| Sanjiang cattle,with characteristics as long torso,good labor,strong adaptability,good meat quality,originate in Aba Wenchuan County,Sichuan Province,which is an excellent local cattle breeds for long-term artificial breeding,and it is an extremely valuable genetic resources.In this study,the genetic diversity of Sanjiang cattle is discussed from the aspects of the whole genome,RAPD molecular marker,Y chromosome-specific ZFY gene,which provided the theoretical basis for the preservation and further breeding of the endangered Sanjiang cattle breeds.The results are obtained as follows.1.Whole-genome sequencing analysis.A total of 77.8 Gb of sequence data were generated,99.31% of the reference genome sequence was covered with an mapping depth of 25.32-fold,778 40 reads and 77 840 344 400 bases were obtained,of which 673 670 505 reads and 67 341 451 555 bases covered 86.55% and 86.51% of bovine reference genomes(UMD 3.1)respectively,paired-end reads mapping were 635 242 898(81.61%),paired-end bases mapping were 63 512 636 924(81.59%).A total of 20 477 130 SNPs and 1 355 308 small indels were identified,of which 2 147 988 SNPs(2.4%)and 90 180(6.7%)indels were found to be new.Of the total number of SNPs,989 686(4.83%)homozygous SNPs and 19 487 444(95.17%)heterozygous SNPs were discovered,homozygous/heterozygous SNPs was 1?19.7.Transitions were 14 800 438,transversions were 6 680 058,transition/transversion(TS/TV)was 2.2.SNPs of splice site mutations were 727,the number of SNPs which the start codon converts into no stop codon were 117,SNPs of premature stop codon were 530,the number of SNPs which stop codon converts into no stop codon were 88.A total of 57 621 non-synonymous SNPs and 83 797 synonymous SNPs were detected,the ratio was 0.69.Non-synonymous SNPs were detected in 9,017 genes,567 genes were assigned as trait-associated genes,which included meat quality,disease resistance,milk production,growth rate,fecundity with the number of 471,77,21,10,and 8 respectively,the function of some genes were overlap.In detection of indels,693 180(51.15%)were deletions and 662 148(48.85%)were insertions,161 198(11.89%)were homozygous and 1 194 110(88.11%)were heterozygous.Most variations were located in intergenic regions and introns.Heterozygosity(H),nucleotide diversity(Pi)and theta W of Sanjiang cattle genome-wide were 7.6 × 10-3,0.003 9,0.004 0,respectively,which indicated that Sanjiang cattle have an abundant genetic diversity.The Tajima'D of Sanjiang cattle population was-0.06 832,which speculated that the population exists an unbalanced selection.2.Genetic diversity of RAPD.The 9 high specificity primers were screened from 26 primers to amplify the whole genome and the genetic diversity of the population was analyzed.The results showed that 51 bands were detected clearly,in which there were 43 polymorphic bands.The polymorphic frequency was 84.31%.In Sanjiang group,polymorphic sites accounted for 36,the polymorphic frequency was 70.59%.In Shuimo group,polymorphic sites accounted for 43,the polymorphic frequency was 84.31%.Nei's gene diversity index was respectively 0.273 1,0.295 8,which indicated that two group had rich gene diversity and closer diversity index.The fragments of different amplified primers ranged from 1~8,average 6.The Shannon's diversity index(I),gene diversity(H)and effective number of alleles(Ne)of Sanjiang cattle population were 0.464 8,0.313 5,1.544 8 respectively.Genetic diversity(Ht),genetic diversity within populations(Hs),Fst(Gst)and gene flow(Nm)were 0.313 0?0.284 4?0.091 3 and 4.977 2 respectively.The result indicate that Sanjiang cattle have a rich genetic diversity of RAPD,which is consistent with the result of whole genome sequencing,and it was of great significance to Sanjiang cattle resources research and variety protection.3.Genetic diversity of ZFY gene,we analyzed ZFY gene sequence of 60 individuals with ZFY-11-1,ZFY-11-2 primer,its sequence polymorphism was analyzed,and the phylogenetic tree was constructed.The 602 bp part sequence of exon 11 was amplified by ZFY-11-1 primer,17 individuals without AvaII restriction sites analysised by on-line tool for restriction were identified as male individuals,which consistent with the sex record results of the samples.ZFY-11-2 primer was used to amplify the exon 11 part sequence of 17 male individuals,the 734 bp sequence was obtained.The splicing sequence length of two sequences was 1 090 bp;The splicing sequence was analysised,with nucleotide frequencies of 24.2%?34.2%?20.7% and 20.9% for T?A?C and G respectively,nucleotide diversity was 0.007 61.22.Polymorphic sites were identified of 17 individuals,including 18 transitions,4 transversions.16 haplotypes were identified and haplotype diversity was 0.993.It indicated that Sanjiang cattle had an abundant genetic diversity of ZFY gene.The Tajima'D was 1.040 88,which speculated that neutral test result was not significant.The phylogenetic and genetic distance were analysised using amplification product of the primers ZFY-11-2,the results were clustered 2,sanjiang cattle and Bos frontalis,Bos gaurus,Bos indicus clustered first,then gathered with other bovine species.Phylogenetic analysis of cytb gene were clustered for the same results,which indicated that Sanjiang cattle and Bos frontalis,Bos gaurus,Bos indicus were high genetic similarity and closer genetic relationship,sanjiang cattle should be Bos indicus. |
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