牙鲆抗淋巴囊肿病相关SSR标记的筛选与鉴定

为了筛选出中国牙鲆群体的抗淋巴囊肿病相关SSR标记,实验采用分群分析法对102个牙鲆个体(感病56个,抗病46个)进行了抗淋巴囊肿病相关SSR标记的筛选。首先构建了抗、感基因池(抗病个体和感病个体各15个),并利用178对微卫星引物对其进行了扫描;对于在抗、感池间扩增出差异条带的引物,用构建基因池的30个个体对其进行了第一次单个体验证;对于在第一次单个体验证中差异显著的引物用102个个体进行了第二次验证。结果显示,有4对引物(scaffold440_22585、scaffold826_5003、scaffold703_4284和scaffold185_597)在抗、感基因池间扩增出了差异条带;经第一次单个体验证,表明scaffold826_5003(P=0.023)和scaffold185_597(P178bp=0.028,P173bp=0.009)的差异条带在抗、感个体中呈显著性差异;经第二次单个体验证,发现scaffold185_597的差异条带在抗、感个体中出现的频率分别为60.9%和14.3%(P=0.001),差异显著;对scaffold185_597在10个抗病个体中扩增出的差异条带进行了克隆并测序,经BLAST比对,证实其为黄海水产研究所水产基因组与细胞工程研究室公布的牙鲆微卫星标记scaffold185_597中的一段,同源性达到96%。研究表明,微卫星标记scaffold185_597可能与牙鲆抗淋巴囊肿病相关。

Screening and identification of SSR markers associated with lymphocystis disease resistance in Japanese flounder(Paralichthys olivaceus)

In order to screen out lymphocystis disease resistance-related SSR markers in Chinese flounder population, 102 individuals were used(56 disease susceptible and 46 disease resistant individuals) in the present study.Firstly, two gene pools were constructed using 15 susceptible individuals and 15 resistant individuals, respectively.The two gene pools were scanned using 178 pairs of microsatellite primers.Secondly, the differential bands amplified in gene pools were verified using 30 individuals which were used to construct the gene pools for the first time.Lastly, a second verification for 102 individuals was performed to verify the difference significance of the differential bands identified in the first verification.Results of BSA analysis demonstrated that some differential bands were amplified using four pairs of primers(scaffold440_22585, scaffold826_5003, scaffold703_4284 and scaffold185_597).The first verification indicated that the differential bands are significantly different in the SSR markers scaffold826_5003(P=0.023) and scaffold185_597(P_178bp=0.028, P_173bp=0.009).And in the second verification, the difference was extremely significant in the totally 102 individuals with the SSR marker scaffold185_597, the frequency of differential bands in disease resistant individuals and disease susceptible individuals was 60.9% and 14.3%, respectively(P=0.001<0.01).The differential bands amplified from ten disease resistant individuals using primer scaffold185_597 were cloned and sequenced.Sequence alignment by BLAST confirmed that the bands were fragments of microsatellite marker scaffold185_597 published by lab for aquatic genomics and cell engineering in Yellow Sea Fisheries Research Institute, homology of which was up to 96%.The present study has shown that the microsatellite marker scaffold185_597 may be associated with lymphocystis disease resistance in Japanese flounder(Paralichthys olivaceus), providing some basis for the molecular marker-assisted selection in Chinese flounder.