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

Authors: WEEDEN, NORMAN - Montana State University; Coyne, Clarice - Clare; LAVIN, MATTHEW - Montana State University; MCPHEE, KEVIN - Montana State University

Submitted to: Genetic Resources and Crop Evolution
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/22/2021
Publication Date: 4/22/2021
Citation: Weeden, N., Coyne, C.J., Lavin, M., McPhee, K. 2021. Distinguishing among Pisum accessions using a hypervariable intron within Mendel’s green/yellow cotyledon gene. Genetic Resources and Crop Evolution. 68:2591-2609. https://doi.org/10.1007/s10722-021-01152-1.
DOI: https://doi.org/10.1007/s10722-021-01152-1

Interpretive Summary: Distinguishing among commercial varieties or accessions in a germplasm repository presents considerable challenges when dealing with hundreds to thousands of such entities. Morphological variation among accessions is usually employed as an initial screen. However, in most species reliable (stable under a variety of environments) morphological diversity is too limited to identify more than a small fraction of the accessions in large collections. Molecular markers have proven more successful at distinguishing among accessions. A single, highly variable DNA sequence that could be used as a ‘barcode’ that readily distinguishes all accessions remains the holy grail for researchers in this subject. The SGR gene that controls the green/yellow cotyledon variation described by Gregor Mendel (1866), revealed a surprisingly high level of variation in size and we investigated its use as a 'barcode'. Sufficient differences were found in the SGR gene to differentiate wild pea but not cultivated types.

Technical Abstract: The third intron of the gene Stay-Green (SGR), which is responsible for the green/yellow cotyledon variation in Pisum sativum, contains a complex of repeat sequences that display considerable indel and single nucleotide (SNP) polymorphism within the genus Pisum. In a sample of 137 Pisum accessions obtained from various germplasm collections 77 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 rate of nucleotide site variation in this sequence was found to be about 1 X 10-8 substitutions per site per year. Although high, this value is comparable to those found for many other sequences in more advanced papilionoid tribes, indicating the primary basis for the high level of variation in the SGR sequence has been the formation and rearrangement of indels. The amplicon was particularly useful for distinguishing wild accessions (those with dehiscent pods and dormant seeds). Among the 51 P. sativum 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 accessions analyzed, only a group of three (one from Israel, one from the JIC and one from the Vavilov Institute in Leningrad) had the same allele. The latter two are known to be replicates of the same original collection. We were able to confirm the identities of several P. sativum accessions from different germplasm collections, as well as identifying three 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 a separate derivation of this lineage. This PCR product should prove valuable in the identification of Pisum accessions, for quality control operations in Pisum germplasm repositories, and for exploring the evolutionary relationships within the genus.