【作者】 郭宝英；

【导师】 谢从新；

【作者基本信息】 华中农业大学 ， 湿地资源与环境， 2009， 博士

【摘要】 黑斑原（魚兆）（Glyptosternum maculatum Regan）隶属鲇形目（Siluriformes）、（魚兆）科（Sisoridae）、原（魚兆）属（Glyptosternum）,主要分布在雅鲁藏布江干流及其支流,是唯一一种分布到雅鲁藏布江中上游的（魚兆）科鱼类。黑斑原（魚兆）在雅鲁藏布江的两支流,尼洋河、拉萨河和谢通门江段三地均存在着产卵场,渔获个体大小差异明显,确定他们是否为三个相对独立的种群,还是一个种群,对于资源保护和合理利用都具有重要价值。本文利用AFLP和SSR两种分子标记方法,研究雅鲁藏布江两支流,尼洋河、拉萨河和谢通门江段黑斑原（魚兆）地理群体的遗传多样性,重要结果如下:1.AFLP与SSR两种标记揭示了黑斑原（魚兆）物种的遗传多样性水平（1）选用81对AFLP引物组合对3个地理群体共96个样本进行了遗传多样性研究,选出的5对多态性引物组合共扩增出332条清晰可辨、可重复的条带,平均每对引物组合扩增出66.4条带;332条扩增出的条带中有51条带是多态的,多态条带范围在8（E-AAC/T-AAG,E-AAG/T-AAT和E-AAT/T-AGA）和17条（E-AAC/T-AAT）之间,平均为10.2条。3个地理群体的遗传距离（D）分布范围在0.0015-0.0042之间。（2）选用28对SSR引物对3个黑斑原（魚兆）地理群体共96个样本进行了遗传多样性研究,选出的6对SSR多态性引物共扩出20个等位基因,平均每个位点为3.2个等位基因,多态信息含量为0.0394-0.4159,平均0.2837,属中度多态,可作为黑斑原（魚兆）遗传标记分析有效的微卫星引物。3个地理群体的遗传距离（D）分布范围在0.0022-0.0778之间。2.AFLP标记和SSR标记的比较AFLP标记共产生332条清晰可辨、可重复的条带,其中51条带是多态的;SSR标记共扩出20个等位基因。这两种分子标记的研究结果比较一致:均揭示了黑斑原（魚兆）群体遗传多样性水平比较低,并且拉萨河群体遗传多样性最低;3个地理群体间无遗传差异。AFLP和SSR都是评价黑斑原（魚兆）遗传多样性有效的分子标记。3.黑斑原（魚兆）微卫星序列的开发在本研究所用的生物素标记的（CA）₁₅探针构建的富集文库克隆中,720个（70%）为初步定为含有微卫星序列（含有两到三条带）的阳性克隆中,从中随机挑取145个克隆去测序,其中139个（89.2%）含有重复次数大于或等于7的克隆,序列比对后发现124个克隆为非同源性碱基序列;重复次数范围为8-165次,平均为52次。对124个微卫星序列进行划分:其中perfect为80个（64.5%）;imperfect为40个（32.3%）;compound perfect为14个（3.2%）,部分序列已经在Genbank中进行了注册。4.黑斑原（魚兆）微卫星标记的筛选共设计了33对微卫星引物,经过PCR扩增,其中有29对对黑斑原（魚兆）有扩增产物,从中筛选出具有多态性产物的25对引物。在黑斑原（魚兆）一个群体36个样本的25个微卫星位点上,等位基因的数目从2到10不等,平均每个位点为4.6个等位基因;观测杂合度（Ho）范围从0到1,平均为0.5481;多态信息含量（PIC）范围为0.2846-0.8173,平均值为0.5793,表明这些标记均适合黑斑原（魚兆）进行群体遗传学分析。5.黑斑原（魚兆）微卫星标记通用性的检测设计的33对微卫星引物扩增黄斑褶（魚兆）（与黑斑原（魚兆）同科不同属）一个群体的18尾样品,其中有14对成功扩增,全部显示多态,并且都能进行多态分析。等位基因数目从2到7不等,平均为3.85;期望杂合度范围为0.3476-0.8571,平均值为0.6054;观测杂合度范围为0-1,平均值为0.5992:信息多态含量范围从0.3208到0.8111,平均值为0.7434。结果显示,从黑斑原（魚兆）筛选出的微卫星标记中已有14对成功地跨属扩增。更多还原

