A conserved molecular basis for photoperiod adaptation in two temperate legumes

Abstract

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, high response to photoperiod (HR), is an ortholog of early flowering 3 (ELF3), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR, with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.

Fig. 1.

Adaptation to photoperiod in pea is controlled by two major-effect QTL. (A) Survey of photoperiod-regulated flowering in Pisum. Plants received an 8-h photoperiod of natural daylight (SD) extended with low-irradiance (10 µmol⋅m⁻²⋅s⁻¹) white light (LD) from mixed fluorescent and incandescent sources. Data are mean ± SE for n = 4. Lines not flowering in SD conditions are indicated by a “+” symbol and had produced a minimum of 55 vegetative nodes before termination of the experiment 180 d after sowing. All lines are P. sativum var. sativum unless indicated. Lines carrying the hr (6C) mutation are shaded in pink, and the two lines used for subsequent genetic analysis are indicated by black arrowheads. (B) Location of QTL controlling SD flowering on linkage groups III and VI in the F₂ of a domesticated (NGB5839) × wild (var. humile; JI1794) cross. The one-LOD and two-LOD confidence intervals around the peak are indicated by dark red and pale red shading, respectively. (C) Genotype means ± SE for interaction of QTL3 and QTL6 in the control of flowering and other developmental traits. Genotypes at QTL3 and QTL6 were inferred from the genotype of peak markers MAX1 (QTL3) and RNAhel (QTL6), with the wild (JI1794) and domesticated (NGB5839) alleles indicated by the suffixes -w and -d, respectively. Significance levels (***P < 0.001; ns, P > 0.05) and proportion of variance explained for the individual locus effects and their interaction (int.) were determined by two-way ANOVA and indicated to the right of each plot.

Fig. 2.

The HR locus affects photoperiod responsiveness and circadian rhythms. (A) Effect of HR on responsiveness to photoperiod and light quality. Plants received 8 h of natural daylight (SD) extended for a further 16 h with low-irradiance (10 µmol⋅m⁻²⋅s⁻¹) white light of high (LDH) or low (LDL) R:FR. Data are mean ± SE for n = 8–10. (B) RT-PCR analysis of expression rhythms of clock genes in HR and hr, showing means ± SE for three biological replicates. Plants grown for 3 wk from sowing under a 12-h photoperiod (150 µmol⋅m⁻²⋅s⁻¹) were transferred to constant white light (10 µmol⋅m⁻²⋅s⁻¹ (Left)) or constant dark (Right).

Fig. 3.

A mutation in ELF3 ortholog is the likely basis for the hr spring phenotype. (A) Details of the PsELF3 5C/6C polymorphism. (B) Complementation of flowering and petiole phenotypes of the Arabidopsis elf3-1 mutant by the 5C (HR) but not the 6C (hr) form of 35S::PsELF3, under 8-h SD conditions. Representative plants are shown for two independent transformants for each construct. (C) Association of the 5C/6C polymorphism with photoperiod responsiveness in a selection of P. sativum germplasm. Plants received an 8-h photoperiod of natural daylight (SD) extended with low-irradiance (10 µmol⋅m⁻²⋅s⁻¹) white light (LD) from mixed fluorescent and incandescent sources. Data are mean ± SE for n = 64 (5C) and n = 20 (6C).

Fig. 4.

Sequence diversity in the HR gene. Neighbor-joining tree representing genetic distances among haplotypes identified in a 3.8-kb region of the HR gene in 110 diverse Pisum lines (P.sativum var. sativum except where indicated). Node support (%) was obtained from 10,000 bootstrap replicates. Numbering of haplotypes corresponds to Table S1 and Fig. S2, with bold letters indicating distinct haplotype groups. Haplotypes present in more than one line are indicated by filled triangles with size proportional to the number of lines represented, with the single haplotype containing the hr mutation designated by an open triangle. Haplotypes including lines that flowered in SD conditions, despite carrying an apparently functional form of HR, are indicated by an asterisk.

Fig. 5.

Mutation in an ELF3 ortholog is also associated with early flowering in lentil. (A) Differing photoperiod responsiveness in Lens culinaris lines ILL5588 and ILL6005. Plants received an 8-h photoperiod of natural daylight extended with 2-h (SD) or 16-h (LD) low-irradiance (10 μmol⋅m⁻²⋅s⁻¹) white light from mixed fluorescent and incandescent sources. Data are mean ± SE for n = 9–10. (B) Cosegregation of flowering time under SD with a marker for LcELF3 in the F₂ generation of cross ILL6005 × ILL5588. The flowering time ranges of the two parental lines are shown as horizontal bars. (C) Details of the mutation in LcELF3 genomic DNA. Early-flowering segregants carry a translationally silent G-to-A substitution in the last nucleotide of exon 3. (D) Details of splicing defect. PCR with the indicated primers (small arrowheads) revealed a 52-bp deletion in the LcELF3 mRNA from ILL6005, corresponding to skipping of exon 3. This was verified by sequencing and results in a frame-shift in translation of exon 4 and termination after four missense amino acids.