- Background: The yak is a species of livestock which is crucial for local communities of the Qinghai-Tibet Plateau and adjacent regions and naturally owns one more thoracic vertebra than cattle. - Recently, a sub-population of yak termed as the Jinchuan yak has been identified with over half its members own a thoracolumbar vertebral formula of T15L5 instead of the natural T14L5 arrangement. - Selective breeding of this trait would have great agricultural value and exploration of the molecular mechanisms underlying this trait would both accelerate this process and provide us insight into the development and regulation of somitogenesis.. - Results: Here we investigated the genetic background of the Jinchuan yak through resequencing fifteen individuals, comprising five T15L5 individuals and ten T14L5 individuals with an average sequencing depth of >. - Two genes, PPP2R2B and TBLR1, were found to harbour the most candidate markers associated with the trait and likely contribute to the unique somitic number and identity according to their reported roles in the mechanism of somitogenesis.. - Conclusions: Our findings provide a clear depiction of the Jinchuan yak ’ s genetic background and a solid foundation for marker-assistant selection. - For example, the average net weight of male Jinchuan yaks with 15 pairs of ribs could be over 12 kg heavier than that of male Jinchuan yaks with 14 pairs of ribs [2], demonstrating the great economic potential of the T15L5 Jinchuan yak. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - 4 Key Laboratory of Sichuan Province for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, China Full list of author information is available at the end of the article Wang et al. - The biological mechanism of VRTN affecting somi- togenesis has been reported to be the acceleration of the segmentation clock through the Notch signaling path- way [17].. - Besides increased vertebral number, the Jinchuan yak also possesses other advantageous characteristics includ- ing high milk production [2] and an earlier age at first calving (85.4% of the Jinchuan yak calved first at three years old, which is one year earlier than average) [18], so one of the aims of this study is to dissect the population genetic characteristics of the Jinchuan yak via comparing with the Qinghai-plateau yak. - We also seek a greater understanding of the mechanisms relevant to the aberrant somitogenesis.. - The average sequencing depths of samples reached above 10X, which ensured the accuracy of the genotypes calling. - The number of variants found in each type was similar in the three populations. - Finally, another trans- membrane protein TMEM192 (discussed in the section Association analysis) was also identified because nine population-level variants associated with the trait resided within 5 kb of the gene.. - However, we should also note that: (1) the proportion of the information con- tained in the PC1 (8.13%) and the PC2 (7.73%) was very close. - (2) the division of individuals according to the PC2 was different from that according to the PC1. - As no principle component separated individuals into two groups corresponding to the YC population and the ZC population well, the region regulating the trait was putatively a small proportion of genomic regions.. - likely reflected in the effective population size (N e ) for the YC population which was the smallest compared with the ZC and GY populations. - Due to the slow LD decay rate of the YC population, it became less difficult to select loci linked to the causal variants determining the trait of increased vertebral number for selective breeding.. - However, the large genetic distance within Jinchuan yak suggested a significant genetic diversity exists in the Jinchuan yaks. - The genetic dis- tance from the green block to the red block was closer than to the yellow block. - And the yellow block was closer to the outgroup cattle than any other blocks were.. - In light of the previously reported phylogeographical study of domestic and wild yaks based on complete mitochondrial sequences [19], it was suggested that the. - The num- bering of the lineages here was consistent with the nomenclature used in the phylogeographical study [19], which concluded that three different lineages evolved al- lopatrically and then reunited into one gene pool before the start of domestication. - Of note, the bar of the plot only represented distances between the nodes except tips. - The distance of the cattle ’ s tip to its corresponding closest node was 0.33 and the average distance of all other yaks ’ tips to their corresponding closest nodes was 0.11. - The lineage to which each animal belongs was labeled on the right of the tree. - Another interesting observation was that the genetic distances between individuals shown in the NJ tree were similar to the distances between individuals reflected by PCA (Fig. - In other words, PC2 grouped individuals from the GY and YC populations into a cluster and individuals from the ZC population into another cluster which was consistent with the result of the NJ tree. - This suggests that the similarity inherited from the same ancestors was second only to the similarity that gradually accumulated through living in the same environment. - others using the