Li Haiyang, Ikram Muhammad, Xia Yanshi, Li Ronghua, Yuan Qinghua, Zhao Weicai, Siddique Kadambot H M, Guo Peiguo
Guangdong Provincial Key Laboratory of Plant Adaptation and Molecular Design, International Crop Research Center for Stress Resistance, School of Life Sciences, Guangzhou University, Guangzhou, 510006 China.
Guangdong Provincial Engineering & Technology Research Center for Tobacco Breeding and Comprehensive Utilization, Guangdong Key Laboratory for Crops Genetic Improvement, Crops Research Institute, Guangdong Academy of Agricultural Sciences (GAAS), Guangzhou, 510640 China.
Physiol Mol Biol Plants. 2022 May;28(5):1077-1089. doi: 10.1007/s12298-022-01187-3. Epub 2022 May 27.
Insertions and deletions (InDels) can be used as molecular markers in genetic studies and marker-assisted selection breeding. However, genetic improvement in tobacco has been hindered by limited genetic diversity information and relatedness within available germplasm. A Chinese tobacco variety, Yueyan-98, was resequenced using restriction-site associated DNA (RAD-seq) approach to develop InDel markers. In total, 32,884 InDel loci were detected between Yueyan-98 and the K326 reference sequence [18,598 (56.55%) deletions and 14,288 (43.45%) insertions], ranging from 1 to 62 bp in length. Of the 6,733 InDels (> 4 bp) that were suitable for polyacrylamide gel electrophoresis, 150 were randomly selected. These 150 InDels were unevenly distributed on 23 chromosomes, and the highest numbers of InDels were observed on chromosomes Nt05, Nt13, and Nt23. The average density of adjacent InDels was 19.36 Mb. Thirty-seven InDels were located in genic regions. Polymerase chain reaction (PCR)-based markers were developed to validate polymorphism; 113 (79.80%) of the 150 InDel markers showed polymorphism and were further used for genetic diversity analysis of 50 tobacco accessions (13 from China, 1 from Mexico, and 36 from the USA). The average expected heterozygosity (He) and polymorphism information content (PIC) values were 0.28 ± 0.16 and 0.38 ± 0.10, respectively. The average Shannon diversity index (I) was 0.34 ± 0.18, with genetic diversity ranging from 0.13-0.57. The 50 accessions were classified into two groups with a genetic similarity coefficient of 0.68. Principal coordinate analysis (PCoA) and population structure analysis showed similar results and divided the population into two groups unrelated to their geographical origins. AMOVA showed 4% variance among the population and the remaining 96% within the population, suggesting low genetic differentiation between two subpopulations. Furthermore, 10 InDels (19 alleles) were significantly identified for tobacco plant height using GLM+Q model at < 0.005. Among these, three markers (Nt-I-26, Nt-I-41, and Nt-I-44) were detected in at least two environments, with phenotypic variance explained (PVE) ranging from 14.03 to 32.68%. The polymorphic InDel markers developed can be used for hybrid identification, genetic diversity, genetic linkage map construction, gene mapping, and MAS breeding programs of tobacco.
The online version contains supplementary material available at 10.1007/s12298-022-01187-3.
插入和缺失（InDels）可作为遗传研究和标记辅助选择育种中的分子标记。然而，烟草的遗传改良受到可用种质内有限的遗传多样性信息和相关性的阻碍。利用限制性位点关联DNA（RAD-seq）方法对中国烟草品种云烟98进行重测序，以开发InDel标记。总共在云烟98和K326参考序列之间检测到32,884个InDel位点[18,598个（56.55%）缺失和14,288个（43.45%）插入]，长度从1到62 bp不等。在适合聚丙烯酰胺凝胶电泳的6,733个InDels（>4 bp）中，随机选择了150个。这150个InDels在23条染色体上分布不均，在Nt05、Nt13和Nt23染色体上观察到的InDels数量最多。相邻InDels的平均密度为19.36 Mb。37个InDels位于基因区域。开发了基于聚合酶链反应（PCR）的标记来验证多态性；150个InDel标记中有113个（79.80%）显示出多态性，并进一步用于50份烟草种质（13份来自中国，1份来自墨西哥，36份来自美国）的遗传多样性分析。平均期望杂合度（He）和多态性信息含量（PIC）值分别为0.28±0.16和0.38±0.10。平均香农多样性指数（I）为0.34±0.18，遗传多样性范围为0.13 - 0.57。50份种质被分为两组，遗传相似系数为0.68。主坐标分析（PCoA）和群体结构分析显示了相似的结果，并将群体分为两组，与它们的地理起源无关。方差分析（AMOVA）显示群体间方差为4%，群体内方差为96%，表明两个亚群体之间的遗传分化较低。此外，使用GLM + Q模型在<0.005水平上显著鉴定出10个与烟草株高相关的InDels（19个等位基因）。其中，三个标记（Nt-I-26、Nt-I-41和Nt-I-44）在至少两个环境中被检测到，表型变异解释率（PVE）范围为14.03%至32.68%。开发的多态性InDel标记可用于烟草的杂种鉴定、遗传多样性分析、遗传连锁图谱构建、基因定位和标记辅助选择育种计划。
在线版本包含可在10.1007/s12298-022-01187-3获取的补充材料。
