Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes as assessed by transcriptome sequencing

Abstract

Background: Alfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species. Result: The transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated.

Development of an Alfalfa SNP Array and Its Use to Evaluate Patterns of Population Structure and Linkage Disequilibrium

“…Minimal diversity appeared to have been lost in cultivated tetraploid alfalfa compared to wild diploid alfalfa based on a transcriptome analysis of 27 genotypes [19]. In this study, we genotyped a larger population of individuals with the SNP array and found a similar result.…”

The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

“…This species is a perennial, cross-pollinated and autotetraploid (2n ¼ 4x ¼ 32) plant with a genome size of 800e900 Mb (Choi et al, 2004). Extensive efforts have been made to develop alfalfa transcriptome data, including 454 sequencing (Han et al, 2011) and Illumina sequencing (Li et al, 2012;Liu et al, 2013;Postnikova et al, 2013;Yang et al, 2011), which have facilitated the development of transcript-based molecular markers for alfalfa, such as expressed sequence tag simple sequence repeat markers (EST-SSRs).…”

Transcriptome Sequencing of Diverse Peanut (Arachis) Wild Species and the Cultivated Species Reveals a Wealth of Untapped Genetic Variability

“…Annotation of assembled RNASeq reads can help in understanding the function of a gene or a transcript. Annotations have been used in several crops for establishing genetic information where limited knowledge exists (Garg et al 2011; Li et al 2012). SNPs in annotated transcripts associated with traits would benefit functional characterization of genes.…”