Identification of Elymus (Triticeae, Poaceae) and its related genera genomes by RFLP analysis of PCR-amplified Adh genes

Abstract

Elymus L. is the largest genus in Triticeae, containing about 150 species with four recognized genome donors (St, H, P, and W). Traditionally, the genome compound of this genus is identified based on cytological data. Recently, molecular phylogenetic analysis was used to investigate its genomic combination. Here we describe a restriction fragment length polymorphism (RFLP) assay based on digesting alcohol dehydrogenase (Adh) amplicons with two restriction enzyme combinations, EcoRI-HindIII and EcoRI-PstI, which easily can be used to distinguish Elymus and its closely related genera genomes. The method includes only four steps: (1) amplifying nuclear Adh genes with universal primers; (2) purifying and cloning PCR products; (3) digesting plasmids with restriction enzymes that identify a given genome; (4) running the digested products on an agarose gel and identify the sample based on the restriction profiles. Results showed that: (1) PCR products ranged from 1,200 to 2,000 bp; (2) Adh2 gene was amplified from all the tested genomes; Adh1 gene was amplified from almost all of the tested genomes except the W genome; Adh3 gene was amplified only from the St genome; (3) the EcoRI-HindIII combination was effective to distinguish different Adh gene types (Adh1, Adh2, and Adh3); (4) the Adh2-EcoRI-PstI fragments could be used to distinguish Elymus and its closely related genera genomes. Therefore, This RFLP assay provides an inexpensive and simple means of identifying Elymus genomes.