B (2), 5B (1), 6B (1), 7B (2) and 6D (1) were significantly associated with T  significantly associated with T was very close to TRAESCS6B02G418100  which was the homolog of phosphorylase  SNPs were mapped in intergenic regions, and 34 (83%) were mapped in genic regions . Among tndosperm . Other cBS00023017_51, Excalibur_c10124_361 and Kukri_c4560_110 had significant (p < 0.05) effects on traits in at least four environments, and could be used as optimal loci in marker-assisted breeding and quality improvement. The haplotype analysis was conducted on these loci and three blocks were detected  revealed one block for BD on chromosome 3B and two blocks for T on chromosome 7B, cultivars with superior haplotypes at these loci showed better starch pasting viscosity than the average of all cultivars surveyed. Validation studies for SNPs in the candidate genes and detected loci will be conducted in the future by designing KASP assays, which can be further used for marker-assisted breeding for improvement of grain starch quality in wheat.It is not feasible that phenotypic selection for improving wheat starch quality at the early stages in wheat breeding. Even at later stages in the breeding process, starch quality was also impacted greatly by environments. Therefore, genetic selection through MAS is a desirable way to improve wheat starch quality in wheat breeding programs. Based on dense SNPs across the whole genome, the GWAS has become a common approach to uncover genetic components of agronomic traits, which provides us with insightful information into genetic architecture of complex traits. In this study, GWAS analysis were performed for RVA parameters with 47,362 SNPs in 192 Chinese spring wheat accessions among six environments. Forty-one SNPs at 25 loci were stably detected in at least two environments, of which 20 SNPs were located in the CDS of 18 annotation genes. Haplotype analysis for regions harboring the SNPs (


Microarray-based genomic selection is a central tool to increase the genetic gain of economically significant traits in dairy cattle. Yet, the effectivity of this tool is slightly limited, as estimates based on genotype data only partially explain the observed heritability. In the analysis of the genomes of 17 Israeli Holstein bulls, we compared genotyping accuracy between whole-genome sequencing (WGS) and microarray-based techniques. Using the standard GATK pipeline, the short-variant discovery within sequence reads mapped to the reference genome (ARS-UCD1.2) was compared to the genotypes from Illumina BovineSNP50 BeadChip and to an alternative method, which computationally mimics the hybridization procedure by mapping reads to 50 bp spanning the BeadChip source sequences. The number of mismatches between the BeadChip and WGS genotypes was low (0.2%). However, 17,197 (40% of the informative SNPs) had extra variation within 50 bp of the targeted SNP site, which might interfere with hybridization-based genotyping. Consequently, with respect to genotyping errors, BeadChip varied significantly and systematically from WGS genotyping, introducing null allele-like effects and Mendelian errors (<0.5%), whereas the GATK algorithm of local de novo assembly of haplotypes successfully resolved the genotypes in the extra-variable regions. These findings suggest that the microarray design should avoid polymorphic genomic regions that are prone to extra variation and that WGS data may be used to resolve erroneous genotyping, which may partially explain missing heritability. DGAT and ABCG2 [On the eve of the genomic era, it was reasoned that progress in the use of DNA technologies for the enhancement of cattle production would be proportional to advances in the knowledge of the underlying mechanisms of the genes involved . Howevernd ABCG2 , which hnd ABCG2 . Giving nd ABCG2 . Genomicnd ABCG2 .Two commercial companies, Illumina and Affymetrix, have developed SNP genotyping technologies on a large scale. The Illumina microarray uses microscopic beads, whereas Affymetrix uses glass or silicon microchip surfaces. Illumina beads are applied into microarray wells allowing multiple (~30) technical replicates. Depending on the targeted variation, two designs of beads are used. For the first design (D1 type), which commonly targets the variation A/G, the beads contain a 50 bp oligonucleotide for which the 3′ end is positioned directly adjacent to the SNP site. The second design (D2 type) requires two types of beads for which the 3′ end of the 50 bp oligonucleotide overlaps with polymorphic site. This design, which is normally used for A/T or C/G variations, allows polymerase extension only when a perfect match occurs. DNA samples are amplified, digested into smaller parts and hybridized to the beads’ probes. Then, an extension reaction is performed, elongating the samples by a labeled nucleotide . AffymetAiming at developing a robust genotyping platform for genomic selection of dairy cattle, a U.S.