朱鹮人工饲养种群的DNA多态性研究
摘要
朱鹮是世界极濒危鸟类，被称为我国的四大国宝之一。1981年在陕西洋县发现的朱鹮种群数量只有7只，目前洋县人工饲养朱鹮种群就是由这7只发展而来的。较小的群体和频繁的近交，导致朱鹮后代遗传多态性的进一步丧失，利用现代分子生物学方法对这一濒危物种遗传多态性和遗传背景进行研究，对于科学地指导朱鹮的保护和扩繁工作具有重要的意义。本研究首次利用mtDNA序列分析技术、微卫星DNA标记以及RMAPD标记技术对洋县43只人工饲养种群的遗传多态性进行了分析，其结果如下：
1. 测定其中42只朱鹮的mtDNA D-loop区298 bp的序列，序列分析表明，42个个体共有4种单倍型，其中单倍型Ⅰ有9个个体，占群体的21.43％；单倍型Ⅱ有8个个体，占群体的19.05％；单倍型Ⅲ有2个个体，占群体的4.76％；单倍型Ⅳ有23个个体，占群体的54.76％。42个个体有2处发生突变，突变的形式为颠换（G?C、A?C）。提示这一朱鹮种群具有较近的母系起源，这与其祖先来自7只野生朱鹮的事实基本吻合。
2. 测定了5只朱鹮的mtDNA Cyt b基因的部分序列共303 bp，没有发现突变位点。暗示朱鹮mtDNA Cyt b基因具有较强的保守性和朱鹮个体间较近的亲缘关系，也暗示了朱鹮的起源比较单一。
3．将朱鹮mtDNA Cyt b基因序列与鹮科、鹳科、红鹳科、鷺科、鶴科共18个个体的mtDNA Cyt b基因进行同源序列分析，并计算了mtDNA Cyt b基因的碱基相似率和遗传距离。序列的同源分析揭示了科间mtDNA Cyt b基因序列由17种单倍型组成，朱鹮与非洲琵鹭的遗传距离最小，为0.0932；与黄苇鳽的遗传距离最大，为0.1665。利用NJ法对18个个体的mtDNA Cyt b基因序列进行聚类，结果表明朱鹮与非洲琵鹭和彩鹳具有较近的亲缘关系（分别为0.0932，0.1170～0.1249），与形态学的观察相一致。
4. 利用27对其它物种的微卫星引物对43只人工饲养的朱鹮DNA进行扩增，发现只有4对微卫星引物扩增产物具有多态性，说明其它物种的微卫星引物可以用于朱鹮遗传多态性研究，但引物的通用性不强。4个基因座的平均杂合度为0.3491，平均多态信息含量为0.3036，平均有效等位基因数为1.773，均显示朱鹮人工饲养种群的遗传多态性较低。
5. 本研究首次使用RMAPD技术对朱鹮人工饲养种群进行研究。实验结果表明，RMAPD的稳定性比RAPD效果好，对朱鹮群体的分析发现：RAPD的遗传多样性指数为1.474，
而RMAPD的遗传多样性指数为3.029，明显高于RAPD。RMAPD更适合进行种内遗传多态性的分析。
6. 利用RMAPD技术对43只人工饲养的朱鹮的遗传多态性分析表明，朱鹮种群平均带纹相似率为0.718，遗传多样性指数为3.664，反映出个体基因组间相似程度较高，遗传多态性较低，需要加强措施对朱鹮予以保护。
Nipponia Nippon, as an endangered bird in the world, has been considered one of four natural treasures in China. There were only 7 Nipponia Nippon individuals discovered in 1981 in Yangxian, Shaanxi Province. Yangxian artificially reared Nipponia Nippon were developed by these 7 birds. It was well known that a scientific guidance in protecting endangered species by modern molecular biological methods became more and more inevitable. Study on polymorphism and genetic background of Nipponia Nippon from Yangxian, Shaanxi province, would help to prevent from the lose of genetic polymorphism and decline of biological fitness in offsprings. In this study, genetic polymorphisms of 43 Nipponia Nippon individuals from Yangxian were evaluated with mtDNA sequence analysis, microsatellite marker and RMAPD. The results were as follows:
298 bp DNA fragment from mtDNA D-loop control region of 42 Nipponia Nippon individuals were sequenced and analyzed. Haplotypes were detected out. Individual numbers and percentage of four haplotypes were 9, 8, 2, 23 and 21.43%, 19.05%, 4.76% 54.76, respectively. Only two mutation loci (namely transversion) were found, which indicated a near maternal origin relationship between these birds. The results were identical with the fact that their ancestors come from seven wild Nipponia Nippon.
