【摘要】：河南信陽大別山區(qū)是黃緣閉殼龜最重要的分布區(qū)之一,但是由于生境破壞、環(huán)境污染和濫殺濫捕等原因,黃緣閉殼龜?shù)囊吧Y源量急劇下降,目前已被IUCN及CITES列為珍稀瀕危物種,瀕危等級(jí)EN。為了給黃緣閉殼龜提供在分子水平上的理論依據(jù),本研究對(duì)黃緣閉殼龜進(jìn)行了微衛(wèi)星標(biāo)記、單核苷酸多態(tài)性標(biāo)記的開發(fā),對(duì)黃緣閉殼龜卵殼膜與糞便進(jìn)行了非損傷DNA試提取,并利用微衛(wèi)星標(biāo)記和線粒體標(biāo)記技術(shù)對(duì)黃緣閉殼龜?shù)娜后w進(jìn)行研究。通過對(duì)黃緣閉殼龜群體的遺傳多樣性和遺傳結(jié)構(gòu)進(jìn)行分析,測(cè)定群體間的遺傳差異,預(yù)測(cè)雜種優(yōu)勢(shì),為種質(zhì)資源庫的構(gòu)建提供理論基礎(chǔ),同時(shí)也為大別山黃緣閉殼龜野生資源的保護(hù)和人工養(yǎng)殖的開展提供建議。本文研究的主要結(jié)果有:1.本研究采用FIASCO法,構(gòu)建了黃緣閉殼龜富集文庫,成功分離設(shè)計(jì)得到136對(duì)微衛(wèi)星引物,并對(duì)微衛(wèi)星位點(diǎn)進(jìn)行評(píng)價(jià),共檢測(cè)出44對(duì)多態(tài)性微衛(wèi)星引物。2.嘗試采用磁珠富集的方法對(duì)瀕危保護(hù)動(dòng)物黃緣閉殼龜基因組中潛在的SNP位點(diǎn)進(jìn)行了開發(fā),共獲得10條確認(rèn)SNP位點(diǎn)的引物序列。3.以幼龜孵化后殘留的卵殼和新鮮的糞便為研究材料,采用非損傷性方法對(duì)黃緣閉殼龜基因組DNA進(jìn)行提取,證實(shí)了從糞便中提取DNA是不可行的,而從卵殼膜中提取DNA是可行的,能為后續(xù)黃緣閉殼龜?shù)姆肿舆z傳提供研究材料。4.利用新開發(fā)的微衛(wèi)星標(biāo)記對(duì)3個(gè)不同來源的43只黃緣閉殼龜群體的遺傳結(jié)構(gòu)進(jìn)行分析,發(fā)現(xiàn)養(yǎng)殖場(chǎng)中部分個(gè)體出現(xiàn)了不同亞群之間的基因交流現(xiàn)象,并將救護(hù)中心類群和師院類群合并為一個(gè)亞群,養(yǎng)殖場(chǎng)的兩個(gè)類群分別作為一個(gè)亞群。5.利用線粒體基因CO1和CO2對(duì)來3個(gè)不同來源的黃緣閉殼龜群體(21只)進(jìn)行了單倍型和系統(tǒng)發(fā)育分析,結(jié)合微衛(wèi)星標(biāo)記分析結(jié)果,把所有個(gè)體分為信陽地區(qū)種群,安徽地區(qū)種群和未知地區(qū)種群三個(gè)不同的種群,并推測(cè)未知地區(qū)種群可能為大別山以外地區(qū)的種群且與其他個(gè)體存在較遠(yuǎn)的進(jìn)化關(guān)系,而信陽地區(qū)種群和安徽地區(qū)種群之間的進(jìn)化關(guān)系更近。
[Abstract]:The Dabie Mountain area of Xinyang, Henan Province is one of the most important distribution areas of the closed shell tortoise. However, due to habitat destruction, environmental pollution and indiscriminate hunting, the wild resources of the closed shell tortoise have declined sharply. At present, it has been listed as rare and endangered species by IUCN and CITES, and EN. has been classified as endangered. In order to provide theoretical basis on molecular level, microsatellite markers and single nucleotide polymorphism (SNP) markers were used to extract DNA from egg shell membrane and feces. Microsatellite markers and mitochondrial markers were used to study the population of the closed shell tortoise. Based on the analysis of genetic diversity and genetic structure of the populations, the genetic differences among populations were determined, and heterosis was predicted, which provided a theoretical basis for the construction of germplasm resource bank. At the same time, it also provides suggestions for the protection of wild resources and the development of artificial breeding of the closed shell turtle in the yellow margin of Dabie Mountain. The main results of this paper are as follows: 1. In this study, FIASCO method was used to construct the enrichment library of the turnip, and 136 pairs of microsatellite primers were designed successfully, and the microsatellite loci were evaluated. A total of 44 pairs of polymorphic microsatellite primers were detected. In this paper, the potential SNP loci in the genome of endangered protected animals were developed by means of magnetic bead enrichment. A total of 10 primer sequences were obtained to confirm the SNP loci. Using egg shell and fresh feces from hatching of young turtle as the research materials, the genomic DNA was extracted by non-invasive method. It was proved that extracting DNA from feces was not feasible, but DNA from egg shell membrane was feasible. It can be used to study the molecular heredity of the subsequent turnip turtle. 4. Using the newly developed microsatellite markers, the genetic structure of 43 populations from three different sources was analyzed. It was found that some individuals in the farm had the phenomenon of gene exchange among different subpopulations. The rescue center group and the teacher's college group were merged into one subgroup, and the two groups in the farm were regarded as one subgroup respectively. Haplotype and phylogenetic analysis were carried out in 21 populations (21 individuals) from three different sources of yellow-bordered turtle by mitochondrial gene CO1 and CO2. Combined with the results of microsatellite marker analysis, all individuals were divided into two populations in Xinyang area. There are three different populations in Anhui and unknown regions, and it is speculated that the population in the unknown region may be the population outside the Dabie Mountains and has a far evolutionary relationship with other individuals. The evolutionary relationship between Xinyang population and Anhui population is closer.
【學(xué)位授予單位】：信陽師范學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q953

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本文編號(hào)：2323478