The responsive characteristics of phytochrome genes to photoperiod, abiotic stresses and identification of their key natural variation sites in foxtail millet (Setaria italica L.)

Abstract

The responsive patterns of phytochrome gene family members to photoperiod and abiotic stresses were comparatively analyzed and the favorable natural variation sites of these genes were identified. This would help understand the mechanism of phytochrome gene family in photoperiod-regulated growth and development and abiotic stress response. In addition, it may facilitate the molecular marker assisted selection of key traits in foxtail millet. In this study, we used RT-PCR to clone three phytochrome genes SiPHYA, SiPHYB and SiPHYC from ultra-late maturity millet landrace variety 'Maosu'. After primary bioinformatics analysis, we studied the photoperiod control mode and the characteristics of these genes in responding to five abiotic stresses including polyethylene glycol (PEG)-simulated drought, natural drought, abscisic acid (ABA), high temperature and NaCl by fluorescence quantitative PCR. Finally, we detected the mutation sites of the three genes among 160 foxtail millet materials and performed haplotype analysis to determine the genes' functional effect. We found that the cloned cDNA sequences of gene SiPHYA, SiPHYB and SiPHYC were 3 981, 3 953 and 3 764 bp respectively, which contained complete coding regions. Gene SiPHYB and SiPHYC showed closer evolutionary relationship. Photoperiod regulated all of the three genes, but showed more profound effects on diurnal expression pattern of SiPHYB, SiPHYC than that of SiPHYA. Under short-day, when near heading, the expression levels of SiPHYA and SiPHYB were significantly lower than that under long-day, indicating their roles in suppressing heading of foxtail millet under long-day. SiPHYB and SiPHYC were responsive to PEG-simulated drought, natural drought, ABA and high temperature stresses together. SiPHYA and SiPHYB responded differently to salt stress, whereas SiPHYC did not respond to salt stress. Re-sequencing of 160 foxtail millet materials revealed that SiPHYB was highly conservative. Two missense mutations of SiPHYA, such as single nucleotide polymorphism (SNP) 7 034 522C→T and SNP7 036 657G→C, led to delaying heading and increasing plant height. One missense mutation of SiPHYC, such as SNP5 414 823G→T, led to shortening heading under short-day and delaying heading under long-day, as well as increasing plant height and panicle length regardless of photo-thermal conditions. Photoperiod showed different regulatory effects on SiPHYA, SiPHYB and SiPHYC. SiPHYB and SiPHYC jointly responded to various abiotic stresses except for the salt stress. Compared with the reference genotype, mutation genotypes of SiPHYA and SiPHYC delayed heading and increased plant height and panicle length.