-based consortium has established a panel of markers for the Illumina technology. The resulting BovineSNP50 BeadChip was commercially released in 2007, offering the detection of 54,001 SNPs, and its marker design was based on the alignment of sequence data from different breeds of cattle to a single cow’s genome assembly: the Hereford cow, L1 Dominette . To dateSperm DNA from 17 bulls was extracted according to the manufacturer’s protocol of the Genomic Mini AX Swab & Semen Spin kit . By direct pedigree analysis, this bull sample was chosen to represent several lineages and, when available, several bulls from the same lineage were included to allow testing for allele segregation. Generally, as indicated by analysis of the kinship-coefficient average and standard deviation, the level of pedigree relationships among these bulls was 0.027 ± 0.04 . Whole-ghttp://broadinstitute.github.io/picard/, accessed on 13 February 2022), and curated BAM files were coordinated, sorted, and indexed by Picard tools. Variant calling and filtration stages were performed with GATK 3.6 [Following Genome Analysis Toolkit (GATK) best practice workflows of the Broad Institute , we applGATK 3.6 .Based on direct read aligning to 50 bp sequences spanning the SNP top genomic sequences in the BovineHD manifest file, we also devised a simpler method to directly call the SNP alleles using the GAP5 sequence assembly visualizer. In this procedure, referred to as the GAP5 method, BWA-MEM was used for read mapping to genomic templates by preparing a temp_cons file  larger fold coverage (~35%). However, as we used this method to genotype older sires, the mean rate of matches to genotypes from the older BeadChip was reduced. To exclude the possibility of sequencing method bias, sire 7733 was sequenced in both platforms, which yielded a similar number of matches indicating no critical bias. Thus, comparing to HiSeq, the number of NovaSeq mismatches for both calling methods was also reduced  sequence reads than NovaSeq, which yielded a mean coverage depth of ~40-fold . Followi reduced , indicat reduced . HoweverTo determine why certain SNP yielded discordant BeadChip and WGS genotypes, we used assembly visualizers to examine their sequences at the read level. As shown in To further examine the effect of a third allele on BeadChip genotyping, we examined cases that did not fit the Mendelian paradigm, as the father allele of a homozygotic genotype was not detected in the son. As shown in p = 2.9 × 10−4) yielded 8% more matching genotypes and significantly (p = 2.1 × 10−7) 8-fold less mismatches with the BeadChip genotypes (The availability of WGS allows the comparison of genotyping accuracy between WGS and BeadChip techniques. In this paper, we present the deep sequencing of 17 genomes of Israeli Holstein sires belonging to the bull panel used for the artificial insemination of dairy cows. In both the HiSeq and NovaSeq platforms, the 151 bp paired-end reads provided detailed information that allowed accurate alignment to the reference genome. Based on this alignment and following the best practices provided by the GATK software, SNPs and indels were simultaneously called via the local de novo assembly of haplotypes in the variable regions and the genotypes were recorded in VCF files . Providienotypes . Thus, aNull alleles can generate significant genotyping inaccuracies that negatively affect the statistical power of genetic studies ,23. In oGenotyping inaccuracies may also account for some of the missing heritability encountered in SNP-array based studies ,26. To cWe developed a method to map short sequence reads to 50 bp genomic contigs. The alignment of short-sequence reads to shorter contig templates somewhat mimics hybridization. At SNP sites, the nearby extra variation hinders hybridization-based allele calling and adjacent extra variation is observable in over 40% of SNPs in BovineSNP50 BeadChip. This variation produces interfering sequence variants that induce null allele-like effects and Mendelian errors, which are a primary reason for failed calling that is not related to technical problems, such as DNA quality and handling.


Dirofilaria immitis) control in both the USA and Australia. ML-resistant D. immitis isolates have been confirmed to exist in the USA and studies have shown that 42 single nucleotide polymorphisms (SNPs) are associated with phenotypic ML-resistance. Currently, ML-resistance has not been reported in any Australian clinical cases of canine heartworm. The aim of the study is to determine whether the 42 SNPs associated with resistance to MLs in the isolates from the USA are present in adult heartworms from a clinical case in Australia. Five adult D. immitis obtained from a dog at post-mortem  were sequenced using the Illumina sequencing technology. The genomic analyses revealed 6 out of the 42 SNPs associated with ML-resistance to be present in our samples, 3 out of the 6 SNPs identified were nonsynonymous SNPs but not in candidate genes for ML-resistance. ML-susceptibility profile was mixed using the 42-SNP and 10-SNP models, but the 5-SNP, 3-SNP and 2-SNP models demonstrated ML susceptibility for all five individuals. In this study, the first whole-genome reference of D. immitis from Australia establishes a new baseline for comparative studies and will be valuable for tracking ML-resistance emergence.For the past 30 years, chemoprophylaxis with macrocyclic lactone (ML) anthelmintics has been the primary strategy for canine heartworm (  •Dirofilaria immitis from Sydney, Australia, sequenced using the Illumina NGS technology.•New baseline for comparative studies for tracking ML-resistance emergence in Australia.•Mixed ML-susceptibility profile using the 42-SNP and 10-SNP models.