麻疯树EST-SSR和SSR标记开发及其应用
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摘要
麻疯树是新兴能源植物,属于大戟科多年生灌木,起源于中南美洲,广泛分布于全球热带亚热带地区。野生或半栽培的麻疯树能够在干热的气候和贫瘠的土壤条件下生长。麻疯树的种仁含有高比例的油酸和亚油酸,这些不饱和脂肪酸具有半干性特性,与石化柴油性能相近,可以开发作为生物柴油利用。近年来,利用麻疯树发展生物柴油使用潜力的经济价值逐渐被人们重视。
但目前对麻疯树的资源收集和相关基础性研究非常有限,本研究通过对大戟科木薯的SSR引物在麻疯树的通用性检测,成功转化241对麻疯树SSR引物,并利用其中56对引物评价了本实验室所收集的45份麻疯树的遗传多样性。同时,利用这些SSR构建了麻疯树的初级分子标记遗传连锁图谱。具体结果如下：
1.本研究共获得302个在麻疯树中有特异扩增产物EST-SSR和SSR标记,241个在5个供试品系中具有多态性。其中EST-SSR标记187个,SSR标记54个,全部多态性标记的引物序列、扩增参数均登录在Genebank数据库共享。这些具有多态性的EST-SSR的核苷酸碱基重复类型分别是双核苷酸重复类型(26.20%)、三核苷酸重复类型(57.75%)、四核苷酸重复类型(8.56%)和五核苷酸重复类型(7.49%)。此外,在具有多态性的SSR中,主要是双核苷酸重复类型(62.96%)。
2.利用36对EST-SSR和20对SSR引物分析45份麻疯树的遗传多样性,总共获得183个多态性位点,基于这些多态性位点将45份麻疯树种质分为6个类群(类群Ⅰ、类群Ⅱ、类群Ⅲ、类群Ⅳ、类群Ⅴ和类群Ⅵ),与其地理起源基本相似。类群内的遗传多样性指数在0.4099到0.5072之间,物种水平的遗传多样性指数为0.5572,表明本实验室收集的麻疯树资源具有较高的遗传多样性水平。
3.用遗传差异较大的云南株系YN049和海南株系H001-31-1杂交,构建了包含240个F1单株的分离群体,试图构建麻风树的分子遗传图谱。初步选用31对EST-SSR引物对作图群体进行了遗传多态性检测,共获得83个多态性位点,其中45个满足1：1或3：1的分离比例。利用JoinMap3.0软件进行连锁群分析,获得3个较大的连锁群,共包含21个SSR标记,该连锁群的总图距为158.67cM,标记间的平均图距为7.56cM,相关工作有待进一步完善。
Jatropha curcas (L.) is a newly energy plant and perennial shrub, belonging to the Euphorbiaceae family. This species is native of Central and South America but widely distributed in the tropics and subtropical area in the world. J. curcas can grow well under xerothermic climatic and sterile soil conditions by the form of wild or semi-cultivated type. The kernel contain high percentage of oleic and linoleic acid, and these unsaturated fatty acid has a semi-drying property, closing to petrochemical diesel fuel property, it may be developed as biodiesel. In recent years, the economic importance of J. curcas has been increasingly recognized as the potential use for biodiesel.
But our understanding on this crop remains very limited as germplasm collection and basal research work are very limited. The present study aimed to identify a set of SSR markers transferability available from Manihot esculenta (cassava), a species in the same Eurphorbiacae family as Jatropha.241 SSR markers are transferred successfully,56 of which are used to study genetic diversity among 45 Jatropha accessions from collections in our germplasm bank and construct a primary molecule marker genetic linkage map of J. curcas. The results are addressed in this paper:
1. A total of 302 EST-SSR and SSR markers were found to be amplifiable in J. curcas. Of them,241 SSR markers were polymorphic in five tested lines. In these polymorphic markers, including 187 EST-SSR markers and 54 SSR markers, their primer sequences and parameters of amplification are logged in the Genebank Database. Analysis of the nucleotide sequences of the polymorphic EST-SSRs and SSRs showed that, in J. curcas, the EST-SSRs corresponded to 57.75% trinucleotide repeats,26.20% dinucleotide repeats,8.56% pentanucleotide repeats, and 7.49% tetranucleotide repeats. In contrast, the SSRs were composed mainly of dinucleotide repeats (62.96%).
2. Thirty-six EST-SSRs and twenty SSRs were chosen for detection of genetic diversity in 45 Jatropha accessions, and a total of 183 polymorphic loci have been revealed in the population. Based on the data of polymorphic loci, these germplasm were grouped into 6 clusters (including clusterⅠ, clusterⅡ, clusterⅢ, clusterⅣ, clusterⅤand clusterⅥ), which generally reflect their differences by the geographic origin. The intergroup genetic diversity index ranged from 0.4099 to 0.5022, and an estimated average of genetic diversity index (Ⅰ) reached to 0.5572 suggested a considerable high genetic diversity in the Jatropha germplasm collection.
3. A SSR linkage map is constructed using two high genetic diversity clones cross Yunnan clone YN049 and Hainan clone H001-31-1. The F1 segregating popultion is composed of 240 seedlings. Initially,31 EST-SSR primers were seclected to detect genetic polymorphism in the popultion. There are 45 loci accord to 1:1 or 3:1 segregation ratio among 83 polymorphic loci. The map is consist of 21 markers and be divided to 3 linkage groups by JoinMap3.0 software. The total distance of the map is 158.67cM. The average interval is 7.56cM. The genetic linkage map must be further completed in the future.
