昆明犬15个微卫星基因座的多态性及其复合扩增研究
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摘要
微卫星DNA，又称短串联重复序列(STR)，是一类具有高度多态性的DNA分子标记，被广泛应用于遗传学、法医学等领域的研究，如基因物理图谱的绘制，动、植物遗传育种的指导及亲权鉴定。昆明犬作为我国自主培育出来的优良品种犬，已形成狼青、黑背、草黄三个稳定的品系。本文第一部分通过筛选15个高度多态的微卫星基因座PEZ1、PEZ2、PEZ3、PEZ5、PEZ6、PEZ8、PEZ12、PEZ15、PEZ20、FHC2010、FHC2054、FHC2079、FHC2087Ub、FHCL2132和FHC2611，自行设计引物，应用聚合酶链式反应(PCR)、非变性聚丙烯酰胺凝胶电泳(PAGE)和银染法检测74头昆明犬和11头德国牧羊犬在15个STR基因座上的基因型，并对所得数据进行遗传学统计分析。结果表明昆明犬在15个STR基因座上的平均杂合度(H)为0.638，平均多态信息含量(PIC)为0.604，累积非父排除率为0.9996，累积个体识别率(DP)为0.999999999997。Hardy-Weinberg平衡检验表明PEZ15、FHC2079和FHC2087Ub偏离Hardy-Weinberg平衡。通过遗传距离D_S和D_A来估计德国牧羊犬与昆明犬三个品系间的进化关系。采用邻近法(NJ)和非加权配对组平均数法(UPGMA)构建进化树。结果表明狼青昆明犬和草黄昆明犬的遗传距离较近，而黑背昆明犬与德国牧羊犬的遗传距离较近。
本文第一部分对昆明犬多态性研究都是采用单基因座PCR扩增，它耗材多、成本高、费时、工作量大，尤其在处理大量样本时。相比之下，由2或2个以上基因座组成的复合扩增体系具有耗样量少、信息量多、高效且成本低的优点而为大多数人所采用。本文第二部分是在调查了15个STR基因座等位基因分布范围的基础上，选择FHC2132来代替FHCL2132，其它基因座不变，对PEZ6，PEZ15，FHC2054三基因座重新设计引物，组成五个复合扩增体系即复合扩增体系Ⅰ(PEZ2，FHC2079，PEZ8)，复合扩增体系Ⅱ(PEZ1，PEZ20，PEZ5)，复合扩增体系Ⅲ(FHC2054，FHC2611，FHC2132)，复合扩增体系Ⅳ(PEZ6，FHC2010，PEZ15)，复合扩增体系Ⅴ(PEZ3，PEZ12，FHC2087Ub)。通过单基因座PCR扩增和三因素三水平的复合扩增正交实验摸索出各复合扩增体系中的最佳引物浓度比例，成功构建了五个复合扩增体系。将五个复合扩增体系用于犬的STR基因型检测，结果表明五个复合扩增体系对犬的STR基因型检测是有效的。这五个复合扩增体系的构建成功为进一步开发犬的荧光复合扩增试剂盒奠定了坚实的基础。五个复合扩增体系的最佳体系组成如下：
As a class of highly polymorphic DNA marker, microsatellite DNA, named as short tandem repeats(STR), is widely applied to genetics and forensic science such as making physical map, guiding the breeding of animal and plant, paternity test and so on. Being an excellent purebred dog, Kunming dog which was bred by Chinese has been bred into three steady inherited breeds wolf cyan, grass yellow and black back Kuning dog. At the first part of this dissertation, fifteen highly polymorphic canine STR loci (PEZ1, PEZ2, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, PEZ15, PEZ20, FHC2010, FHC2054, FHC2079, FHC2087Ub, FHCL2132, FHC2611) were chosen and the primers were designed by ourselves. By PCR (Polymerase Chain Reaction) and non-denatured PAGE (Polyacrylamide Gel Electrophoresis) and silver-staining, we analyzed the genotype of 74 Kunming dogs at 15 STR loci and computed the statistical data in population genetics and forensic science. For the fifteen canine STR loci, it shows that the average Heterozygosity (H), the average Ploymorphism Information Content (PIC), the Cumulative probability of paternity exclusion (CPE) and the Total Power of Discrimination (TDP) is 0.638, 0.604, 0.9996 and 0.999999999997 respectively. Twelve loci correspond to Hardy-Weinberg equilibrium except PEZ15, FHC2079 and FHC2087Ub. The evolutionary relationships among three kinds of Kunming dog and German shepherd were estimated by genetic distance Ds and Da-With two methods Neighbour-Joining (NJ) and unweighted pair group method with arithmetic (UPGMA), we constructed evolution tree among them. According to the pairwise genetic distances, wolf cyan Kunming dog and grass yellow Kunming dog had shorter diverse evolutionary histories and black back Kunming dog and German shepherd had shorter diverse evolutionary histories.
