Reconstruction of the full-length transcriptome of cigar tobacco without a reference genome and characterization of anion channel/transporter transcripts.

BACKGROUND: Cigar wrapper leaves are the most important raw material of cigars. Studying the genomic information of cigar tobacco is conducive to improving cigar quality from the perspective of genetic breeding. However, no reference genome or full-length transcripts at the genome-wide scale have been reported for cigar tobacco. In particular, anion channels/transporters are of high interest for their potential application in regulating the chloride content of cigar tobacco growing on coastal lands, which usually results in relatively high Cl- accumulation, which is unfavorable. Here, the PacBio platform and NGS technology were combined to generate a full-length transcriptome of cigar tobacco used for cigar wrappers. RESULTS: High-quality RNA isolated from the roots, leaves and stems of cigar tobacco were subjected to both the PacBio platform and NGS. From PacBio, a total of 11,652,432 subreads (19-Gb) were generated, with an average read length of 1,608 bp. After corrections were performed in conjunction with the NGS reads, we ultimately identified 1,695,064 open reading frames including 21,486 full-length ORFs and 7,342 genes encoding transcription factors from 55 TF families, together with 2,230 genes encoding long non-coding RNAs. Members of gene families related to anion channels/transporters, including members of the SLAC and CLC families, were identified and characterized. CONCLUSIONS: The full-length transcriptome of cigar tobacco was obtained, annotated, and analyzed, providing a valuable genetic resource for future studies in cigar tobacco.

Construction of a SNP Fingerprinting Database and Population Genetic Analysis of Cigar Tobacco Germplasm Resources in China.

Cigar tobacco is an important economic crop that is widely grown around the world. In recent years, varietal identification has become a frequent problem in germplasm preservation collections, which causes considerable inconvenience and uncertainty in the cataloging and preservation of cigar germplasm resources, in the selection of parental lines for breeding, and in the promotion and use of high quality varieties. Therefore, the use of DNA fingerprints to achieve rapid and accurate identification of varieties can play an important role in germplasm identification and property rights disputes. In this study, we used genotyping-by-sequencing (GBS) on 113 cigar tobacco accessions to develop SNP markers. After filtering, 580,942 high-quality SNPs were obtained. We used the 580,942 SNPs to perform principal component analysis (PCA), population structure analysis, and neighbor joining (NJ) cluster analysis on the 113 cigar tobacco accessions. The results showed that the accessions were not completely classified based on their geographical origins, and the genetic backgrounds of these cigar resources are complex and diverse. We further selected from these high-quality SNPs to obtained 163 SNP sites, 133 of which were successfully converted into KASP markers. Finally, 47 core KASP markers and 24 candidate core markers were developed. Using the core markers, we performed variety identification and fingerprinting in 216 cigar germplasm accessions. The results of SNP fingerprinting, 2D barcoding, and genetic analysis of cigar tobacco germplasm in this study provide a scientific basis for screening and identifying high-quality cigar tobacco germplasm, mining important genes, and broadening the basis of cigar tobacco genetics and subsequent breeding work at the molecular level.