•5-SNP, 3-SNP and 2-SNP models demonstrated ML susceptibility. Canine heartworm disease affects dogs in Australia each year, particularly in Queensland  associated with failure to reduce microfilariae following ML prophylaxis have been identified  obtained at necropsy from a deceased dog donated to the Sydney School of Veterinary Science, The University of Sydney, in September 2020, as part of the Sydney School of Veterinary Sciences Animal Donation Programme. The adult dog came from Sydney, New South Wales, Australia, but the history of travel and rigorous treatment with ML products is unknown. Individually extracted nematodes from the heart were washed 3 times in phosphate-buffered saline (pH = 7.4) and 1 cm from between the first and second third of the anterior portion of the body was excised using a sterile scalpel blade for DNA isolation. The genomic DNA from samples JS5873 to JS5876 (n = 5) was extracted with a Monarch® Genomic DNA Purification Kit  as per the manufacturer's protocol.Samples tested include five adult 2.2D. immitis DNA samples (A-C: 4.72–90.15 ng/μl and D-E: 0.27–1.06 ng/μl) were sent to Novogene (HK) Co., Ltd for indexing, library construction and whole-genome sequencing; JS5873 to JS5875 (A-D) were sequences at an expected depth of 10G while JS5876 and JS5877 were sequenced at a depth of 1G. Sequencing was performed with the HiSeq platform from Illumina® with PE150 paired-end reads.Extracted 2.3https://doi.org/10.25833/8egx-j055. From the 42 SNPs, a subset of SNP loci  which appears to be promising markers for predicting ML resistance are used to predict the phenotypic response of JS5873 to JS5877 to ML treatment  (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw sequencing reads were trimmed to have a Phred quality score (Q score) of 30 and filtered to have a minimum read length of 150 bp using Trimmomatic (D. immitis reference genome (www.nematodes.org/genomes/dirofilaria_immitis/) (http://samtools.sourceforge.net/). Pileup files were created from the sorted BAM datasets using the “Generate pileup” tool and the consensus base and coverage at each of the 42 SNPs were extracted. FreeBayes, a haplotype-based variant detector was used to determine the total number of variants between the genomes of JS5873 to JS5877 and the nDi.2.2. D. immitis reference genome (All bioinformatic tools used to identify DNA polymorphic sites at the 42 SNP loci in JS5873 to JS5877 adult .org.au) . Qualitymmomatic . The filmmitis/)  using thmmitis/) . Bam file genome .2.4Wolbachia endosymbiont of D. immitis, the filtered paired-end read sets were mapped to the wDi.2.2 reference genome using the pipeline described above (www.nematodes.org/genomes/dirofilaria_immitis/)  . FreeBayhttps://github.com/chrishah/MITObim) (D. immitis complete mtDNA (NC_005305) as bait (c oxidase subunit 1 gene (cox1) was compared to cox1 haplotypes from www.nematodes.org/genomes/dirofilaria_immitis/)  for each nematode was assembled from fastq data using the MITObim tool (MITObim)  as previMITObim) , with th as bait . The cytmmitis/) .2.5https://parasite.wormbase.org/Dirofilaria_immitis_prjeb1797/Info/Index/) were used to annotate the nDi.2.2. D. immitis reference genome and visualised in IGV (Integrative Genomics Viewer) to assess whether the SNP was synonymous or nonsynonymous. The type of SNP, synonymous or nonsynonymous was determined by assessing whether the SNP altered the amino acid sequence of the annotated protein.Gene annotations in the GFF3 (Generic Feature Format 3) file available at   . Average 207,665 .Table 1SD. immitis samples were found in JS5873 to JS5877 samples  was included in 50,985 to 3,725,184 sequence reads mapped to the reference wDi genome  with the reference D. immitis mtDNA (NC_005305) from Australia  and MX03 (Mexico).To confirm the identity of ustralia , mDi_Ath4D. immitis in Sydney, Australia, based on the genotyping data available from the USA. For nearly three decades, ML anthelmintics have been used as the primary strategy for canine heartworm prevention in the USA and Australia but ML-resistant isolates have only been recovered in the USA , which could affect the viability of the SNP markers previously identified to be promising in detecting ML-resistant cases  endosymbionts that could become resistant to the antibiotic doxycycline - an established component for treatment of heartworm-infected dogs (Wolbachia-endosymbiont has been shown to include 67 loci specifically found in resistant D. immitis isolates but not in susceptible D. immitis USA isolates (D. immitis (D. immitis sampling and genome sequencing are needed to understand why there is almost twice the number of SNPs in Australian wDi.The filarial nematode ted dogs . The whoisolates . Around  immitis . Further5D. immitis originating from Australia. While finding evidence of ML-resistance in D. immitis is still pending, whole-genome sequencing and bioinformatics provide an affordable and scalable approach for future studies. Efforts to design reliable and sensitive in vitro tests for resistance remains unsuccessful (D. immitis.This is the first study to analyse whole-genome reference sequences of adult ccessful . Therefo10.13039/100007456Canine Research Foundation and Dogs Victoria, Australia.This research was supported by the Daisy Ching-Wai Lau: Methodology, Validation, Formal analysis, Data curation, Writing - Original Draft, Writing - Review & Editing. Sara Collaery: Methodology, Formal analysis, Writing - Original Draft. Michelle Liang: Methodology, Formal analysis, Writing - Original