【Abstract】 Glyptosternum maculatum（Regan）（Sisoridae,Siluriformes） is one of the GlyptosternoM catfish species distributed only in the middle-up reach of the Yarlung Zangbo River,Tibet,China.Nevertheless,three spawning grounds have been proposed, one each in the Nyang river,Lhasa river（two branches of the Yarlung Zangbo River） and Shetongmon reach.The individuals collected from these three populations are obviously fifferent in both body weight and body length.It is very important to make sure whether the geographical populations evolved independent or not for the conservation and proper utilization of Glyptosternum maculatum resources.In the present study,AFLP and SSR molecular methods were utilized to study the genetic diversity of the different Glyptosternum maculatum populations,the important results as bellows,1.The genetic diversity of Glyptosternum maculatum revealed by AFLP and SSR on species level（1） 81 AFLP primer combinations in total had been used to select the polymorphic primer combinations in Glyptosternum maculatum samples,then five ones selected as polymorphic primers,detected 332 products,51 of them（15.4%） were polymorphic in at least 1 population,no band was found to be specific for a population.The number of polymorphic bands ranged from 8（E-AAC/T-AAG,E-AAG/T-AAT and E-AAT/T-AGA） to 17（E-AAC/T-AAT） with an average of 10.2.The genetic distance between the three populations ranged from 0.0015 to 0.0042.（2） 28 SSR primer pairs had been used to select the polymorphic ones,at the end,6 polymorphic primer pairs amplified 20 alleles in all 96 Glyptosternum maculatum samples,on the average of 3.2,the information content（PIC） ranged from 0.0394 to 0.4159,on the average of 0.2837,indicating that these markers could be used to analyzed the genetic diversity of Glyptosternum maculatum.The genetic distance between the three populations ranged from 0.0022 to 0.0778.2.The comparison between SSR and AFLP:The AFLP markers detected 332 products,among which there were 51 polymorphic bands;The SSR markers amplified 20 alleles in total,which suggested that AFLP marker could provide more genetic information than SSR marker.Nevertheless,the results obtained by the two marker systems were very similar:they both revealed that the genetic diversity of Glyptosternum maculatum was very low,especially at the area of Lhasa,in addition to,there was no genetic variance existed among the populations.Both AFLP and SSR markers were effective to evaluate the genetic diversity of Glyptosternum maculatum.3.The development of microsatellite for Glyptosternum maculatumAll of the enriched library clones constructed by biotin-（CA）₁₅,there were 720 clones containing mierosatellite sequence（two or three bands in PCR results）,145 clones were randomly selected to sequence,among which,139 sequences had the microsatellte sequence repeats above 7,15 sequences were the same.The repeats ranged from 8 to 165, on the average of 52.Among 124 microsatellite sequences,there were 80 of perfect,40 of imperfect and 14 of compound categories,part of sequences had been registered in Genebank.4.The screening of SSR markers for Glyptosternum maculatumThirty-three microsatellite primers were designed with their amplification conditions optimized,there were 29 primers amplified products and 25 one were polymorphism.The number of alleles per locus（A） was 2-10 with an average of 4.6,the observed heterozygosity（H_o） ranged from 0 to 1.The ranges of PIC varied from 0.2846 to 0.8173 with an average of 0.5793,which indicated their applicability for genetic analysis.5.The cross-amplification in other related taxa of SSR markers for Glyptosternum maculatumAll of the 14 primer pairs were revealed polymorphism for cross-species amplification in P.sulcatus（McClelland） using eighteen individuals.The number of alleles per loci ranged from 2 to 7（3.85 on the average） with the expected heterozygosity （H_E） ranging from 0.3476 to 0.8571（0.6054） and the observed heterozygosity（H_o） varying from 0 to 1（0.5992）.The results suggested that there were 14 SSR marker developed from Glyptosternum maculatum successfully amplifying in other taxa Pseudechermeis sulcatus（McClelland）.更多还原