303 bp DNA fragments of Cyt b gene sequence in mtDNA of five Nipponia Nippon individuals were analyzed and no mutation was found, which implied Cyt b gene were highly homological and genetic relationship of them was close. The results indicated the origin of Nipponia Nippon was comparative singleness.
Homology of eighteen Cyt b gene sequences in mtDNA region among Threskiornithidae, Ciconiidae, Phoenicopteridae, Ardeidae and Gruidae were analyzed and their BSR and genetic distances calculated. Seventeen haplotypes were discoveried. Genetic distances between Nipponia Nippon and other species ranged from 0.0932 to 0.1665. The clustering result by Neighbour Joint clusterinhg method indicated Nipponia Nippon, Plegadis falcinellus and Platalea alba were firstly clustered together, which was identical with the results of morphological observation.
Genomic DNA samples from 43 Nipponia Nippon individuals were amplified with 27 paires of microsatellite primers and polymorphism was found on 4 microsatellite loci, which demonstrated some microsatellite primers from other species, such as bovine and sheep, could be used in Nipponia Nippon genomic DNA analysis, but the universality was limited. Average locus heterozygosity, PIC, effective allelic numbers of four loci were 0.349, 0.304, 1.77, respectively, which indicated genetic polymorphism of Nipponia Nippon was poor.
RMAPD was a novel molecular marker technique. It was more stable and polymorphic than RAPD. Hence RMAPD was considered better than microsatellite marker and RAPD in genetic polymorphism detection within population.
Genetic polymorphism of 43 Nipponia Nippon individuals was analyzed with RMAPD marker. Results showed that BSR and genetic diversity indexes were 0.718, 3.664, respectively, which indicated genetic structure of the Nipponia Nippon was simple and poor genetic variations exists in the Nipponia Nippon group from Yangxian.
1. 测定其中42只朱鹮的mtDNA D-loop区298 bp的序列，序列分析表明，42个个体共有4种单倍型，其中单倍型Ⅰ有9个个体，占群体的21.43％；单倍型Ⅱ有8个个体，占群体的19.05％；单倍型Ⅲ有2个个体，占群体的4.76％；单倍型Ⅳ有23个个体，占群体的54.76％。42个个体有2处发生突变，突变的形式为颠换（G?C、A?C）。提示这一朱鹮种群具有较近的母系起源，这与其祖先来自7只野生朱鹮的事实基本吻合。
2. 测定了5只朱鹮的mtDNA Cyt b基因的部分序列共303 bp，没有发现突变位点。暗示朱鹮mtDNA Cyt b基因具有较强的保守性和朱鹮个体间较近的亲缘关系，也暗示了朱鹮的起源比较单一。
3．将朱鹮mtDNA Cyt b基因序列与鹮科、鹳科、红鹳科、鷺科、鶴科共18个个体的mtDNA Cyt b基因进行同源序列分析，并计算了mtDNA Cyt b基因的碱基相似率和遗传距离。序列的同源分析揭示了科间mtDNA Cyt b基因序列由17种单倍型组成，朱鹮与非洲琵鹭的遗传距离最小，为0.0932；与黄苇鳽的遗传距离最大，为0.1665。利用NJ法对18个个体的mtDNA Cyt b基因序列进行聚类，结果表明朱鹮与非洲琵鹭和彩鹳具有较近的亲缘关系（分别为0.0932，0.1170～0.1249），与形态学的观察相一致。
4. 利用27对其它物种的微卫星引物对43只人工饲养的朱鹮DNA进行扩增，发现只有4对微卫星引物扩增产物具有多态性，说明其它物种的微卫星引物可以用于朱鹮遗传多态性研究，但引物的通用性不强。4个基因座的平均杂合度为0.3491，平均多态信息含量为0.3036，平均有效等位基因数为1.773，均显示朱鹮人工饲养种群的遗传多态性较低。
5. 本研究首次使用RMAPD技术对朱鹮人工饲养种群进行研究。实验结果表明，RMAPD的稳定性比RAPD效果好，对朱鹮群体的分析发现：RAPD的遗传多样性指数为1.474，
而RMAPD的遗传多样性指数为3.029，明显高于RAPD。RMAPD更适合进行种内遗传多态性的分析。
6. 利用RMAPD技术对43只人工饲养的朱鹮的遗传多态性分析表明，朱鹮种群平均带纹相似率为0.718，遗传多样性指数为3.664，反映出个体基因组间相似程度较高，遗传多态性较低，需要加强措施对朱鹮予以保护。
