RAPD markers for ascochyta resistance, phylogenetic studies and cultivar identification In lentil

Abstract

Random amplified polymorphic DNA (RAPD) markers were used in three genetic studies in lentil (Lens culinaris Medikus). The first study involved development of RAPD markers linked with genes for resistance to ascochyta blight, caused by Ascochyta fabae f. sp. lentis Gossen et al. Seventy F2-derived F3 (F2:3) lines were field screened for ascochyta blight reaction in each of two hybrid populations, Indianhead x Eston and ILL 5588 x Eston, that were segregating for resistance to ascochyta blight. Resistance to ascochyta blight in ILL 5588 lentil was conferred by a single dominant gene (Ral1), whereas resistance in Indianhead lentil was conferred by a single recessive gene (ral2). An efficient DNA extraction procedure and a PCR protocol that yielded RAPD markers with high resolution were developed for lentil. Bulked segregant analysis was used to produce four bulks of DNA from resistant vs. susceptible F2 plants in each of these two populations which were then screened for RAPD markers using 400 random oligonucleotide primers. One RAPD marker, UBC2271290, was linked to the recessive gene, ral2, in Indianhead lentil in repulsion phase with a map distance of 14.1 ± 4.5 cM. No RAPD marker was linked with the Ral1 gene in ILL 5588 lentil. In the second study, RAPD markers were used to study phylogeny of the genus Lens. DNA, extracted from 23 accessions of all five taxa of the genus Lens (culinaris, orientalis, nigricans, odemensis and ervoides), was screened for RAPD polymorphisms, using 11 random oligonucleotide primers. Two accessions of the differentiated cytotype of L. nigricans were also included. One hundred and forty eight polymorphic RAPD markers were resolved. A dendrogram for these RAPD markers, using the unweighted pair group method, clearly separated all accessions into their supposedly related taxa. Lens orientalis was the undisputed progenitor of the cultivated lentil, Lens culinaris. A low level of RAPD polymorphism was observed in Lens culinaris and L. ervoides. The differentiated cytotype of L. nigricans was well separated from the normal cytotype of L. nigricans and was closely associated with the L. odemensis accessions, indicating its close genetic similarity to L. odemensis. Principal component analysis (PCA) also indicated a similar relationship among these accessions, but resulted in a better resolution of the groupings. In the third study, the genetic polymorphism of seven Canadian lentil cultivars were studied using RAPD markers. Four lentil cultivars, CDC Gold, CDC Matador, Eston and Indianhead each had a unique, cultivar-specific RAPD marker, allowing their identification.