南方红豆杉EST-SSR标记的开发及其在父本分析中的应用
发布时间：2018-09-06 17:27
【摘要】：南方红豆杉(Taxus chinensis var.mairei)是我国特有珍稀濒危树种,其基因组信息匮乏,分子遗传学研究相对滞后。目前,南方红豆杉可有效利用的分子标记数量极其有限,远不能满足遗传学和育种的研究要求。本研究以NCBI数据库中的4种红豆杉转录组序列为基础,利用生物信息学软件,开发南方红豆杉EST-SSR分子标记,检验多态性位点在红豆杉种间扩增情况,并将其应用于南方红豆杉父本分析。相关研究结果对红豆杉属物种保护和分子育种具有重要意义。主要研究结果如下:(1)分析了 4种红豆杉转录组序列特征。以NCBI数据库中4种红豆杉转录组序列为基础,利用生物信息学软件对转录组序列进行分析,分别得到南方红豆杉(Taxus chinensis var.mairei)、东北红豆杉(Taxus cuspidata)、欧洲红豆杉(Taxus baccata)和曼地亚红豆杉(Taxus media)SSR位点2 344、2 361、1 357和2 312个。在2-6bp重复单元的微卫星中,南方红豆杉和曼地亚红豆杉以六核苷酸重复基元为主,其次为三核苷酸;东北红豆杉和欧洲红豆杉以三核苷酸为主,其次为六核苷酸。4种红豆杉,二核苷酸、三核苷酸、六核苷酸优势重复基序类型均为AG/CT、AAG/CTT、AAGAGG/CCTCTT。(2)开发得到112个红豆杉属多态性位点。以4种红豆杉位点信息为基础,选择重复单元数大于或等于9的位点合成150对引物。基因分型结果表明,112对引物具有多态性,多态性比率达到74.67%。其中,南方红豆杉、东北红豆杉、欧洲红豆杉和曼地亚红豆杉中各开发得到59、17、13和23个多态性位点。(3)开发得到103个南方红豆杉多态性位点。利用16个单株样本对103个EST-SSR位点进行遗传参数分析,共检测到462个等位基因,每位点等位基因数(Na)变化范围为2-11,平均每个位点的等位基因数为4.41。各位点观测杂合度(Ho)的变化区间为0-1,平均值为0.459;期望杂合度(He)的变化区间为0.0625-0.891,平均值为0.513;近交系数(FIS)的变化区间为-0.654-1,平均值为0.085。多态性信息含量(PIC)变化区间为0.059-0.849,平均多态性信息含量为0.453。14个位点存在无效等位基因(Oosterhout值),频率变化区间为0.135-0.470;22个位点偏离哈代-温伯格平衡(Hardy-Weinberg equilibrium)。(4)分析了 107个位点在5种红豆杉中的种间扩增情况。其平均扩增率为96.07%。其中,只有一个位点(Ma-25497-83A)没有扩增,4个位点(C-52641-193B,Ma-1402-721B,Ma-186-234D,Ma-6383-968B)的扩增率小于 50%,100 个位点的种间扩增率达到100%。(5)检验了开发位点的有效性和准确性。48株红豆杉样本的主成分分析(PCA)结果与种的分类相一致,6种红豆杉属的聚类分析结果与其它标记结果基本类似。(6)筛选10个多态性丰富的位点对三个南方红豆杉群体进行父本分析。CERVUS软件在95%的置信度可确定祁阳群体、攸县群体、八大公山群体的自由授粉父本各12个(占总子代的37.50%)、21个(占总子代的84.00%)、7个(占总子代的18.91%),三个群体候选父本的累积排除率分别达到了 98.32%,93.80%和97.40%。父本分析结果表明南方红豆杉有效花粉散布距离为10-25m。
[Abstract]:Taxus chinensis var. mairei is a rare and endangered tree species in China. Its genome information is scarce and molecular genetics research is relatively lagging behind. At present, the number of molecular markers available in Taxus chinensis var. mairei is extremely limited, far from meeting the requirements of genetics and breeding. In this study, four species of Taxus chinensis var. mairei in NCBI database were used. Based on the transcriptome sequence of Taxus chinensis, EST-SSR molecular markers were developed by using bioinformatics software to test the amplification of polymorphic loci in Taxus chinensis, and applied to the analysis of male parents of Taxus chinensis. (1) The transcriptome sequence characteristics of four Taxus species were analyzed. Based on the transcriptome sequences of four Taxus species in NCBI database, the transcriptome sequences were analyzed by bioinformatics software. The transcriptome sequences were obtained from Taxus chinensis var. mairei, Taxus cuspidata, Taxus baccata and Taxus mandiana, respectively. There were 2 344,2 361,1 357 and 2 312 SSR loci in Taxus media. Among the 2-6 BP repeat units, Taxus chinensis var. mairei and Taxus mandii were dominated by hexanucleotide repeat units, followed by trinucleotides; Taxus chinensis var. mairei and Taxus chinensis var. mairei were dominated by trinucleotides, followed by hexanucleotides. The dominant repeat motifs of acid and hexanucleotide were AG/CT, AAG/CTT, AAGAGG/CCTCTT. (2) 112 polymorphic loci of Taxus were developed. Based on the information of four Taxus loci, 150 pairs of primers were synthesized by selecting loci with repeat units greater than or equal to 9. Among them, 59, 17, 13 and 23 polymorphic loci were developed respectively in Taxus chinensis var. mairei, Taxus cuspidata var. mairei, Taxus cuspidata var. mairei and Taxus mandii. (3) 103 polymorphic loci were developed. A total of 462 alleles were detected in 103 EST-SSR loci using 16 single plant samples. The variation range of allele number (Na) was 2-11, and the average number of alleles per locus was 4.41. The variation range of observed heterozygosity (Ho) was 0-1, with an average value of 0.459; the variation range of expected heterozygosity (He) was 0.0625-0.891, with an average value of 0.513; the variation range of inbreeding coefficient (FIS) was - 0.654-1, with an average value of 0.085. The variation range of PIC was 0.059-0.849, the average polymorphism information content was 0.453.14 loci had invalid alleles (Oosterhout value), the frequency range was 0.135-0.470; 22 loci deviated from Hardy-Weinberg equilibrium. (4) The amplification of 107 loci among 5 Taxus species was analyzed. The amplification rate was 96.07%. Only one locus (Ma-25497-83A) was not amplified. The