RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)

Abstract

RNA-Seq is an efficient way to comprehensively identify single nucleotide polymorphisms (SNPs) and alternative splicing (AS) events from the expressed genes. In this study, we conducted transcriptome sequencing of four Asian lotus (Nelumbo nucifera) cultivars using Illumina HiSeq2000 platform to identify SNPs and AS events in lotus. A total of 505 million pair-end RNA-Seq reads were generated from four cultivars, of which 86% were mapped to the lotus reference genome. Using the four sets of data together, a total of 357,689 putative SNPs were identified with an average density of one SNP per 2.2 kb. These SNPs were located in 1,253 scaffolds and 15,016 expressed genes. A/G and C/T were the two major types of SNPs in the Asian lotus transcriptome. In parallel, a total of 177,540 AS events were detected in the four cultivars and were distributed in 64% of the expressed genes of lotus. The predominant type of AS events was alternative 5' first exon, which accounted for 41.2% of all the observed AS events, and exon skipping only accounted for 4.3% of all AS. Gene Ontology analysis was conducted to analyze the function of the genes containing SNPs and AS events. Validation of selected SNPs and AS events revealed that 74% of SNPs and 80% of AS events were reliable, which indicates that RNA-Seq is an efficient approach to uncover gene-associated SNPs and AS events. A large number of SNPs and AS events identified in our study will facilitate further genetic and functional genomics research in lotus.

Fig 1. Summary of gene number identified from the Asian lotus transcriptome.

(A) Venn diagram of genes expressed in the four cultivars. (B) Correlations between detected genes number and reads number in the four cultivars.

Fig 2. Number and type of SNPs identified from the Asian lotus transcriptome.

(A) Venn diagram of SNPs discovered from the four cultivars. (B) Comparison of SNP number between our study (I) and the previous study of Zhang et al. [16] who discovered SNP by RAD-Seq technology (II). (C) Summary of SNP types identified from the Asian lotus transcriptome.

Fig 3. SNP distribution among scaffolds and genes.

(A) Distribution of expressed SNPs on the top ten scaffold. (B) Number of SNP per gene.

Fig 4. GO analysis of all the expressed genes in lotus and the genes contained SNP.

Fig 5. Numbers of AS events and distribution in the lotus Genome.

(A) Venn diagram of AS events discovered from the four datasets. (B) Correlations of AS event number with expressed gene number. (C) Correlations of AS frequency with expressed gene number. (D) Distribution of AS events in the lotus genome.

Fig 6. Statistics of the different AS events and GO analysis of AS genes.

(A) Diagram of different types of AS events. (B) Proportion of different types of AS events. (C) GO analysis of AS genes using all expressed genes for lotus as the background.

Fig 7. Validation of AS events in the Asian lotus transcriptome.

(A) Validation of AS in NNU_25229-RA. (B) Validation of IR in NNU_21858-RA. (C) Validation of AE in NNU_17825-RA.