Draft. Stephanie Mcleod: Methodology, Formal analysis, Writing - Original Draft. Selina Peou: Methodology, Formal analysis, Writing - Original Draft. Andy Tran: Methodology, Formal analysis, Writing - Original Draft. Jan Šlapeta: Conceptualization, Validation, Investigation, Resources, Data Curation, Writing - Review & Editing, Supervision, Funding acquisition. All authors read and approved the final manuscript.https://doi.org/10.25833/8egx-j055.Raw fastq sequence data was deposited at SRA NCBI BioProject PRJNA681066. SNP data tables and additional data are available at LabArchives: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Bos taurus chromosome 1—BTA1), paraoxonase (BTA4) and γ-glutamyl transferase (BTA17) in the individual breed analysis that coincided with the position of the genes coding for these proteins, suggesting that their expression is under cis-regulation. A meta-analysis of both breeds identified additional significant associations with paraoxonase on BTA 16 and 26. Finding genetic associations with variations in the levels of these biomarkers suggests that the selection for high or low levels of expression could be achieved rapidly. Whether the level of expression of the biomarkers correlates with the response to stressful situations has yet to be determined.Stress in livestock reduces productivity and is a welfare concern. At a physiological level, stress is associated with the activation of inflammatory responses and increased levels of harmful reactive oxygen species. Biomarkers that are indicative of stress could facilitate the identification of more stress-resilient animals. We examined twenty-one metabolic, immune response, and liver function biomarkers that have been associated with stress in 416 Italian Simmental and 436 Italian Holstein cows which were genotyped for 150K SNPs. Single-SNP and haplotype-based genome-wide association studies were carried out to assess whether the variation in the levels in these biomarkers is under genetic control and to identify the genomic loci involved. Significant associations were found for the plasma levels of ceruloplasmin ( The intensification of animal production systems has imposed a wide range of stressors on animals, including heat and metabolic stress, which are likely to increase as a result of climate change . RoutineResponses to stress are highly complex and cannot easily be measured; therefore, identifying biological processes that act as proxy traits and finding quantifiable biomarkers for stress will accelerate the scientific understanding of stress responses and facilitate the selection for more resilient animals ,18,19. SIn this study, we tested biomarkers related to metabolism, inflammation and liver function in the blood, milk and hair samples of lactating cows as genetic proxies for the stress response in two cattle breeds , which are characterised by different production performance, immune-metabolic status, selective breeding histories and genetic backgrounds ,33,34. SAll experimental procedures and the care of animals complied with the Italian legislation on animal ethics  and adhered to the bioethical rules of the University of Udine. Ethical approval for this study was given by the qualified veterinarian responsible for animal welfare of the Department of Agricultural and Environmental Science of the University of Udine. Blood, milk and hair samples were collected from 500 Italian Simmental (IS) and 451 Italian Holstein (IH) cows reared on 10 commercial farms in Udine province . The animals sampled had no clinical symptoms. Sampling was organised on the day of the official milk recording. A 50 mL sample of milk was collected from each cow during the morning milking into a tube containing bronopol  preservative, and on the same day, hair samples were collected and stored in a paper envelope, both for the measurement of biomarkers, as described below. At the same time, blood samples were collected from the coccygeal vein during the morning milking before feeding, into two 10 mL vacutainers, one containing EDTA, for DNA extraction, and the second containing Li-heparin, for haematochemical profiling, as anticoagulants. Li-heparin samples were kept at 6–8 °C for no more than 2 hours until centrifugation at 3000 RPM for 15 min; then, plasma was collected and frozen (−80 °C) until its analysis. The local Farmer and Breeder Association  providedPlasma was assessed for biomarkers associated with metabolism , nonesterified-fatty-acids (NEFA), creatinine and urea), innate immune response  and liver function (aspartate transaminase (AST/GOT), γ-glutamyl-transferase (GGT), total bilirubin, albumin, globulin, total protein and paraoxonase). Blood assays were performed as previously described  using thMilk samples were analysed for protein, fat, lactose, urea and somatic cell count (SCC) using a FT-NIR FOSS 6000 . BHB was analysed in milk using the same test as for plasma. Cortisol, a biomarker of chronic stress, was assayed in milk and in hair, both following the procedure described by Sgorlon et al. .The distribution of phenotypic data was tested for normality using a Shapiro–Wilk test. Non-normal phenotypes were adjusted by either the truncation of outliers  or square-root transformation. The effect of factors potentially affecting phenotypic data was assessed by the analysis of variance (ANOVA) implemented in R , and sigGenomic DNA was isolated from whole