INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow-leafed lupin (Lupinus angustifolius L.)

Abstract

Narrow-leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization-insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423-bp deletion in the cis-regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis-regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423-bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162-bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up-regulating gene expression. The 1,208-bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.

Keywords: FLOWERING LOCUS T (FT); cis-regulation; insertion/deletion (INDEL); variant series.

Figure 1

A schematic illustrating the positions of insertion/deletion genotypes in the 5' regulatory region of LanFTc1 relative to the start codon (ATG) of the coding sequence (CDS). The wild‐type LanFTc1 sequence (ku) was obtained from P27255, a wild Moroccan accession, and the 1,423‐bp deletion (Ku) from the Tanjil reference genome. A 5,162‐bp deletion (Julius or Jul) was found in several European breeding lines and cultivars, including Krasnolistny (Russian cultivar), P29039 (Belarussian breeding line), and Emir (Polish cultivar). A 1,208‐bp deletion was identified in P22660, a wild accession from Israel. Critical regions of the regulatory region, if deleted, enable high (C_H, shaded dark grey) and moderately high (C_M, shaded light grey) levels of LanFTc1 expression, respectively, relative to the wild‐type sequence

Figure 2

Average rate to flowering (reciprocal of degree‐days to flowering) in mildly and fully vernalizing conditions for narrow‐leafed lupins possessing various insertion/deletions (INDELs) in the 5' regulatory region of LanFTc1 in two phenotyping trials. (a) Trial 1 included narrow‐leafed lupins carrying the 0‐bp deletion (n = 29), 1,208‐bp deletion (n = 1), and 1,423‐bp deletion (n = 9). (b) Trial 2 included narrow‐leafed lupins with the 0‐bp deletion (n = 2), 1,208‐bp deletion (n = 1), 1,423‐bp deletion (n = 8), and 5,162‐bp deletion (n = 6). The least significant difference (LSD) value is provided to compare responses within and between vernalization treatments in each phenotyping trial ([a] LSD = .00020; [b] LSD = .00011)

Figure 3

Relative expression of LanFTc1 at various degree‐days from 4‐leaf stage (approximately 277 degree‐days) to first flowering in deletion categories of narrow‐leafed lupin (red circle, 0‐bp deletion wild‐type; yellow triangle, 1,208‐bp deletion; blue square, 1,423‐bp deletion; and green diamond, 5,162‐bp deletion) with (a) and without (b) vernalization. The quadratic regression (QR) model captured 94.7% of variance and indicated significant intercept, linear, and quadratic slope differences (p < .001) between category/vernalization treatment combinations. The final sampling time is at first flower and therefore varies widely between treatment combinations

Figure 4

Linkage (represented as r ²) of insertion/deletions (INDELs; black triangles) and single nucleotide polymorphisms (SNPs; grey circles) identified in the narrow‐leafed lupin LanFTc1 wild‐type genomic sequence, represented by Moroccan accession P27255, with vernalization responsiveness. The positions of polymorphisms are indicated relative to the base pair (bp) position along the wild‐type LanFTc1 genomic sequence (GenBank ID KT862491) and a schematic of the LanFTc1 genomic features, including regulatory regions (solid black line), untranslated regions (solid white bar with black border), exons (solid black bar with black border), and introns (solid grey bar with black border). An r ² value of 1.0 represents perfect linkage with vernalization response phenotype. Asterisks denote significant associations between polymorphisms and the vernalization responsiveness phenotype (Bonferroni adjusted p values: *.01 < p < .05; **.001 < p < .01; ***p < .001)