Allelic characterization and protein structure analysis reveals the involvement of splice site mutation for growth habit differences in Lablab purpureus (L.) Sweet.

Background: Interrelationship between growth habit and flowering played a key role in the domestication history of pulses; however, the actual genes responsible for these traits have not been identified in Indian bean. Determinate growth habit is desirable due to its early flowering, photo-insensitivity, synchronous pod maturity, ease in manual harvesting and short crop duration. The present study aimed to identify, characterize and validate the gene responsible for growth habit by using a candidate gene approach coupled with sequencing, multiple sequence alignment, protein structure prediction and binding pocket analysis.

Results: Terminal flowering locus was amplified from GPKH 120 (indeterminate) and GNIB-21 (determinate) using the primers designed from PvTFL1y locus of common bean. Gene prediction revealed that the length of the third and fourth exons differed between the two alleles. Allelic sequence comparison indicated a transition from guanine to adenine at the end of the third exon in GNIB 21. This splice site single-nucleotide polymorphism (SNP) was validated in germplasm lines by sequencing. Protein structure analysis indicated involvement of two binding pockets for interaction of terminal flowering locus (TFL) protein with other proteins.

Conclusion: The splice site SNP present at the end of the third exon of TFL locus is responsible for the transformation of shoot apical meristem into a reproductive fate in the determinate genotype GNIB 21. The splice site SNP leads to absence of 14 amino acids in mutant TFL protein of GNIB 21, rendering the protein non-functional. This deletion disturbed previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from an external loop. This finding may enable the modulation of growth habit in Indian bean and other pulse crops through genome editing.