blood using the GenElute Mammalian Genomic DNA Miniprep Kit . A total of 951 cows were genotyped: 152 IS cows were genotyped with the BovineHD Genotyping Beadchip , containing 777,000 SNPs, and 348 IS and 451 IH cows were genotyped with the GeneSeek GGP Bovine 150K array , containing 138,892 SNPs. SNP positions were updated to the most recent bovine reference sequence, ARS-UCD1.2 . SNP mar2) calculated in BEAGLE. Imputed SNP markers showing an r2 lower than 0.75 were excluded from further analyses. After imputation, genotypes were quality-checked. Individuals with extremely low or high observed heterozygosities  or admixed  ,,58,30,58The final SNP dataset comprised 343 IH individuals genotyped at 121,432 SNPs, and 350 IS cows genotyped at 119,858 SNP. The dataset used for phasing, to identify haplotypes for the GWAS, comprised 116,904 SNP for IH that identified 872,520 haplotype alleles (HapAlleles) in 120,913 haplotype blocks (HaploBlocks), and 113,592 SNPs for IS that identified 961,249 HapAlleles in 117,452 HaploBlocks. Both single-SNP and haplotype GWAS analyses identified genome-wide significant associations for only three phenotypes: GGT, CP and PON . The herCP gene  on BTA1. In the same region, the haplotype analysis detected haplotypes reaching the genome-wide suggestive threshold  associated with CP in IH at 118,970 kb , within hreshold . For IS,gene see .PON1 . Haplotypes that were close to significance were identified in the same region but did not reach the genome-wide threshold  and GGT1 . For IS, three SNPs had genome-wide significance for GGT levels, in the same BTA17 region as that detected in IH between 69,823 and 71,484 kb . The two most significant SNPs associated with PON on BTA4  closely flank the PON1 gene . On BTA17, the peak spanning 172 kb from ARS-BFGL-NGS-117653  to ARS-BFGL-NGS-74168  includes both GGT5  and GGT1 . The most significant SNP close to these two genes was ARS-BFGL-NGS-26126 .A meta-analysis  combininCP, PON1, GGT1 and GGT5, the significance of the suggestive signal on BTAs 16 and 26 associated with the level of PON increased. The SNP on BTA16 had an increased p value but did not reach genome-wide significance, while the SNP on BTA26 did reach genome-wide significance threshold in the meta-analysis. The SNP with the highest significance on BTA16 was BovineHD1600012174 at 42,784,481. There are five genes within a 500 kb interval flanking this SNP; the closest is castor zinc finger 1 (CASZ1) followed by peroxisomal biogenesis factor 14 (PEX14), and the other genes are TAR DNA binding protein (TARDP), spermidine synthase (SRM), mannan binding lectin serine peptidase 2 (MASP2) and exosome component 10 (EXOSC10). The signal on BTA26 became the most significant association for PON, higher than the SNP within the PON1 gene. The most significant SNP on BTA26 in the meta-analysis is BovineHD2600003408, at 13,116,737, in the 5′ of the domain containing E3 ubiquitin protein ligase 2 (HECTD2) gene. A second SNP near to the significant SNP, BovineHD2600003402, at 13,083,916 bp also maps within HECTD2. Two other genes map within 500 kb flanking this SNP: Polycomb group ring finger 5 (PCGF5) and protein phosphatase 1 regulatory subunit 3C (PPP1R3C). A single isolated significant SNP (BTA-113142-no-rs) on BTA17 at 42,821,258 that was above the significance threshold falls in an intergenic gene-poor region and no gene maps in the 500 kb flanking interval.In addition to the significant SNP close to 2 > 0.3 were identified in IH and 633 in IS  that changes an asparagine into a histidine was classified as deleterious by the SIFT algorithm (SIFT score = 0.03). The other variants were classified as tolerated by SIFT. A single missense variation was identified within the GGT1 gene boundaries in IH, which was classified as tolerated by SIFT. No variant of high or moderate effect was identified by VEP in IS within the CP gene.Of the variants within the genes, those with a moderate-to-high effect on the protein were identified using VEP. Five missense and two frameshift mutations were found in the CP, GGT1, GGT5 and PON1 promoter regions identified 193 variants  is within a binding site recognised by both Serum Response Factor (SRF) and ETS Transcription Factor (ELK1). The second is at position 118,900,683 bp (−698 bp from the start site) within a Forkhead Box J2 (FOXJ2) binding site. The LD between these variants and the most significant SNP was above the threshold set for linkage disequilibrium (r2 > 0.3). In Holstein, variants in the same upstream region had a low LD (r2 < 0.3) with the most significant CP SNP detected in Italian Holstein.On BTA1, two variants in linkage with the most significant SNP for CP in Italian Simmental were mapped within transcription binding sites upstream of the GGT1 at position 71,471,816 bp (−161 bp from GGT1 start site), within an Activating Transcription Factor 1 (ATF1) binding site; at position 71,472,255 (−600 bp from the start site) in a Vitamin D Receptor Transcription Factor (VDR) binding site; at 71,472,377 bp (−722 bp from the start site), a site recognised by two transcription factors, MYC-Associated Zinc Finger Protein (MAZ) and Sp1 Transcription Factor (SP1); at position 71,473,234 bp (−1579 bp from the start site) and in a binding site for both Chicken Ovalbumin Upstream Promoter Transcription Factor 1 (COUP-TF1) and Estrogen Receptor 1 (ER-α) factors. A single variant in LD with