彭泽鲫两个雌核发育克隆的RAPD分析

MOLECULAR ANALYSIS BETWEEN TWO GYNOGENETIC CLONES OF PENGZE CRUCIAN CARP,CARASSIUS AURATUS OF PENGZE,BASED ON DETECTION OF RAPD MARKERS

采用随机扩增多态DNA技术,对彭泽鲫种群内两个不同雌核发育克隆进行比较分析。在使用的30个随机引物中有28个引物的扩增效果良好,两个克隆各5个样本共产生清晰可辨的扩增带2154条(其中克隆H为1088条,克隆L为1066条)。克隆H群体内的平均遗传距离为0.018070±0.015489,克隆L群体内的平均遗传距离为0.017827±0.015203,两个克隆群体间的平均遗传距离为0.352511±0.009692,群体内和群体间的遗传距离清晰地表明了同一雌核发育克隆群体的遗传相似性和不同克隆群体间的遗传异质性。由NJ聚类法构建的分支树状图也很好地反映了两个雌核发育克隆群体及个体间的相互关系。在28个引物对两个克隆的扩增带中,一共可找到91条多态性片段,一些引物如Opj15、Opp8、Opq11和Opq14等,产生了非常丰富、稳定的多态性标记,可以作为两个雌核发育克隆的有效鉴别指标。彭泽鲫两个雌核发育克隆在分子水平上的差异进一步证实了彭泽鲫种群的遗传多样性,与银鲫相类似,彭泽鲫种群的遗传多样性对单性脊椎动物的进化理论研究和彭泽鲫的选种育种实践都具有重要的意义。

Pengze crueian carp （ Carassius auratus of pengze）, a popular cultural fish in China, is an endemic bisexual population with natural gynogenetic reproduction mode. Interestingly, natural populations of this fish consist of a minor portion （no more than 20% ） of males. It was reported that Pengze crucian carp had a high genetic kinship to the clone A in the silver crucian carp, and belonged to one of clones of the silver crucian carp. However, the previous investigations on the morphology and cytogenetics indicated that there were two distinguishable gynogenetic clones of pengze crucian carp, named clone H and clone L, respectively. It is necessary for using direct molecular evidence to prove if the population of Pengze crucian carp includes some gynogenetic clones. In this study, the two clones（ H and L） of gynogenetic pengze crucian carp were analyzed by the random amplified polymorphic DNA （RAPD） technique, using 30 10-nucleotide-long random primers, and twenty-eight primers produced well-amplified DNA fragments. Similar banding patterns were observed among individuals within each gynogenetic clone, whereas obvious differences were detected between the two gynogenetic clones. Using these informative band patterns produced by the 28 primers, all individual, a total of 2,154 distinguishable DNA fragments（215 per individual）, including universal and specific, with data coded as a vector of 1 or 0, representing band presence or absence, respectively. Genetic distance matrices among these individuals of two clones were estimated based on the method of Lynch. The results indicated that the genetic distances of clone H are 0. 013761-0. 044084, clone L are 0. 014085-0. 046948, and between clone H and clone L are 0. 333333-0.368910. The average genetic distances within clone H and L were 0.018070 ± 0.015489 and 0.017827 ± 0.015203, respectively, and which was 0. 352511 ± 0.009692 between the two clones. Average genetic distances within and between the two clones clearly indicated their intraclonal homogeneity and interclonal heterogeneity. A total of 91 polymorphic fragments were scored from 28 primers after excluding bands that were monomorphic for the two clones. These polymorphic fragments showed the genetic diversity of the population of Pengze crucian carp. Some primers, for example Opj15, Opp8, Opqll, Opql4 and so on, produced abundant and reproducible banding patterns that could be efficiently used to discriminate between the two gynogenetic clones. At the same time, according to the genetic distance among these individuals of two clones, a phylogenetic tree was constructed using Neighbor-Joining program in MEGA3.1. The dendrogram of 10 individuals in the two clones showed their origin and relationship. All individuals in the clone H were clustered into one group, and all individuals from the clone L were clustered into another group. The difference based on detection of RAPD molecular markers provided more evidence for the genetic diversity of Pengze crucian carp, and identified two gynogenetic clones were obviously existent in the population of Pengze crucian carp. This study results further indicate that it is a general phenomena with natural gynogenetic fishes including a lot of gynogenetic clones. As the silver crucian carp, the genetic diversity of the gynogenetic Pengze crucian carp will likely benefit evolutionary genetics studies of unisexual vertebrates and selective breeding of Pengze erucian carp.