Study On MtDNA Genetic Diversity And Phylogeny Evolution Of 9 Indigenous Sheep Breeds In The Middle-Western Of China

Ear samples from 470 individuals of 9 different sheep breeds were collected from Henan province (Henan big tail sheep, n=52), Shanxxi province (Hanzhong sheep, n=50), Xinjiang province (Bashibai sheep, n=48; Tashikuergan sheep, n=47), Gansu province (Lanzhou big tail sheep, n=42; Ganjia sheep, n=47; Qiaoke sheep, n=56; Oula sheep, n=48 and Minxian black sheep, n=48). And the skudde (n=30) sheep from Changping livestock farm of Beijing city as a control. The genetic diversities of cytochrome b, COI, ND2, ND4, 12S rRNA and 16S rRNA genes of mtDNA were analyzed by PCR-SSCP method among these individuals, and variable sites were found through sequencing analysis which led to the diversities. The complete sequences of D-loop and cytochrome b gene were first used to study the phylogeny evolution and population diversities among Chinese local sheep breeds, the results showed that: 1. The results of PCR-SSCP and DNA sequencing on mtDNA coding region 1.1 10 pairs of special primers of the six genes were designed to analyze the diversity in the 10 breeds. The amplified products of only 5 pairs primers were polymorphic. Based on the analysis of PCR-SSCP, the different bands were respectively named haplotype A, B and C according to the electrophoresis results. The three haplotypes existed in Chinese 9 sheep breeds on the 4 polymorphic loci except SP2 locus, and the frequency of haplotype A was higher than that of B and C. But only haplotype A and B in Skudde sheep were detected, and the frequency of B was higher than that of A. 1.2 The polymorphism of SP2 locus represented in Skudde sheep, but not in other breeds. The differentiation was caused by the substitution of C←→T at 27(the substitution in the gene COⅠwas at 252) site of the locus through further sequencing analysis. Haplotype A was T nucleotide at this site, and B was C nucleotide. It could be used to distinguish Chinese indigenous breeds from foreign ones. 1.3 SP4 locus had three haplotypes on the 8 Chinese sheep breeds except Tashikuergan and Skudde sheep. The polymorphism of SP5 locus was lower, because it was found noly haplotype A and B in Henan big tail sheep, Hanzhong sheep and Tashikuergan sheep, and the frequency of haplotype C was lower in the other sheep breeds. There were no polymorphism in Hanzhong sheep and Skudde sheep for SP9 primer, and Lanzhou big tail sheep only existed haplotype A and C. Relative to other sites, the polymorphism of SP10 locus was higher. 1.4 32, 25, 47 and 23 individuals of the 10 sheep breeds were sequenced on SP4, SP5, SP9 and SP10 loci respectively. The sequencing results showed that: the nucleotides diversity of SP4 was 4.9%. The substitutions of 155(T? C) and 171(A? G) sites was occupied by haplotype C, and 162(C? T) belonged to A, 129(T? C) to haplotype A and C. The nucleotide diversity of SP5 was 4.17%. The substitution of T to C at 195 site could be used to distinguish haplotype B from A and C, but it didn't change the amino acid. The nucleotide polymorphism of SP9 was higher than SP4 and SP5, it was 6.32%. The substitutions at 20, 59 and 137 sites which only occurred in haplotype C could be treated as the characteristic substitution of haplotype.C. 2. The analysis results of D-loop and cytochrome b gene sequences 2.1 Taking the reference NC001941 as standard, the 82 mitochondrial DNA sequences from the 10 domestic sheep breeds were grouped into 78 haplotypes, and the haplotype diversity was 92.7%, The nucleotides composition of all the D-loop haplotypes was 32.9%A, 29.8%T, 22.9%C and 14.4%G, and the content of G+C was 37.3%. 274 polymorphism sites were obtained, including 35 sites transitions but only 8 transversions except some deletions and insertions. The nucleotide diversity was 3.058%. The sequence similarity percentage between sheep and goat was 80%. 18 haplotypes of cytochrome b gene were obtained from 20 individuals among the 10 domestic sheep breeds, and the haplotype diversity was 97.1%. The mean nucleotide compositions were 27.1%T, 28.5%C, 31.4%A and 13.0%G, and G+C was 41.5%. 43 polymorphic sites were obtained from the 1140 bp cytochrome b gene sequence, including 40 transition sites and 3 transvertion sites, the nucleotide diversity was 0.602%. Genetic differentiation (Gst) is the index to measure the gene differentiation among populations. If it is lower, then the relationship between populations is closer. From the Gst analysis of cytochrome b gene sequences it could be concluded that the relationship between haplotype A and B was the closest, B and C was the farthest. 2.2 The length of the D-loop sequences was considerable variable between 1103 bp and 1225 bp, and the majority of that was 1180 bp. Except for minor insertions and deletions, the observed length variations were caused by different copy numbers of a 75 bp tandem repeat sequence. But it could not be considered as the characteristic sequence of haplotypes. 2.3 The NJ, UPGMA and ME phylogenetic trees was constructed by using MEGA 2.0 with goat as outgroup, three major lineages appeared in the 10 domestic sheep, which termed A, B and C lineage. Whether PCR-SSCP or DNA sequence analysis, haplotype C didn't exist in Skudde sheep breeds. The result of median joining method was the same as the above. In order to improve the reliability of the results, all the D-loop complete sequences were used to clustering which was O.musimon, O. vignei, O.aries, B.taurus and B.indicus from GenBank together with the sequences obtained in this study. The results showed that: the relationship between O.musimon and O.aries was nearer, because they were all in B lineage. But O.vignei separated from other O.aries, it didn't belong to the 3 lineages. B.indicus and B.taurus were first gathered, and then to clustering with goat as outgroup. The outgroup lastlygathered with O.aries. It further proved the reliability of the above result, that is to say the modern domestic sheep have three maternal origins. In addition, it also testified that the D-loop sequence of goat amplified by sheep D-loop primer was exact. It indicated that the relationship between goat and sheep was homogeneous. The phylogenetic analysis of cytochrome b gene sequences also validated the above-mentioned ideas. The lineage C was indubitably existent from another aspect. All the analysis results indicated that the modern domestic sheep have the third maternal origin, and the genesis might be in China.