GGT5 was detected upstream of the gene at position 71,455,325 (−1956 bp from the transcription starting site), within a SMAD Family Member 4 (SMAD4) transcription factor binding site. These variants were in strong LD with the most significant SNP (r2 > 0.4) with the highest values observed for variants mapping in MAZ and SP1 (r2 = 0.641) and in ATF1 binding sites (r2 = 0.641). In IS, variants in the same upstream region had a weak LD (r2 < 0.3) with the most significant GGT SNP detected.On BTA17, five variants were in LD with the most significant SNP for GGT in IH. Four were within transcription binding sites upstream of 2 > 0.3) with the most significant SNP for PON.On BTA4, no variants in transcription binding sites were identified above the LD threshold  paraoxonase (BTA4) and γ-glutamyl transferase (BTA17). The levels of these three biomarkers had an estimated heritability between 0.06 (s.e. 0.14), for CP in IH, and 0.69 (s.e. 0.13), for PON in IS, although the small number of animals analysed does not allow an accurate estimation of heritability. In each case, a well-defined chromosomal region reached genome-wide significance, which included the gene itself , in both breeds analysed, the Italian Holstein and Italian Simmental. This suggests that the control of expression of these biomarkers is regulated by a variation near or close to the gene, and there are no major direct effects of other genetic loci on their expression. The most significant SNP for these biomarkers explains 7.33% and 8.87% of the phenotypic variance for CP in IH and IS, respectively; 7.98% and 6.69% for PON in IH and IS, respectively; and 14.64% and 8.08% for GGT in IH and IS, respectively. Genetic selection could rapidly change allele frequencies of these genes and, hence, the expression of the biomarker, which would be valuable if they can be shown to be associated with improved stress response.The three biomarkers for which significant genetic associations were found are all primarily produced by the liver with functions related to protection from oxidative stress. Oxidative stress is associated with various diseases, including diabetes, atherosclerosis and cancer , and in CP gene is located on BTA1 and is approximately 60 kb long. Ceruloplasmin is an a-2 glycoprotein involved in copper transport and iron metabolism, and it is an antioxidant produced by the liver [CP knockout mice, the impact of CP deficiency results in the accumulation of iron in the hippocampus that impacts brain neurochemistry and behaviour. The CP knockout mice showed elevated anxiety levels compared with normal mice [The he liver ,68. Levehe liver . Genetiche liver . In CP kmal mice . This sumal mice ,72,73.PON1 knock-out mice on a high-fat, high-cholesterol diet develop atherosclerosis more rapidly, and the LDL in their artery walls is more highly oxidised than in controls [PON1-overexpressing transgenic mice, there are fewer lesions and the oxidation status of aorta is improved. Oxidative stress and the consequent lipid oxidation are thought to play a role in psychiatric disorders. In humans, decreased PON activity and specific polymorphisms in the gene have been associated with clinical conditions, including depression, generalised anxiety disorder (GAD) and schizophrenia [PON1 gene promoter region have been found to be associated with plasma PON levels in Brazilian Holstein [The gene encoding the acute-phase protein PON is located on BTA4 and is approximately 33 kb long. PON is a high-density lipoprotein-associated enzyme that protects lipoproteins against oxidation. controls . In contophrenia . The conophrenia  and decrophrenia , e.g. asophrenia ,73. Six Holstein .GGT1 and GGT5 genes are located on BTA17 and are approximately 22 kb long. The GGT enzyme is a biomarker of liver function. GGT is a membrane-bound glycol-protein, the production of which increases during the acute-phase response to stress-induced inflammation and oxidative stress [GGT1 knock-out mice showed chronic GSH deficiency, growth retardation, skeletal abnormalities and cataracts, and they died younger [GGT1 is in a large linkage block containing numerous genes, including GGT1, GGT5, GSTT1 and GSTT. GGT1 knock-out mice have no glutathione activity in the spleen, small intestine and kidney, suggesting that GGT5 does not compensate for the lack of GGT1.The e stress . Under ce stress . GGT1 kn younger . GGT1 isCP, PON1, GGT1 and GGT5. However, two additional signals were identified on BTAs 16 and 26 associated with the expression of PON. The signal on BTA16 was above the genome-wide suggestive, the one on BTA26 above the significant threshold in the meta-analysis, in which data from both breeds were combined in the GWAS. Indeed, the significant SNP on BTA26 had a higher p value than the SNP associated with PON on BTA4. The significant BTA26 SNPs are within the domain containing the E3 ubiquitin protein ligase 2 (HECTD2) gene, which is involved in the function of the innate immune system and is a crucial regulator of cytokine secretion [CASZ1). CASZ1 is involved in blood pressure regulation [peroxisomal biogenesis factor 14 (PEX14), which also plays a role in countering oxidative stress [PEX14 seems to have been under selection during domestication [The only SNPs above the genome-wide threshold for significance in the individual-breed GWAS were those associated with ecretion . The clogulation , neuronagulation  and