绿豆高密度分子遗传图谱的构建

doi:10.3864/j.issn.0578-1752.2014.11.003

摘要/Abstract

摘要： 【目的】在前期研究的基础上，进一步利用绿豆基因组SSR、EST-SSR、STS和普通菜豆基因组SSR等标记构建绿豆遗传连锁图谱，为绿豆重要性状相关基因的定位、克隆及分子标记辅助选育新品种等研究搭建技术平台。【方法】利用澳大利亚引进的Berken（高感豆象绿豆栽培种）× ACC41（高抗豆象绿豆野生种）及其重组自交系（recombinant inbreed line，RIL）群体，对6 686对引物进行PCR扩增及多态性筛选，包括6 100对绿豆基因组SSR、149对EST-SSR、13对STS和424对普通菜豆基因组SSR引物，将亲本间多态性引物，进一步分析重组自交系群体。结合前期研究的分子标记数据，利用Mapmarker/Exp 3.0软件构建遗传图谱，并设置LOD≥3.0，最大图距50.00 cM。用Joinmap 4.0软件进行图谱整合。【结果】用2个亲本共筛选了6 686对SSR引物，共有3 691对引物有稳定的扩增产物，得到有多态的引物有588对。其中，通过磁珠富集法开发的绿豆SSR引物6 100对，有效扩增3 459对，有效扩增率56.7%，得到多态性引物559对；通过转录组测序开发的绿豆MGCP引物149对，有效扩增126对，有效扩增率84.6%，得到多态性引物21对；通过磁珠富集法开发的菜豆SSR引物424对，有效扩增97对，有效扩增率22.9%，得到多态性引物6对；绿豆STS引物13对，有效扩增9对，有效扩增率69.2%，得到多态性引物2对。表明不同来源和种类的SSR引物在RIL群体亲本中的有效扩增率有明显差别，绿豆EST-SSR引物（84.6%）最高，绿豆STS引物（69.2%）和SSR引物（55.7%）次之，菜豆SSR引物（22.9%）最低。获得一张含有585个标记（499个SSR标记、74个RFLP标记、9个STS标记和3个RAPD标记）的绿豆遗传图谱，图谱总长732.9 cM，包括11个连锁群，每个标记间的平均距离为1.25 cM，平均长度为66.63 cM。每个连锁群长度为45.2—112.8 cM，每条染色体上面的标记数为35—92个，平均53.18个。标记位点数最多的连锁群LG1含92个标记，长度为112.8 cM；标记位点数最少的连锁群LG11仅含有35个标记，长度为48.7 cM。对图谱的585个标记位点进行χ2测验，在P＜0.05和P＜0.01条件下，分别有79个和151个标记表现为偏分离，占总标记位点数的39.3%。【结论】构建了一张目前国内外发表的标记数最多、密度最高的绿豆遗传连锁图谱。

关键词: 绿豆 , 遗传连锁图谱 , 标记

Abstract: 【Objective】On the basis of previous studies, a genetic map of mungbean was constructed by using genome SSR, EST-SSR, STS primers of mungbean and common bean to build a platform for positioning important traits related genes, cloning and molecular marker-assisted breeding of new varieties of mungbean.【Method】A total of 6 686 SSR, EST-SSR, STS primers of mungbean and common bean, including 6 100 genome SSR, 149 EST-SSR, 13 STS primer pairs of mungbean, and 424 genome SSR primers of common bean, were used for PCR amplification to screen polymorphic markers between Australia-imported Berken (highly susceptible cultivar)×ACC41 (highly resistant wild species) and a RIL derived from these two genotypes were tested with the polymorphic markers. Combined with the molecular marker data of previous studies, Mapmarker/Exp 3.0 software was used for map construction and set LOD≥3.0, the maximum figure at 50.00 cM. Finally, Joinmap 4.0 software was used for map integration.【Result】In this study, from the two parents, 6 686 SSR primers were screened, a total of 3 691 pairs of primers were amplified stable products, 588 pairs of polymorphic primers were obtained. Among them, mungbean SSR primers 6 100 pairs, effective amplification 3 459 pairs, the effective rate of 56.7%, obtained 559 pairs of polymorphic primers; Mungbean EST-SSR primers 149 pairs, effective amplification 126 pairs, the effective rate of 84.6%, obtained 21 pairs of polymorphic primers; Common bean SSR primers 424 pairs, effective amplification 97 pairs, the effective rate of 22.9%, obtained 6 pairs of polymorphic primers; Mungbean STS primers 13 pairs, effective amplification 9 pairs, the effective rate of 69.2%, obtained 2 pairs of polymorphic primers; These results indicated that different sources and types of SSR primers to amplify the effective rate of the parent were significantly different. Mungbean EST-SSR primers (84.6%) were the highest, mungbean STS primers (69.2%) and mungbean SSR primers (55.7%) followed, common bean SSR primers (22.9%) were the lowest. An integrated genetic linkage map of mungbean containing 585 markers was constructed (including 499 SSR markers, 74 RAPD markers, 9 STS markers and 3 RAPD markers). The total length of the map was 732.9 cM and covered 11 linkage groups. The average distance between markers was 1.25 cM. The average distance of each linkage group spanned 66.63 cM. The average number of markers was 53.18 for each of 11 chromosomes. The length of each linkage group ranged from 35 to 92 markers was from 45.2 cM to 112.8 cM. LG1 linkage group contained the largest number of 92 markers, the length was 112.8 cM. LG11 linkage group contained the minimum number of 35 markers, the length was 48.7 cM. Of the 585 markers loci mapping conducted χ2 tests under P＜0.05 and P＜0.01 conditions, respectively, 79 and 151 markers showed a segregation distortion, the total number of sites marked 39.3%.【Conclusion】A linkage map of mungbean with a maximum and the highest density genetic markers was constructed compared with that published at home and abroad at present.

Key words: mungbean , genetic linkage map , marker

吴传书1, 2, 王丽侠2, 王素华2, 陈红霖2, 吴健新2, 程须珍2, 杨晓明1, 3. 绿豆高密度分子遗传图谱的构建[J]. 中国农业科学, 2014, 47(11): 2088-2098.

WU Chuan-Shu-1, 2 , WANG Li-Xia-2, WANG Su-Hua-2, CHEN Hong-Lin-2, WU Jian-Xin-2, CHENG Xu-Zhen-2, YANG Xiao-Ming-1, 3 . Construction of a Genetic Linkage Map in Mungbean[J]. Scientia Agricultura Sinica, 2014, 47(11): 2088-2098.

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