amplification rate of four loci (C-52641-193B, Ma-1402-721B, Ma-186-234D, Ma-6383-968B) was less than 50%. The inter-species amplification rate of 100 loci was 100%. (5) The validity and accuracy of the developed loci were tested. The results of cluster analysis of 6 taxa were similar to those of other markers. (6) 10 polymorphic loci were screened for paternity analysis of three populations of Taxus chinensis var. mairei. The cumulative exclusion rates of the three male candidates were 98.32%, 93.80% and 97.40% respectively. The results of the paternal analysis showed that the effective pollen dispersal distance of Taxus chinensis var. mairei was 10-25m.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S791.49
，
本文编号：2227043
[Abstract]:Taxus chinensis var. mairei is a rare and endangered tree species in China. Its genome information is scarce and molecular genetics research is relatively lagging behind. At present, the number of molecular markers available in Taxus chinensis var. mairei is extremely limited, far from meeting the requirements of genetics and breeding. In this study, four species of Taxus chinensis var. mairei in NCBI database were used. Based on the transcriptome sequence of Taxus chinensis, EST-SSR molecular markers were developed by using bioinformatics software to test the amplification of polymorphic loci in Taxus chinensis, and applied to the analysis of male parents of Taxus chinensis. (1) The transcriptome sequence characteristics of four Taxus species were analyzed. Based on the transcriptome sequences of four Taxus species in NCBI database, the transcriptome sequences were analyzed by bioinformatics software. The transcriptome sequences were obtained from Taxus chinensis var. mairei, Taxus cuspidata, Taxus baccata and Taxus mandiana, respectively. There were 2 344,2 361,1 357 and 2 312 SSR loci in Taxus media. Among the 2-6 BP repeat units, Taxus chinensis var. mairei and Taxus mandii were dominated by hexanucleotide repeat units, followed by trinucleotides; Taxus chinensis var. mairei and Taxus chinensis var. mairei were dominated by trinucleotides, followed by hexanucleotides. The dominant repeat motifs of acid and hexanucleotide were AG/CT, AAG/CTT, AAGAGG/CCTCTT. (2) 112 polymorphic loci of Taxus were developed. Based on the information of four Taxus loci, 150 pairs of primers were synthesized by selecting loci with repeat units greater than or equal to 9. Among them, 59, 17, 13 and 23 polymorphic loci were developed respectively in Taxus chinensis var. mairei, Taxus cuspidata var. mairei, Taxus cuspidata var. mairei and Taxus mandii. (3) 103 polymorphic loci were developed. A total of 462 alleles were detected in 103 EST-SSR loci using 16 single plant samples. The variation range of allele number (Na) was 2-11, and the average number of alleles per locus was 4.41. The variation range of observed heterozygosity (Ho) was 0-1, with an average value of 0.459; the variation range of expected heterozygosity (He) was 0.0625-0.891, with an average value of 0.513; the variation range of inbreeding coefficient (FIS) was - 0.654-1, with an average value of 0.085. The variation range of PIC was 0.059-0.849, the average polymorphism information content was 0.453.14 loci had invalid alleles (Oosterhout value), the frequency range was 0.135-0.470; 22 loci deviated from Hardy-Weinberg equilibrium. (4) The amplification of 107 loci among 5 Taxus species was analyzed. The amplification rate was 96.07%. Only one locus (Ma-25497-83A) was not amplified. The amplification rate of four loci (C-52641-193B, Ma-1402-721B, Ma-186-234D, Ma-6383-968B) was less than 50%. The inter-species amplification rate of 100 loci was 100%. (5) The validity and accuracy of the developed loci were tested. The results of cluster analysis of 6 taxa were similar to those of other markers. (6) 10 polymorphic loci were screened for paternity analysis of three populations of Taxus chinensis var. mairei. The cumulative exclusion rates of the three male candidates were 98.32%, 93.80% and 97.40% respectively. The results of the paternal analysis showed that the effective pollen dispersal distance of Taxus chinensis var. mairei was 10-25m.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S791.49
，
本文编号：2227043
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