Distinguishing among Pisum accessions using a hypervariable intron within Mendel’s green/yellow cotyledon gene

Abstract

Distinguishing among commercial varieties or accessions in a germplasm repository presents considerable challenges when dealing with common crops or large collections. In the genus Pisum L. the third intron of the gene STAYGREEN (SGR), responsible for the green/yellow cotyledon variation, contains a complex of repeat sequences that display considerable indel and single nucleotide (SNP) polymorphism. In a sample of 137 Pisum L. accessions obtained from various germplasm collections 76 alleles were identified, ranging from about 600 to 1500 nucleotides in length. In most cases alleles differed by insertion/deletion (indel) polymorphisms in addition to single base changes. The amplicon was particularly useful for distinguishing wild accessions (those with dehiscent pods and dormant seeds). Among the 51 P. sativum L. accessions examined with a definite wild phenotype there were 44 SGR alleles. When this allelic variation was combined with location data for the wild accessions, each allele could be assigned a unique geographical location. Similarly, of the 10 P. fulvum Sibth. & Sm. accessions analyzed, only a group of three had the same allele, two of which are known to be replicates. We were able to confirm the identities of several P. sativum accessions from different germplasm collections, as well as identifying two cases where supposedly synonymous accessions gave different intron sequences. In addition, a group of wild pea accessions from Greece, Sicily, and Portugal exhibited a unique repeat sequence not found in any other wild accession, suggesting that these accessions constitute a distinct lineage. The rate of nucleotide site variation in this fragment of the SGR locus was found to be about 2.22 × 10–8 substitutions per site per year. This value is similar to those found for many other sequences in herbaceous papilionoid tribes, although it is at the faster end of the distribution of herbaceous legume substitution rates. We propose that both a fast substitution rate as well as the formation and rearrangement of indels have contributed to the high level of variation we detected at SGR. This PCR product should prove valuable in the identification of Pisum accessions, for quality control operations in germplasm repositories, and for exploring the evolutionary relationships within the genus.