Molecular dissection of the genetic architecture of phenology underlying Lupinus hispanicus early flowering and adaptation to winter- or spring sowing

Abstract

Spanish lupin, Lupinus hispanicus Boiss. et Reut. is an untapped grain legume species characterized by moderate frost resistance, tolerance to poor soil and waterlogging, high yield stability, and remarkable seed protein content. It has been recognized as a good candidate for domestication to broaden the palette of crop diversity. One of the key characteristics that need to be precisely addressed during domestication is the vernalization responsiveness of flowering, which is advantageous in autumn sowing due to improved frost tolerance, whilst undesired in spring sowing as it delays flowering. Ahead of L. hispanicus breeding, in the present work, we aimed to recognize existing phenotypic variability of flowering time and vernalization response and to evaluate the genetic architecture of early and late phenology by DArT-seq genotyping and genome-wide association study (GWAS) in world germplasm collection of the species. Controlled environment phenotyping revealed high variability of flowering time and vernalization responsiveness and significant correlations with population structure. DArT-seq genotyping yielded 23 728 highly polymorphic markers distributed extensively across all 26 chromosomes. GWAS identified a number of markers significantly associated with flowering time with or without pre-sowing vernalization, including those overlapping with the two major quantitative trait loci reported previously for white lupin species. Microsynteny-based analysis of the genetic content of L. hispanicus genome regions carrying significantly associated markers highlighted several candidate genes from photoperiodic and vernalization pathways. To summarize, the present study identified germplasm resources for autumn- and spring-sown cultivation of L. hispanicus and provided tools for marker-assisted selection towards required flowering phenology.

Keywords: Lupinus hispanicus; Flowering induction; GWAS; Legume crop; Legumes; Vernalization.

Fig. 1

Correlation heatmap reporting Pearson correlation coefficients between years for each trait. Observations were performed during 2022 and 2023 growing seasons in a greenhouse at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland (52°26′ N 16°54′ E). The bar below the heatmap indicates the color legend of correlation coefficients (from 0 to 1). Asterisk (*) indicates significant correlations in the following scheme: ***, p < 0.00001; **, 0.00001 ≤ p < 0.0001; *, 0.0001 ≤ p ≤ 0.05.

Fig. 2

Visualization of population structure analysis of 173 Lupinus hispanicus accessions. The panels show (A) STRUCTURE diagrams under different K-values (K4, K5 and K6), (B) country of origin and (C) mean total number of days from sowing to the floral bud emergence and start of flowering for non-vernalized (NB and NF) and vernalized (VB and VF) plants. In total, 5959 SNP and 17,769 PAV markers were used for population structure analysis. Phenotypic observations were performed during 2022 and 2023 growing seasons in a greenhouse at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland (52°26′ N 16°54′ E). The deeper the shade of blue on the NB, NF, VB and VF panels, the later the phenotype according to the scale provided below the panels.

Fig. 3

The Heatmap reporting correlation coefficient values and p-values calculated for assignment to 6 clusters and 8 combinations of trait, year and vernalization. Asterisk (*) indicates significant correlations in the following scheme: ****, p < 0.00001; ***, 0.00001 ≤ p < 0.0001; **, 0.0001 ≤ p ≤ 0.001; *, p < 0.05.; “ “, non-significant.

Fig. 4

Marker effects for 13 markers significantly associated with at least four variables. Observed traits include the number of days from sowing to the floral bud emergence, start of flowering and pod maturity for non-vernalized and vernalized plants. Phenotypic observations were performed during 2022 and 2023 growing seasons in a greenhouse at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland (52°26′ N 16°54′ E). Color scale is presented from blue (minimum value) through white (zero) to red (maximum value). BK stands for BLINK, whereas FU for FarmCPU.

Fig. 5

Allelic effects on the number of growing degree days (GDDs) for thirteen DArT-seq markers, M092420 (A), N114410 (B), M074938 (C), M103926 (D), M103410 (E), M102006 (F), M079035 (G), M055339 (H), M065429 (I), M075705 (J), M104942 (K), M079300 (L), and M089187 (M) tagging novel QTL regions. R stands for the reference allele (0), V for an alternative allele (2), whereas H for a heterozygote (1). Observed traits include the number of days from sowing to the floral bud emergence (bud), start of flowering (flower) and pod maturity (pod) for non-vernalized (Nbud, Nflower, Npod) and vernalized plants (Vbud, Vflower, Vpod). Phenotypic observations were performed during 2022 growing season in a greenhouse at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland. Diamonds indicate mean values.

Fig. 6

Allelic effects on the number of growing degree days (GDDs) for thirteen DArT-seq markers, M092420 (A), N114410 (B), M074938 (C), M103926 (D), M103410 (E), M102006 (F), M079035 (G), M055339 (H), M065429 (I), M075705 (J), M104942 (K), M079300 (L), and M089187 (M) tagging novel QTL regions. R stands for the reference allele (0), V for an alternative allele (2), whereas H for a heterozygote (1). Observed traits include the number of days from sowing to the floral bud emergence (bud), start of flowering (flower) and pod maturity (pod) for non-vernalized (Nbud, Nflower, Npod) and vernalized plants (Vbud, Vflower, Vpod). Phenotypic observations were performed during 2023 growing season in a greenhouse at the Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland. Diamonds indicate mean values.

Fig. 7

Linkage disequilibrium (LD) plots for L. luteus genomic regions carrying DArT-seq markers significantly associated with L. hispanicus plant phenology. The pairwise r2 values between markers are shown. Red indicates high measures of LD, while deep violet indicates low LD. Lengths of analyzed segments is provided on the marker bars.