same type of data which indicated that our data truly reflected the genetic characteristics of the objects we selected.. - To explore the source of the generation of an extra vertebra and advantageous productive traits of Jinchuan yaks from an evolutionary perspective, selective sweep analysis was conducted between Jinchuan yaks and Qinghai-plateau yaks. - The size of the regions under selective sweep in the Qinghai-plateau yak was less than half that of the Jinchuan yak. - A possible reason for this phenomenon was that the habitat of the Qinghai-plateau yak seldom changed but the habitat of the Jinchuan yak might have undergone a major change.. - were in- volved in the ‘protein digestion and absorption pathway’, 14 genes (ERBB4, ADCY3, ADCY1, P2RX1, PDE1C, CCKBR, HRH1, PLCB1, ADRA1A, RYR2, ATP2A3, GRM1, LOC102275229, LOC102287375) were located in. - In this plot, the darker the shade of the blue points, the greater the number of points present at that location.. - The upper and right histograms indicate the number of points in the corresponding intervals. - Table 3 Enrichment analysis of the genes hit by the top 5% of points. - Among them, 330 loci were selected as candi- date markers for selective breeding because the population- level genotypes of these loci of the YC population were dif- ferent from those of the ZC-GY population. - A total of 61 genes harboring these loci or being within 5 kb of these loci were identified as possible causal genes of the T15L5 trait.. - According to the reports, PPP2R2B [21] and TBL1XR1 [22, 23] were most likely to be related to the T15L5 trait (the relationship between these two genes and the development of somites are depicted in the discussion section).. - The selection of a specific trait is equivalent to the se- lection of the causal alleles/genotypes which manifest this trait. - The genes carrying the most loci were also located on these three chromosomes, which indicated that QTLs determining the T15L5 trait of the Jinchuan yak were likely linked with or located within these genes.. - A comparison of the sequences of the PPP2R2B gene from yak, cattle (Bos taurus) and goat (Capra hircus) un- covered a fact that the PPP2R2B gene sequence of yak was assembled into two contigs separately. - But the length of yak’s PPP2R2B gene is 129,107 bp which is made up of the last eight exons. - The simi- lar lengths and sequences of the last eight exons between yak, cattle, and goat indicated the correctness and reliabil- ity of the annotated part of yak. - No population-level vari- ants were observed in the head part of yak’s PPP2R2B gene. - The reference genome of the latter two species has already reached the chromosome level but the reference genome of yak was still fragmented, which reminded us of the necessity of improving the yak’s reference genome for further understanding the molecular mechanism under- lying the yak’s good characteristics.. - 7), those harbor- ing the most loci were preferred so the loci that resided in the genes (PPP2R2B, TBL1XR1, and TMEM192) and the chromosomes (1, 7 and 17) had a high priority. - According to the criteria mentioned above, 13 loci were selected to be genotyped in 51 extra Jinchuan yaks with known thoracolumbar vertebral formula.. - According to the concordance between the genotypes of 15 rese- quenced samples obtained from the whole genome rese- quencing and the target sequencing, 3 loci were excluded. - The definition of representative capacity was the percentage of individuals with a correctly determined phenotype relative to the total individuals who were observed to be this phenotype.. - In the evaluation of the ability of these 7 loci to predict 20 thoracolumbar vertebrae, 6 out of 51 individuals were expected to be 20 thoracolumbar vertebrae and 2 out of 6 were observed to be 20 thoracolumbar vertebrae.. - 6 The violin plot of the haplotype diversity. - The boxplot of each population is shown according to the total number of haplotypes for each locus. - The two parts were asymmetry and separately represented the distribution of the number of the haplotypes of loci on one side. - 7 Seven factors were considered in the selection of the 10 most reliable markers. - A number from 1 to 7 was assigned to each circle from the outside to the inside. - The third circle is representative of the gene names. - while the histogram shown in the second circle aligned with the third circle which represented the number of loci in this gene. - Each gene corresponded to two bars: the left blue bar indicated the number of loci resided in this gene and the right purple bar indicated the loci located at the outside of the gene but within 5 kb of the gene. - The number of haplotypes for a locus was represented by the length of this locus ’ bar at the 5th, 6th, and 7th circle corresponding to the YC-ZC population, the YC population and the ZC population, respectively. - Table 4 The evaluation of the ability of 7 loci predicting 20 vertebrae. - around 20.00% of the total genetic diversity of individ- uals with T15L4/T15L5 traits.. - The objective of this study was to explore the genetic background of the Jinchuan yak and the possible mecha- nisms of aberrant somitogenesis according to the whole genome resequencing data in order to find markers which could be