核桃早实基因的RAPD标记及其序列分析研究

 【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-08900 DAN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1.99 cM，序列全长958 bp，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。

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

Abstract：【OBJECTIVE】To identify the genetic distance between RAPD marker OPB-08900 and precocious gene of walnut (Juglans regia L.), and character the sequence of OPB-08900; 【METHOD】The template DNA extracted from precocious maternal parent Lüyuan、later mature paternal parent Lüfeng and 154 F1 hybrid plants were screened with OPB-08 primer (5′GTCCACACGG 3′). The genetic distance between OPB-08900 and precocious gene of walnut was calculated using Haldane function .DNA fragments of OPB-08900 derived from precocious F1 hybrid No.2 was retrieved from agarose gel and ligated into pMD18-T Vector, and then transformed into E. coli JM109. Sequencing of positive clones was carried out .The restriction sites of sequence OPB-08958 analyzed by DNAMAN and BLAST in nr database of GenBank;【RESULTS】The RAPD-PCR result showed that specific fragment OPB-08900 was presented in precocious maternal parent Lüyuan and 69 F1 hybrid plants, but was absented in later mature paternal parent Lüfeng and 82 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 result showed the RAPD marker OPB-08900 was 958bp, so RAPD marker OPB-08900 should be renamed OPB-08958，GenBank accession number is DQ673614.There were 97 restriction sites in the sequence of OPB-08958, digested by 36 restriction enzymes; The sequence of OPB-08958 was not homologous with any plants in GenBank;【CONCLUSION】The precocious trait of walnut is controlled by more than 2 genes which were dominance. RAPD marker OPB-08958 linked to one of the precocious genes and the sequences of OPB-08958 is unique to Walnut genome.