利用EST-SSR、IT-ISJ分子标记研究四倍体与八倍体柳枝稷的遗传多样性

Genetic diversity assessment of tetraploid and octoploid switchgrass using EST-SSR and IT-ISJ molecular markers

柳枝稷是重要的能源植物之一,为研究四倍体Alamo与通过秋水仙素加倍后的八倍体之间的遗传差异,本研究以65份四倍体Alamo与其诱变的八倍体为研究材料,通过EST-SSR与IT-ISJ两种分子标记进行遗传分析。结果表明,EST-SSR引物共扩增出245条带,多态性比率为99.59%,观测等位基因Na为1.9878,有效等位基因Ne为1.1718,基因多样性指数H为0.1308,Shannon信息指数I为0.2388,其中四倍体Alamo的各遗传参数(226条,88.93%,1.8816,1.1823,0.1349,0.2398)多高于八倍体Alamo(217条,92.24%,1.9184,1.1652,0.1228,0.2234)。IT-ISJ引物共扩增出165条条带,多态性比率为96.36%,观测等位基因Na为1.9878,有效等位基因Ne为1.5777,基因多样性指数H为0.3354,Shannon信息指数I为0.4999,四倍体Alamo各项指数(145条,94.55%,1.9333,1.6366,0.3602,0.5280)均高于八倍体Alamo(139条,84.24%,1.7515,1.4377,0.2573,0.3864)。通过UPGMA分析表明,65个柳枝稷材料中四倍体与八倍体没有明显区分开,表明其没有产生明显的遗传分化。通过AMOVA分析表明,EST-SSR和IT-ISJ的遗传变异分别有94.78%和80.76%发生在倍性内,有5.22%和19.24%发生在倍性间,表明柳枝稷倍性间没有明显的差异。

Switchgrass is an important bioenergy plant. In this study, the genetic diversity of 65 samples of tetraploid Alamo and mutated octoploid Alamo （using colchicine） was assessed using expressed sequence tag-simple sequence repeat （EST-SSR） and intron-targeted intron-exon splice junction （IT-ISJ） markers. The results showed that EST-SSR primers amplified 245 fragments; the percentage of polymorphic loci was 99.59%, the number of alleles （Na） was 1.9878, the effective number of alleles （Ne） was 1.1718, Nei’s gene diversity （H） was 0.1308, and Shannon’s information index （I） was 0.2388. These genetic parameters for the tetraploid Alamo were as follows： 226 fragments; 88.93% polymorphic loci; Na, 1.8816; Ne, 1.1823; H, 0.1349; and I, 0.2398. These values were higher than those obtained for octoploid Alamo： 217 fragments; 92.24% polymorphic loci; Na, 1.9184; Ne, 1.1652; H, 0.1228; and I, 0.2234. The IT-ISJ primers amplified 165 fragments and the percentage of polymorphic loci was 96.36%, Na was 1.9878, Ne was 1.5777, H was 0.3354, and I was 0.4999. These parameters for tetraploid Alamo were as follows： 145 fragments; 94.55% polymorphic loci; Na, 1.9333; Ne, 1.6366; H, 0.3602; and I, 0.5280. These values were higher than those obtained for octoploid Alamo： 139 fragments; 84.24% polymorphic loci; Na, 1.7515; Ne, 1.4337; H, 0.2573; and I, 0.3864. A clustering analysis with the unweighted pair-group method with arithmetic means did not clearly separate 68 samples of tetraploid Alamo and octoploid Alamo, which had a low level of genetic differentiation. An analysis of molecular variance showed that 94.78% and 80.76% of genetic diversity occurred within ploidy levels, while 5.22% and 19.24% of genetic diversity occurred between ploidy levels.