Studies on development of genome markers and application to colonies in the common marmoset

1.设计了200套引物，以2004年存档的NCBI数据库为基础开发微卫星DNA标记。在200个标记中，有87个标记与5只狨猴存在遗传多态性。随机选取42个标记，从育种角度对不同绒猴群体进行了研究。结果，等位基因数量和频率的差异逐一被观察到。结果表明，微卫星DNA标记可用于绒猴群体的遗传评价和育种。在这项研究中开发的80%的标记被分配到狨猴染色体上，这些染色体是用每个用于DNA探针的人类染色体确定的。选择13个标记进行遗传监测，作为质量控制程序之一。用荧光标记引物扩增。PCR产物成功测序，以确定其在一个碱基水平上的大小。对绒猴MHCⅱ类基因DQA1的外显子2和DQB1的外显子3进行测序，并在其序列中检测到单核苷酸多态性。用不同的电泳技术对蛋白质和酶的同工酶进行了研究，发现TRF （transfer）和GPI （glucossephosphate isomerase）各有2个等位基因。用10种人血型（ABO、MN和Rh）的抗血清研究红细胞抗原。4 .除抗a抗血清血凝反应阳性外，其余均为阴性。对线粒体DNA约1kb的d环进行测序，检测可能是限制性内切酶识别位点的snp。结果，分别用5个限制性内切酶和1个限制性内切酶的引物检测到8个单倍型。G.利用体外培养的外周血淋巴细胞和脾细胞成功制备了用于核型分析的染色体标本。

1.Two hundred primer sets were designed to develop microsatellite DNA markers based on the NCBI database archived in 2004. Of 200 markers, 87 showed genetic polymorphisms with five marmosets. Using 42 markers of them selected randomly, different marmoset colonies were studied from a view point of breeding. As a result, differences in the number of alleles and their frequencies were observed one by one. It was revealed that the microsatellite DNA markers are useful for genetic evaluation and breeding of marmoset colonies. Eighty percent of the markers developed in this study were assigned on marmoset chromosomes that are determined with each human chromosome used for DNA probe. Thirteen markers were selected for genetic monitoring which is one of the quality control program. They were amplified using fluorescence-labeled primers. The PCR products were successfully sequenced to determine their sizes at one-base level.2.Exon 2 of the DQA1 and exon 3 of DQB1, which are the marmoset MHC class II genes, were sequenced and SNPs (single nucleotide polymorphisms) were detected in their sequences.3.Isozymes of proteins and enzymes were studied with various electrophoresis techniques and two alleles were observed in each of TRF (transferring) and GPI (glucosephosphate isomerase).4.Red blood cell antigens were studied using 10 antisera for human blood groups (ABO, MN and Rh). Only anti-A antiserum showed positive hemagglutination, but the others showed negative reaction.5.About 1kb D-loop of mitochondrial DNA was sequenced to detect SNPs which may be restriction enzyme recognition sites. As a result, 8 haplotypes were detected using one primer set with five restriction enzymes and another set with one restriction enzyme. G. Preparation of chromosome specimen used for karyotype analysis was successfully done using cultured peripheral lymphocytes and spleen cells.