Nucleotide Diversity of Common Bean Phaseolin (α-phs) Gene and Its Association With Seed Protein Content

Abstract

Phaseolin (α-Phs) is the most abundant protein reserve in seeds of common beans accounting for 40 – 50 % of the total seed protein. Despite having low methionine (0.5 - 0.80 %) content, phaseolin is the primary source of amino acids in common bean seeds. More than 40 genetic variants of phaseolin differing in amino acid composition have been reported. Therefore, phaseolin gene diversity could be used as a strategy to improve the nutritional value of protein in common beans. To date, no information is available on the relationship between natural nucleotide polymorphisms of α-Phs gene and seed protein content in common beans. This study was conducted to determine natural nucleotide polymorphism in α-Phs gene and their association with protein content in dry seeds of common bean. Eleven selected common bean accessions were planted in plastic pots in the greenhouse. Young leaves of 4-week-old plants were used for extraction of genomic DNA, followed by polymerase chain reaction (PCR) amplification using primers specific to different fragments of the phaseolin gene. Amplified PCR products were sequenced, sequences edited and analysed for nucleotide polymorphisms to infer levels of genetic variability, genetic diversity indices and other evolutionary analyses including haplotype diversity, neutrality tests, linkage disequilibrium and recombination events using DNA Sequence Polymorphism (DnaSP) software. Amino acid/codon changes occurring on sequenced α-Phs gene of the common bean accessions were elucidated using Codon Code Aligner software. Dry mature seeds of the selected common bean accessions were harvested and analysed for the total protein content using Lowry protein method. The association of α-Phs gene sequence polymorphisms and protein content was determined. The full-length sequence of α-Phs gene revealed a total of 41 genetic variants which consisted of 24 single nucleotide polymorphisms (SNPs) and 17 indels/parsimony informative sites. Ninety percent of the segregated sites in the coding region of the gene resulted in non- synonymous mutations. The coding region polymorphisms classified the α-Phs gene into 9 distinct haplotypes. The full-length sequence had a nucleotide diversity of π = 0.00271. Some mutated positions of the α-Phs gene were in positive or negative linkage disequilibrium and 6 paired informative sites had a history of recombination. The computed Tajima’s D was significantly less than 0 indicating presence of purifying selection. The association analysis revealed that three non-synonymous indels on the coding region were significantly associated with protein content. The findings from this study indicate that the polymorphisms detected in α- Phs gene can be used for discrimination of the genetic relationships among common bean germplasm. The genetic variants associated with seed protein content in the common bean accessions, could be explored in molecular breeding as well as potential genetic markers in the improvement of protein content in common beans.