In the first part of this dissertation, we adopted singleplex PCR which was time-consuming and expended largely with high cost and heavy work, especially when there were a lot of samples to be tested. In contrast with singleplex PCR, multiplex PCR consisted of two or more loci, is more used for the merits that it is low-cost and timesaving with a little sample DNA and plentiful information. At the second part of this dissertation, we substituted FHC2132 for FHCL2132 and redesigned the primers for PEZ6, PEZ15 and FHC2054 after investigating the range of alleles of 15 loci severally. We have five multiplex systems as follow: I (PEZ2, FHC2079, PEZ8), II
本文第一部分对昆明犬多态性研究都是采用单基因座PCR扩增，它耗材多、成本高、费时、工作量大，尤其在处理大量样本时。相比之下，由2或2个以上基因座组成的复合扩增体系具有耗样量少、信息量多、高效且成本低的优点而为大多数人所采用。本文第二部分是在调查了15个STR基因座等位基因分布范围的基础上，选择FHC2132来代替FHCL2132，其它基因座不变，对PEZ6，PEZ15，FHC2054三基因座重新设计引物，组成五个复合扩增体系即复合扩增体系Ⅰ(PEZ2，FHC2079，PEZ8)，复合扩增体系Ⅱ(PEZ1，PEZ20，PEZ5)，复合扩增体系Ⅲ(FHC2054，FHC2611，FHC2132)，复合扩增体系Ⅳ(PEZ6，FHC2010，PEZ15)，复合扩增体系Ⅴ(PEZ3，PEZ12，FHC2087Ub)。通过单基因座PCR扩增和三因素三水平的复合扩增正交实验摸索出各复合扩增体系中的最佳引物浓度比例，成功构建了五个复合扩增体系。将五个复合扩增体系用于犬的STR基因型检测，结果表明五个复合扩增体系对犬的STR基因型检测是有效的。这五个复合扩增体系的构建成功为进一步开发犬的荧光复合扩增试剂盒奠定了坚实的基础。五个复合扩增体系的最佳体系组成如下：
As a class of highly polymorphic DNA marker, microsatellite DNA, named as short tandem repeats(STR), is widely applied to genetics and forensic science such as making physical map, guiding the breeding of animal and plant, paternity test and so on. Being an excellent purebred dog, Kunming dog which was bred by Chinese has been bred into three steady inherited breeds wolf cyan, grass yellow and black back Kuning dog. At the first part of this dissertation, fifteen highly polymorphic canine STR loci (PEZ1, PEZ2, PEZ3, PEZ5, PEZ6, PEZ8, PEZ12, PEZ15, PEZ20, FHC2010, FHC2054, FHC2079, FHC2087Ub, FHCL2132, FHC2611) were chosen and the primers were designed by ourselves. By PCR (Polymerase Chain Reaction) and non-denatured PAGE (Polyacrylamide Gel Electrophoresis) and silver-staining, we analyzed the genotype of 74 Kunming dogs at 15 STR loci and computed the statistical data in population genetics and forensic science. For the fifteen canine STR loci, it shows that the average Heterozygosity (H), the average Ploymorphism Information Content (PIC), the Cumulative probability of paternity exclusion (CPE) and the Total Power of Discrimination (TDP) is 0.638, 0.604, 0.9996 and 0.999999999997 respectively. Twelve loci correspond to Hardy-Weinberg equilibrium except PEZ15, FHC2079 and FHC2087Ub. The evolutionary relationships among three kinds of Kunming dog and German shepherd were estimated by genetic distance Ds and Da-With two methods Neighbour-Joining (NJ) and unweighted pair group method with arithmetic (UPGMA), we constructed evolution tree among them. According to the pairwise genetic distances, wolf cyan Kunming dog and grass yellow Kunming dog had shorter diverse evolutionary histories and black back Kunming dog and German shepherd had shorter diverse evolutionary histories.
In the first part of this dissertation, we adopted singleplex PCR which was time-consuming and expended largely with high cost and heavy work, especially when there were a lot of samples to be tested. In contrast with singleplex PCR, multiplex PCR consisted of two or more loci, is more used for the merits that it is low-cost and timesaving with a little sample DNA and plentiful information. At the second part of this dissertation, we substituted FHC2132 for FHCL2132 and redesigned the primers for PEZ6, PEZ15 and FHC2054 after investigating the range of alleles of 15 loci severally. We have five multiplex systems as follow: I (PEZ2, FHC2079, PEZ8), II
引文
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[7] http://www.ncbi.nlm.nih.gov/disease/
[8] Breen M, Thomas R, Binns MM, et al. Reciprocal chromosome painting reveals detailed regions of conserved synteny between the karyotypes of the domestic dog (Canis familiaris) and human [J].Genomics, 1999, 61:145-155
[9] Wayne RK, Ostrander EA. Origin, genetic diversity, and genome structure of the domestic dog [J].Bioessays, 1999, 21: 247-257
[10] Jonasdottir TJ, Mellersh CS, Moe L, et al. Genetic mapping of a naturally occurring hereditary renal cancer syndrome in dogs [J].Proc Natl Acad Sci USA, 2000,97: 4132-4137
[11] Moe L, Lium B. Hereditary multifocal renal cystadenocarcinomas and nodular dermatofibrosis in 51 German shepherd dogs [J].J Small Anim Pract, 1997, 38: 498-505
[12] Sargan DR. Research in canine and human genetic disease [J].J Med Genet, 1995, 32(9): 751-754
[13] Cargill EJ, Clark LA, Steiner JM, et al. Multiplexing of canine microsatellite markers for whole-genome screens [J].Genomics, 2002, 80: 250-253
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[15] Skinner D M, Beattie W G, Blatter F R, et al. The sequence of a hermitcrab satellite DNA in (TAGG)_n/(ATCC)_n[J].Biochemistry, 1974,13:3930-3937
[16] Tautz D. Hypervariability of simple sequences as a general source for polymorphic DNA markers [J]. Nucleic Acids Rec, 1989,17: 6463 - 6471
[17] Bar W, Brinkmann B, Budowle B, et al. DNA recommendations: Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems [J].Int J Leg Med, 1997,110(4): 175-176
[18] Gill P, Kimpton C, d'Aloja E, et al. Report of the European DNA profiling group (EDNAP)—towards standardisation of short tandem repeat (STR) loci [J]. Forensic Sci Int, 1994,65: 51-59
[19] Gill P, Brinkmann B, d'Aloja E, et al. Considerations from the European DNA profiling group (EDNAP) concerning STR nomenclature [J].Forensic Sci Int, 1997, 87:185-192
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