Genome-wide identification and development of InDel markers in tobacco (Nicotiana tabacum L.) using RAD-seq

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 P < 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.