东北梅花鹿野生种群分布与 遗传变异的分子鉴定

梅花鹿东北亚种(Cervus nippon hortulorum)曾被认为已野外灭绝,近年来在黑龙江东南部和吉林东部临近边境地区发现少量分布,其生境隔离、面积狭小,破碎化严重。亟需对其种群的遗传变化,特别是遗传多样性和近交衰退等种群遗传信息开展进一步评价,增强保护与管理的针对性。本研究在大、小兴安岭和长白山设计9个重点研究区域,共收集673份疑似梅花鹿粪样样本。首先基于线粒体DNA Cyt b基因测序技术开展物种鉴定,并对鉴定为梅花鹿的阳性样本利用微卫星技术进行个体识别。结果证实,东北梅花鹿仅在老爷岭东部山脉有分布,106份梅花鹿粪便DNA中识别出33只个体(穆棱保护区20只,老爷岭保护区13只)。33个Cyt b基因序列共检测出6个变异位点和5个单倍型,单倍型多样性指数(Hd)为0.621,核苷酸多样性指数(Pi)为0.006 7;微卫星检出种群平均等位基因数(Na)7.1个,观测杂合度(Ho)0.604,期望杂合度(He)0.712,固定系数(Fis)0.152。结果表明,东北梅花鹿种群遗传多样性丰富,但也存在一定程度的杂合度不足和近亲繁殖;种群近期经历了瓶颈效应,未发生种群扩张;群体间无遗传分化,可作为一个管理单元加以保护。建议,对东北梅花鹿稀有单倍型个体重点监测和保护,恰当时期考虑圈养种群野外放归,以提高野外个体间基因交流和快速种群恢复。

Molecular Identification of the Population Distribution and Genetic Variation of Cervus nippon hortulorum

Cervus nippon hortulorum was once considered to be extinct in the wild. In recent years, some small size populations were found in the southeastern part of Heilongjiang province and the eastern part of Jilin province, nearing the border. Since the habitats of C. n. hortulorum are narrow, isolated and fragmented, it is an urgent need to further evaluate the genetic changes of the population, especially the genetic diversity and inbreeding decline, so as to enhance the pertinence of conservation and management. In this study, 673 suspected fecal samples of sika deer were collected from 9 key research areas in Daxing''an, Xiaoxing''an and Changbai Mountains (Fig. 1). Firstly, species identification was carried out based on DNA Cyt b gene sequencing technology, and the positive samples were supplied for further individual identification by microsatellite technology. At last, Microchecker 2.2.3 software was used to detect the invalid allele or allele deletion of each locus; and Genalex 6.0 software was used to calculate the population average allele number (Na), observed heterozygosity (Ho), expected heterozygosity (He) and fixed coefficient (Fis). A total of 33 individuals (20 in Muling nature reserve and 13 in Laoyeling nature reserve) were identified from 106 fecal DNA samples of sika deer (Fig. 2 and Table 1). Six variation sites and five haplotypes were detected in the Cyt b sequence of these 33 individuals. The values of Hd, Pi, Na, Ho, He and Fis were 0.621, 0.006 7, 7.1, 0.604, 0.712, and 0.152, respectively (Table 1). The results showed that the population genetic diversity of Northeastern sika deer was rich, but there was also a certain degree of heterozygosity deficiency and inbreeding (Table 1); the population experienced bottleneck effect in recent years, without population expansion (Table 1 and Fig. 5); there was no genetic differentiation between populations, which could be protected as a management unit (Table 2 and Fig. 3, 4). It is suggested that the individuals with rare haplotypes should be taken as the key point in monitoring and protection, and the artificial bred populations should be released in the field at the right time, so as to improve the gene exchange between individuals in the field and accelerate the population restoration.