inflgulation , all fune stress . Interestication , where dCP, 373 in IH and 86 in IS, of which two were in common. None of the variants were above the LD threshold for PON1, in either breed. In IH, 76 variants were in GGT1 and 33 in GGT5, most of which were within the coding region or introns  overlapped Serum Response Factor (SFR) and Elk-1 binding sites. ELK-1 and SRF form a tertiary complex [The  complex  and have complex . The secm BTA1:1100034 ovePON1 coding region, among which six have been associated with increased PON expression, e.g. in relation to calving stress [2 < 0.3) with BovineHD0400003479, the second most significant SNP associated with PON in the present study. The first SNP is in a GATA3 binding site. GATA3 is a transcription factor involved in the development and survival of sympathetic neurons [PON1 transcription binding sites in cattle [There have been 54 variants described upstream of the g stress , of whic neurons . The sec neurons . We also neurons . Howevern cattle ; howeverGGT1 gene has 69 variants upstream of the coding region of which eight were in LD with the significant SNP for serum GGT levels. We identified transcription factor binding sites at four of these at 71,471,816, 71,472,255, 71,472,377 and 71,473,234 bp on BTA17. The SNP at BT17:71471816 is within an activating transcription factor (AFT) binding site, the SNP at BT17:71472255 is within a Vitamin D Receptor (VDR), and the SNP at BT17:71472377 is within a Myc-associated zinc-finger protein (MAZ) binding site and a Specific protein 1 (Sp1) binding site. MAZ expression is increased in chronic inflammation [PON1.The ammation . Sp1 is ammation . The SNPGGT5 gene has 45 variants upstream of the coding region, of which three are in LD with the most significant SNP in the single-SNP GWAS. Only one (BTA17:71455325) is within a transcription binding site, which is SMAD4. Smad-dependent signalling regulates the expression of the TGF-β family of cytokines, which in turn, negatively regulates neuronal morphogenesis during brain development and plays a role in neuronal disease in humans [The n humans ,96.We assessed variations in potential biomarkers of stress and the genetic control of their expression. Three biomarkers, all associated with oxidative stress, showed strong genetic associations that indicated that their expression was primarily under cis-acting control. In the case of PON, two additional genetic loci were identified that were associated with the level of PON expression. Candidate genes at these loci are also implicated in oxidative stress. In this study, the genetic association between levels of biomarkers was examined in non-stressed animals. It would be interesting to assess changes in the levels of these biomarkers in stressed animals, and in particular, if genetic variations predict the response of animals to stress, which would facilitate the selection of animals that respond better to stressful situations and hence remain healthier and more productive.


PDGFRA) and chromosome 19 (CDC6) have been found associated to trypanotolerance in West African taurines. Screening for FST outliers in trypanosome positive/negative animals we detected seven variants putatively under selection. Finally, we identified a minimum set of highly ancestry informative markers for routine admixture testing. The results of this study contribute to a better understanding of the genetic basis of trypanotolerance in Baoulé cattle and their crossbreeds. Furthermore, we provide a small informative marker set to monitor admixture in this valuable indigenous breed. As such, our results are important for conserving the genetic uniqueness and trypanotolerance of Baoulé cattle, as well as for the improvement of Baoulé and Zebu crossbreds in specific community-based breeding programs.The genomes of crossbred (admixed) individuals are a mosaic of ancestral haplotypes formed by recombination in each generation. The proportion of these ancestral haplotypes in certain genomic regions can be responsible for either susceptibility or tolerance against pathogens, and for performances in production traits. Using a medium-density genomic marker panel from the Illumina Bovine SNP50 BeadChip, we estimated individual admixture proportions for Baoulé x Zebu crossbred cattle in Burkina Faso, which were tested for trypanosome infection by direct ELISA from blood samples. Furthermore, we calculated local ancestry deviation from average for each SNP across 29 autosomes to identify potential regions under selection in the trypanotolerant Baoulé cattle and their crossbreds. We identified significant deviation from the local average ancestry (above 5 and 10% genome-wide thresholds) on chromosomes 8 and 19 in the positive animals, while the negative ones showed higher deviation on chromosomes 6, 19, 21, and 22. Some candidate genes on chromosome 6 ( Bos taurus taurus and Bos taurus indicus subspecies are the two most important cattle types in West Africa  genotyping, inferring selection signatures from differences in local admixture levels has received considerable attention in human genetics . SimilarThe genomic ancestry proportions between trypano-susceptible indicine Zebu and the trypano-tolerant taurine Baoulé cattle in Burkina Faso were assessed based on microsatellites and 155 SNPs in 23 candidate regions . In thisThis study was carried out in the province of Poni in the South-western