Nipponia Nippon, as an endangered bird in the world, has been considered one of four natural treasures in China. There were only 7 Nipponia Nippon individuals discovered in 1981 in Yangxian, Shaanxi Province. Yangxian artificially reared Nipponia Nippon were developed by these 7 birds. It was well known that a scientific guidance in protecting endangered species by modern molecular biological methods became more and more inevitable. Study on polymorphism and genetic background of Nipponia Nippon from Yangxian, Shaanxi province, would help to prevent from the lose of genetic polymorphism and decline of biological fitness in offsprings. In this study, genetic polymorphisms of 43 Nipponia Nippon individuals from Yangxian were evaluated with mtDNA sequence analysis, microsatellite marker and RMAPD. The results were as follows:
298 bp DNA fragment from mtDNA D-loop control region of 42 Nipponia Nippon individuals were sequenced and analyzed. Haplotypes were detected out. Individual numbers and percentage of four haplotypes were 9, 8, 2, 23 and 21.43%, 19.05%, 4.76% 54.76, respectively. Only two mutation loci (namely transversion) were found, which indicated a near maternal origin relationship between these birds. The results were identical with the fact that their ancestors come from seven wild Nipponia Nippon.
303 bp DNA fragments of Cyt b gene sequence in mtDNA of five Nipponia Nippon individuals were analyzed and no mutation was found, which implied Cyt b gene were highly homological and genetic relationship of them was close. The results indicated the origin of Nipponia Nippon was comparative singleness.
Homology of eighteen Cyt b gene sequences in mtDNA region among Threskiornithidae, Ciconiidae, Phoenicopteridae, Ardeidae and Gruidae were analyzed and their BSR and genetic distances calculated. Seventeen haplotypes were discoveried. Genetic distances between Nipponia Nippon and other species ranged from 0.0932 to 0.1665. The clustering result by Neighbour Joint clusterinhg method indicated Nipponia Nippon, Plegadis falcinellus and Platalea alba were firstly clustered together, which was identical with the results of morphological observation.
Genomic DNA samples from 43 Nipponia Nippon individuals were amplified with 27 paires of microsatellite primers and polymorphism was found on 4 microsatellite loci, which demonstrated some microsatellite primers from other species, such as bovine and sheep, could be used in Nipponia Nippon genomic DNA analysis, but the universality was limited. Average locus heterozygosity, PIC, effective allelic numbers of four loci were 0.349, 0.304, 1.77, respectively, which indicated genetic polymorphism of Nipponia Nippon was poor.
RMAPD was a novel molecular marker technique. It was more stable and polymorphic than RAPD. Hence RMAPD was considered better than microsatellite marker and RAPD in genetic polymorphism detection within population.
Genetic polymorphism of 43 Nipponia Nippon individuals was analyzed with RMAPD marker. Results showed that BSR and genetic diversity indexes were 0.718, 3.664, respectively, which indicated genetic structure of the Nipponia Nippon was simple and poor genetic variations exists in the Nipponia Nippon group from Yangxian.
引文
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