Maturity2, a novel regulator of flowering time in Sorghum bicolor, increases expression of SbPRR37 and SbCO in long days delaying flowering

Abstract

Sorghum bicolor is a drought-resilient facultative short-day C4 grass that is grown for grain, forage, and biomass. Adaptation of sorghum for grain production in temperate regions resulted in the selection of mutations in Maturity loci (Ma1 -Ma6) that reduced photoperiod sensitivity and resulted in earlier flowering in long days. Prior studies identified the genes associated with Ma1 (PRR37), Ma3 (PHYB), Ma5 (PHYC) and Ma6 (GHD7) and characterized their role in the flowering time regulatory pathway. The current study focused on understanding the function and identity of Ma2. Ma2 delayed flowering in long days by selectively enhancing the expression of SbPRR37 (Ma1) and SbCO, genes that co-repress the expression of SbCN12, a source of florigen. Genetic analysis identified epistatic interactions between Ma2 and Ma4 and located QTL corresponding to Ma2 on SBI02 and Ma4 on SBI10. Positional cloning and whole genome sequencing identified a candidate gene for Ma2, Sobic.002G302700, which encodes a SET and MYND (SYMD) domain lysine methyltransferase. Eight sorghum genotypes previously identified as recessive for Ma2 contained the mutated version of Sobic.002G302700 present in 80M (ma2) and one additional putative recessive ma2 allele was identified in diverse sorghum accessions.

Fig 1. Circadian expression of genes regulating flowering in S. bicolor in 100M and 80M under long days.

(A) Expression of SbPRR37 in 100M (solid black lines) and 80M (dashed red lines). The expression peaks of SbPRR37 are reduced in 80M. This is consistent with earlier flowering in 80M because SbPRR37 represses the expression of the sorghum FT-like genes. (B) Expression of SbCO in 100M and 80M. Expression peaks of SbCO are also reduced in 80M. This is consistent with earlier flowering in 80M because under long days SbCO is a repressor of flowering. All expression values are normalized to SbUBC and are the mean of 3 biological replicates.

Fig 2. Expression of the S. bicolor FT-like genes SbCN8, SbCN12, and SbCN15 in long days at the expected peak of expression.

Expression of SbCN12 is elevated in 80M, which is consistent with earlier flowering in that genotype. All expression values are normalized to SbUBC and are the mean of 3 biological replicates. Fold change was calculated as 2^{-[Ct(100M)-Ct(80M)]}.

Fig 3. Quantitative trail locus (QTL) map of flowering time in the Hegari/80M F₂ population.

Two QTL were identified for variation in flowering time in the F₂ population derived from Hegari (Ma₁Ma₂Ma₃ma₄) and 80M (Ma₁ma₂Ma₃Ma₄). This population was expected to segregate for Ma₂ and Ma₄. Each recessive Ma allele causes earlier flowering. The QTL on LG10 corresponds to Ma₄ because F₂ individuals carrying the Hegari allele contributed to accelerated flowering. F₂ individuals carrying the 80M allele at the QTL on LG02 flowered earlier, so this QTL corresponds to Ma₂.

Fig 4. Interaction plots for the Ma2 QTL and the Ma4 QTL.

There is a known interaction between Ma₂ (represented by marker c2_68327634) and Ma₄ (represented by marker c10_3607821). This interaction was identified by multiple QTL mapping (MQM). Dominant alleles of the Ma genes delay flowering. In a recessive ma4 background (AA at c2_68327634), the effect of Ma₂ on days to flowering is reduced. A represents the 80M allele and B represents the Hegari allele at each QTL. Reciprocal plots are shown.

Fig 5. Fine-mapping of the Ma2 QTL.

The Ma₂ QTL spans from 67.3 Mpb to 69.1 Mbp (light blue bar). Five F₂ individuals that were heterozygous across the Ma₂ QTL were self-pollinated to generate heterogeneous inbred families (HIFs) totaling 1000 F₃ individuals. Genotype and phenotype analysis of these HIFs narrowed the QTL region to ~600 kb (darker blue bar). Two additional rounds of fine-mapping narrowed the QTL region to ~500 kb (vertical dashed lines). This region contained 76 genes. The genotypes of relevant HIFs and the parents are shown to the left and their corresponding days to flowering are shown to the right. Blue regions correspond to the 80M genotype and red regions correspond to the Hegari genotype. Purple regions are heterozygous.

Fig 6. Alignment of Sobic.002G302700 with its closest homologs in several plant species.

Sobic.002G302700 is highly conserved across plant species. It is annotated as a Set and MYND (SMYD) protein. SMYD proteins have lysine methyltransferase activity. The MYND region is highlighted in red. The nine conserved Cys residues typical of SMYD proteins are indicated by asterisks.

Fig 7. A model of the flowering time regulatory pathway in S. bicolor.

Ma₂ and Ma₄ work codependently to enhance the expression of SbPRR37 and SbCO. In LD, SbPRR37 and SbCO in turn repress the expression of the SbCN genes, especially SbCN12, to repress the floral transition.