administrative region of Burkina Faso. We selected three study sites with different management and breeding systems of Baoulé cattle and Baoulé x shorthorn Zebu crossbreds, including 27 villages from the Bouroum-Bouroum, Kampti and Loropéni departments . In the A total of 737 blood samples, including 387 Baoulé and Baoulé x Zebu crossbreds from Bouroum-Bouroum, and 350 crossbreeds from Kampti and Loropéni were collected in EDTA tubes during the health monitoring activities of the “Characterization and Sustainable Utilization of Local Cattle Breeds” (LoCaBreed) project. DNA extraction from EDTA blood samples was performed with the MasterPureTM DNA Purification Kit for Blood Version II  following the manufacturer’s protocol. The trypanosomosis status was recorded by indirect ELISA test to diagnose positive or negative trypanosome infection in the blood samples  resultinP-value 1 × 10 − 6. After quality control, 28,034 SNPs and 776 animals were available for subsequent analyses.The genotyping of the 737 DNA samples with the Illumina Bovine SNP50 BeadChip was performed at Neogen . Additional genotypes from 30 purebred Zebu and 35 crossbreds  were incK = 2). The admixture bar plots for ancestry proportions were created in R with the barplot function  is a program for estimating locus-specific ancestries in admixed individuals, using allele frequencies of the reference populations . We applFST outlier loci putatively under selection between the trypanosome positive (n = 244) and negative (n = 266) crossbred animals using a cut-off at p < 0.05 corrected for a false discovery rate . Significance of the Pearson’s r was assessed with the P-value from the Pearson Correlation Coefficient Calculator  in chromosomes 8 and 19 for trypanosome positive crossbreds , and in FST values and disregarded the pure-bred Baulé and Zebu. Among these crossbred animals we grouped them in trypanosome positive and negative animals to avoid detection of breed differences unrelated to trypanosome tolerance. We detected seven variants with a FDR corrected threshold of p < 0.05  and 0.990  (p < 0.001).To reliably detect hybrids even with a small set of SNPs applicable for routine genetic monitoring, we selected the most ancestry informative markers resulting in the highest differentiation between Baoulé and Zebu cattle. The 100 SNPs with the highest divergence presented and 0.79 . We estiand 0.79 . The Pea/top100) . All PeaThe high amounts of global admixture observed in the taurine cattle population in the three studied departments of Burkina Faso indicated mixed genetic backgrounds of the cattle in Bouroum-Bouroum, Kampti and Loropeni , 3. The The uncontrolled mating in extensive production systems, which are typically practiced in West Africa, can lead to the introgression of Zebu genes in the small taurine cattle population, which represents a threat to their genetic integrity , and migB. t. indicus), Eurasian taurine, and African taurine (both B. t. taurus) ancestry proportions.In a recently admixed population, ancestral populations have been mixing for a relatively small number of generations, resulting in a new population with different proportions of their genome derived from the original parental populations . Local aWe applied an approach of significance testing and performed a permutation test of circularizing the genome by concatenating the SNPs of all autosomes in a single string, cutting this string once and rearranging the two resulting segments, as proposed by VAV1, PIK3R5, RAC1, VAV2, GAB2, and INPP5D genes in chromosome 8 to be genes under selection in Muturu and N’Dama cattle breeds in response to trypanosomes infection. Surprisingly, we also found higher Baoulé ancestry in chromosome 8 (35–50 Mb) also in trypanosome positive cattle, which could indicate that these regions harbor beneficial Baoulé haplotypes, which are not connected to trypanosomosis tolerance. These regions might harbor genes of general importance for adaptation to the environment. Some canadidate genes on chromosome 6 at 71373513-71421283 (PDGFRA) and chromosome 19 at 41185975-41196948 (CDC6) for trypanotolerance in West African taurines have been found on these chromosomes  genotyping assay. Such an easy and fast genotyping array can be implemented at any laboratory equipped with Real-Time PCR machine and can be used for routine monitoring of hybridization in Baoulé cattle. FST values showing the highest levels of allele frequency differentiation between the two parental breeds similar to our methodology. These AIMs provided close estimation with pedigree information. Correlation coefficient between breed level based on admixture estimates from 25 SNP data obtained in this study .Quality controlled Bovine 50k SNP chip data, including 31,612 SNPs of the 802 animals included in this study, were uploaded to DRYAD. The dataset has been assigned a unique identifier , Burkina Faso.JS conceived the original idea of the study and together with GM, PB, BY, and NK further developed the idea and decided on the set of analysis. BY and PB did the statistical analysis and wrote the text. BY, DO, BZ, AS, SO-K, SM, HT, and ATr collected the data for the analysis and together with JS, GM, PB, NK, ATa, PO-W, MW, ATr, and OM critically reviewed the text. All authors approved the final version of the manuscript.NK was employed by company SUISAG, Switzerland. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.