核桃早实基因的RAPD标记及其序列分析研究

RAPD Marker Linked to Precocious Gene of Walnut (Juglans regia L.) and Its Sequence Analysis

【目的】确定RAPD标记OPB-08900与核桃早实基因的遗传距离,对该标记测序,分析其序列特征。【方法】以核桃早实优系绿园和晚实、速生优系绿丰及绿园×绿丰杂交组合的154株F1杂种后代材料的DNA为模版,OPB-08(5′-GTCCACACGG-3′)为引物,PRAD-PCR技术检测与核桃早实基因相关的RAPD标记OPB-08900在供试亲本和F1群体的分离状况,Haldane函数计算遗传距离;对OPB-08900DAN片段克隆测序;DNAMAN软件分析序列酶切位点,BLAST分析序列同源性。【结果】RAPD标记OPB-08900在早实亲本绿园上标记带出现,在70株早实后代的69株上出现;但在晚实亲本绿丰上该标记带未出现,在84株晚实后代中有82株未出现;供试杂交群体早实和晚实性状的分离比例符合1﹕1的分离比例,RAPD标记OPB-08900与核桃早实基因共分离,OPB-08900与核桃早实基因的遗传距离为1.99cM,序列全长958bp,该标记应记为OPB-08958,GenBank序列号为DQ673614;有36个限制性内切酶对OPB-08958序列有97个酶切位点,OPB-08958序列和植物基因组序列没有同源性。【结论】核桃的早实性状受多基因控制,RAPD标记OPB-08958与核桃早实基因之一相连锁,其DNA序列为核桃基因组所独有。

【Objective】The aim is to characterize the OPB-08900 sequence and to identify the genetic distance between RAPD marker OPB-08900 and precocious gene of walnut（Juglans regia L.）.【Method】The template DNA extracted from precocious maternal parent Lüyuan,later mature paternal parent Lüfeng and their 154 F1 hybrid plants was screened with OPB-08 primer（5＇-GTCCACACGG-3＇）.The genetic distance between OPB-08900 and the precocious walnut gene was calculated using Haldane function.DNA fragments of OPB-08900 derived from precocious F1 hybrid No.2 were retrieved from the agarose gel.After ligated into pMD18-T Vector,the DNA was transformed into E.coli JM109 and the positive clones were screened.The restriction sites and homologue of the sequence OPB-08958 were analyzed respectively by DNAMAN and BLAST in nr database of GenBank.【Result】 The RAPD-PCR results showed that specific fragment OPB-08900 was presented in precocious maternal parent Lüyuan and 69 precocious F1 hybrid plants,but it was absent in later mature paternal parent Lüfeng and 82 later mature F1 hybrid plants.The RAPD marker OPB-08900 was co-segregating with precocious trait of walnut and the genetic distance between OPB-08900 and precocious gene was 1.99 cM.The sequencing results showed that the RAPD marker OPB-08900 was 958 bp,so RAPD marker OPB-08900 should be renamed OPB-08958 with a GenBank accessioned number of DQ673614.There were 97 restriction sites in the sequence of OPB-08958,which could be digested by 36 restriction enzymes.The sequence of OPB-08958 was not homologous with any accessioned plants in GenBank.【Conclusion】The precocious trait of walnut might be controlled by more than 2 genes.RAPD marker OPB-08958 might link to one of the precocious genes and be unique to walnut genome.