Genetic Diversity And Sex Determination Of Roe Deer In The Northeastern China

Roe deer is widely distributed in the Northeast of China. The population in the wild, however, declined significantly due to excessively hunting of the animal. Therefore, understanding the genetic diversity and population's hereditary structure of the roe deer, and protecting the limited wild roe deer resource well are urgent tasks.At present, genetic marker technique is a rather accurate method in molecule level to study hereditary variation in wild animals. There are not reports about the Northeastern roe deer's genetic marker study in literature, only a few publications are found about hereditary variation studies using DNA sequencing technique in European roe deer and Siberia roe deer. In this study, RAPD marker and mitochondria DNA sequencing techniques were used to study the intra-nuclear and extra-nuclear genetic material variation, and to analyze the phylogeny of the deers. By the Sry gene PCR-amplification and sequencing, sex determining mechanism in roe deer was investigated and then the sexual structure of the wild roe deer population was evaluated.In RAPD molecular marker analysis. 20 arbitrary primers from 80 ones were used to amplify 32 roe deer's samples. Two to eight obvious bands were observed from each primer amplified. One hundred and fourteen bands expressed polymorphism among 123 bands tested and polymorphic percentage was 92.7%. It all showed that the Northeastern roe deer had high genetic diversity in percentage of polymorphic bands (PPB), Shannon's information index and Nei's gene diversity. Among 3 geographic populations, Daxing'an Mountain population had the lowest genetic diversity compared with Changbai Mountain and Wandashan Mountain populations. The UPGMA cluster analysis could distinguish the individual of 3 geographic populations clearly. The principal coordinates analysis (PCA) of polymorphic bands showed this result directly. In population genetic differentiation analysis, Nei's genetic distance, the coefficient of gene differentiation, the partitioning of the Shannon diversity and analysis of molecular variance (AMOVA) were used and the results showed that the genetic differentiation degree between populations was lower and the hereditary variation primarily came from within population.Mitochondrial DNxA. (mtDNA) is extra-nuclear genetic material, it is usually used in genetic diversity analysis and phylogeny study because of its brief molecule structure and rapid evolutionary rate. In this study, mtDNA D-loop sequence of 16 roe deer samples were determined, four sequence has abandoned after identify. A 614bp sequence fragment from 12 individuals was obtained. There were 40 polymorphic sites in comparable 562bp sequence, the rate of polymorphic sites was 7.12%. Among them 20 were singleton variable sites, 20 were parsimony informative sites, 38 were transition, and 2 were transversion. Besides, there were 2 undetected sites, 3 sites with alignment gaps or missing. Altogether 12 haplotypes were detected, sequence differentia averaged at 2.00% between haplotype. Nucleotide diversity (n)was lower, only 0.0204. According to AMOVA analysis, 58.09% variance came from within population. It is consistant with result of RAPD. MtDNA D-loop data supports to classify the Northeastern roe deer to Capreolus pygargus's subspecies ( Capreolus pygargus mantschuricus) , but to be an independent species from Capreolus capreolus. The relationship between populations can be clearly demonstrated from phylogenetic tree and network profiles for haplotypes.In this study, the mtDNA Cytb gene 445bp fragment of the Northeastern roe deer was sequenced, combining GenBank correlation sequence data, the phylogenetic tree of deer in 18 species, in 12 genus of 2 families was reconstructed. The topology structures of gene tree using maximum parsimony method, maximum likelihood method and Bayesian are almost the same. This result indicates that the Capreolus is the earlier group divided from Odocoileinae; Hydropotinae is the earliest species in four subfamilies of deer family animals, secondly is Odocoileinae and Muntiacinae, Cervinae is the highest in the evolution.The roe deer's Sry gene has been PCR-amplified using a pair primer being screened out from human SRY gene. The amplified results suggests sex specificity. The PCR product of roe deer Sry gene was cloned and sequenced, and the partial nucleotide sequences (185bp) of the motif region was determined. This sequence has high homology about 96.76% with sika deer. The results of the Sry-PCR amplified analysis of 42 samples, showed that the sexual ratio of natural population is almost 1:1 (22:20).