但目前对麻疯树的资源收集和相关基础性研究非常有限,本研究通过对大戟科木薯的SSR引物在麻疯树的通用性检测,成功转化241对麻疯树SSR引物,并利用其中56对引物评价了本实验室所收集的45份麻疯树的遗传多样性。同时,利用这些SSR构建了麻疯树的初级分子标记遗传连锁图谱。具体结果如下：
1.本研究共获得302个在麻疯树中有特异扩增产物EST-SSR和SSR标记,241个在5个供试品系中具有多态性。其中EST-SSR标记187个,SSR标记54个,全部多态性标记的引物序列、扩增参数均登录在Genebank数据库共享。这些具有多态性的EST-SSR的核苷酸碱基重复类型分别是双核苷酸重复类型(26.20%)、三核苷酸重复类型(57.75%)、四核苷酸重复类型(8.56%)和五核苷酸重复类型(7.49%)。此外,在具有多态性的SSR中,主要是双核苷酸重复类型(62.96%)。
2.利用36对EST-SSR和20对SSR引物分析45份麻疯树的遗传多样性,总共获得183个多态性位点,基于这些多态性位点将45份麻疯树种质分为6个类群(类群Ⅰ、类群Ⅱ、类群Ⅲ、类群Ⅳ、类群Ⅴ和类群Ⅵ),与其地理起源基本相似。类群内的遗传多样性指数在0.4099到0.5072之间,物种水平的遗传多样性指数为0.5572,表明本实验室收集的麻疯树资源具有较高的遗传多样性水平。
3.用遗传差异较大的云南株系YN049和海南株系H001-31-1杂交,构建了包含240个F1单株的分离群体,试图构建麻风树的分子遗传图谱。初步选用31对EST-SSR引物对作图群体进行了遗传多态性检测,共获得83个多态性位点,其中45个满足1：1或3：1的分离比例。利用JoinMap3.0软件进行连锁群分析,获得3个较大的连锁群,共包含21个SSR标记,该连锁群的总图距为158.67cM,标记间的平均图距为7.56cM,相关工作有待进一步完善。
Jatropha curcas (L.) is a newly energy plant and perennial shrub, belonging to the Euphorbiaceae family. This species is native of Central and South America but widely distributed in the tropics and subtropical area in the world. J. curcas can grow well under xerothermic climatic and sterile soil conditions by the form of wild or semi-cultivated type. The kernel contain high percentage of oleic and linoleic acid, and these unsaturated fatty acid has a semi-drying property, closing to petrochemical diesel fuel property, it may be developed as biodiesel. In recent years, the economic importance of J. curcas has been increasingly recognized as the potential use for biodiesel.
But our understanding on this crop remains very limited as germplasm collection and basal research work are very limited. The present study aimed to identify a set of SSR markers transferability available from Manihot esculenta (cassava), a species in the same Eurphorbiacae family as Jatropha.241 SSR markers are transferred successfully,56 of which are used to study genetic diversity among 45 Jatropha accessions from collections in our germplasm bank and construct a primary molecule marker genetic linkage map of J. curcas. The results are addressed in this paper:
1. A total of 302 EST-SSR and SSR markers were found to be amplifiable in J. curcas. Of them,241 SSR markers were polymorphic in five tested lines. In these polymorphic markers, including 187 EST-SSR markers and 54 SSR markers, their primer sequences and parameters of amplification are logged in the Genebank Database. Analysis of the nucleotide sequences of the polymorphic EST-SSRs and SSRs showed that, in J. curcas, the EST-SSRs corresponded to 57.75% trinucleotide repeats,26.20% dinucleotide repeats,8.56% pentanucleotide repeats, and 7.49% tetranucleotide repeats. In contrast, the SSRs were composed mainly of dinucleotide repeats (62.96%).
2. Thirty-six EST-SSRs and twenty SSRs were chosen for detection of genetic diversity in 45 Jatropha accessions, and a total of 183 polymorphic loci have been revealed in the population. Based on the data of polymorphic loci, these germplasm were grouped into 6 clusters (including clusterⅠ, clusterⅡ, clusterⅢ, clusterⅣ, clusterⅤand clusterⅥ), which generally reflect their differences by the geographic origin. The intergroup genetic diversity index ranged from 0.4099 to 0.5022, and an estimated average of genetic diversity index (Ⅰ) reached to 0.5572 suggested a considerable high genetic diversity in the Jatropha germplasm collection.
3. A SSR linkage map is constructed using two high genetic diversity clones cross Yunnan clone YN049 and Hainan clone H001-31-1. The F1 segregating popultion is composed of 240 seedlings. Initially,31 EST-SSR primers were seclected to detect genetic polymorphism in the popultion. There are 45 loci accord to 1:1 or 3:1 segregation ratio among 83 polymorphic loci. The map is consist of 21 markers and be divided to 3 linkage groups by JoinMap3.0 software. The total distance of the map is 158.67cM. The average interval is 7.56cM. The genetic linkage map must be further completed in the future.
引文
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