used to select individuals with the spe- cific thoracolumbar vertebral formula (T15L5) trait. - In the future, more loci could be included to increase the rep- resentative capacity of the genetic diversity at least in theory. - The predominant type of PP2A is a heterotrimer phos- phatase, which consists of a scaffolding subunit A, a regulatory subunit B and a catalytic subunit C [24], and one of the substrates it could bind is APC [25], which is also the component of the beta-catenin degradation complex. - Four gene families exist for the B subunit and the PPP2R2B gene identified in our study is in the PPP2R2 gene family [24]. - Msgn1 is reported to function as a direct target gene of Wnt3a performing the duty of transferring the spatial signal from the Wnt3a/beta-catenin gradient to the Notch Table 5 The evaluation of the ability of 7 loci predicting 15 thoracic vertebrae. - The period of the segmentation clock is regulated by intercellular coupling through the Notch sig- naling program and disruption of this coupling has been re- ported to increase the period of zebrafish somitogenesis and its embryonic somite length [27]. - Altogether, the mutation of PPP2R2B gene was inferred to be the cause of the increased vertebral number of the Jinchuan yak. - The variants of the PPP2R2B gene were pri- marily located in introns, similar to another study that also identified one genetic variant in the intron of the PPP2R2B gene and found it to be associated with altered breast can- cer risk and recurrence [28].. - The establishment and maintainence of the somitic Hox code and segmental identity during em- bryonic development requires the SMRT-dependent re- pression of RAR (retinoic acid receptors) [22]. - Therefore, the mutation of the TBLR1 gene might influence the normal function of SMRT and hence the segmental identity. - As for the relationship between TBLR1 and the somite number, the necessity of mutual recruitment between TBLR1 and β-catenin for the activation of the transcription of the wnt3a target genes [29, 30] (Fig. - It might be explained by the fact that no extreme difference exists in the ecological environ- ments between the Tibetan Plateau and the surrounding. - Investigating the mechanism determining this trait and developing methods selecting such individuals would provide us better understanding of the mechan- ism regulating this trait and much more production yields. - The report of the unique somitogenesis of yak and the discussion of possible mechanisms underlying this trait will inspire further exploration concerning the somito- genesis and plateau hypoxia. - The interpretation of popu- lation genetics characteristics of this unique sub- population of the yak builds a foundation for both se- lective breeding and evolutionary development. - Therefore the genetic characteristics at the popu- lation level of the Valley yak relative to the Plateau yak were also explored in this study. - Pair-end libraries were constructed according to the protocol of the. - (b) The length of ‘N’ bases in either of a pair-end reads exceeded 10% of the total bases of the corresponding read. - 5) in either of a pair-end reads that accounted for over 50% of total bases of the corresponding read. - The clean reads were then mapped to the yak reference genome (BosGru_v2.0 [32]) with the BWA-MEM (v using the par- ameter ‘-a’. - The visualization of the tree was implemented by ggtree [41].. - The enrich- ment analysis of the candidate genes that were covered by the windows with the top 5% Fst and log2 (pi ratio) simultaneously were implemented with DAVID [44]. - The yak reference genome (BosGru_v2.0 [32]) we used in this study was aligned to a chromosomal level assem- bly of the yak reference genome (Accession ID:. - This chromosomal level assembly was obtained by scaffolding the reference genome we used here in virtue of the reference genome of closely related species cattle.) with LASTZ [45] to anchor scaffolds to their possible corresponding chromosomes. - The geno- types of loci residing within 5 kb of the 330 loci were ex- tracted into 330 groups and phased with Beagle [46] to help us evaluate the haplotype diversity of the region where each locus resided. - The reads generated here were mapped to the reference genome to call variants and generate the genotypes of these loci.. - The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - All methods were performed in accordance with the guidelines and regulations approved by The Animal Care and Use Committee of the Southwest Minzu University. - Verbal consent was obtained from the breeding farms at which the slaughtered animals were sampled, which was sufficient to inform owners of animals and approved by the Animal Care and Use Committee of the Southwest Minzu University. - All procedures and experiments were approved by the Animal Care and Use Committee of the Southwest Minzu University.. - Hox genes specify vertebral types in the presomitic mesoderm. - Early development of the vertebral column. - Whole-genome resequencing of Ujumqin sheep to investigate the determinants of the multi-vertebral trait.. - Variations in the skeletal structure of the pig. - Intercellular coupling regulates the period of the segmentation clock
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