The photoperiod/temperature‐sensitive genic male sterile (P/TGMS) character is important for use of heterosis in hybrid wheat. In the present study, the fertility of 172 doubled haploid (DH) lines derived from the cross between a P/TGMS wheat line BS366 and a restorer wheat line Baiyu149 was investigated under both fertile and sterile environments during three cropping seasons. The phenotype was recorded based on the international seed setting rate. A total of 167 SSR and 1,278 SNP markers were used to construct a linkage map, with a total length of 3,748.94 cM and an average marker interval of 2.59 cM. Three QTLs were identified and designated as QF.bhw‐2DS, QF.bhw‐4BS and QF.bhw‐7Al, explaining 6.9%–12.8%, 19.7%–25.6% and 7.2%–8.8% of the phenotypic variances, respectively. These results lay a good basis for application of male sterility‐related molecular markers in improvement of two‐line hybrid wheat breeding system.

Background Formin, a highly conserved multi-domain protein, interacts with microfilaments and microtubules. Although specifically expressed formin genes in anthers are potentially significant in research on male sterility and hybrid wheat breeding, similar reports in wheat, especially in thermo-sensitive genic male sterile (TGMS) wheat, remain elusive. Results Herein, we systematically characterized the formin genes in TGMS wheat line BS366 named TaFormins (TaFHs) and predicted their functions in inducing stress response. In total, 25 TaFH genes were uncovered, majorly localized in 2A, 2B, and 2D chromosomes. According to the neighbor-joining (NJ) method, all TaFH proteins from wheat and other plants clustered in 6 sub-groups (A-F). The modeled 3D structures of TaFH1-A/B, TaFH2-A/B, TaFH3-A/B and TaFH3-B/D were validated. And different numbers of stress and hormone-responsive regulatory elements in their 1500 base pair promoter regions were contained in the TaFH genes copies. TaFHs had specific temporal and spatial expression characteristics, whereby TaFH1, TaFH4, and TaFH5 were expressed highly in the stamen of BS366. Besides, the accumulation of TaFHs was remarkably lower in a low-temperature sterile condition (Nanyang) than fertile condition (Beijing), particularly at the early stamen development stage. The pollen cytoskeleton of BS366 was abnormal in the three stages under sterile and fertile environments. Furthermore, under different stress levels, TaFHs expression could be induced by drought, salt, abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), indole-3-acetic acid (IAA), polyethylene glycol (PEG), and low temperature. Some miRNAs, including miR167, miR1120, and miR172, interacts with TaFH genes; thus, we constructed an interaction network between microRNAs, TaFHs, phytohormone responses, and distribution of cytoskeleton to reveal the regulatory association between upstream genes of TaFH family members and sterile. Conclusions Collectively, this comprehensive analysis provides novel insights into TaFHs and miRNA resources for wheat breeding. These findings are, therefore, valuable in understanding the mechanism of TGMS fertility conversion in wheat.

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academy of agriculture and Forestry Sciences/the municipal Key laboratory of molecular genetics of hybrid Wheat, Beijing, pR china; b laboratory of plant molecular genetics and proteomics, college of life Science, capital normal university, Beijing, china

One sentence summary: Differential transcriptome analysis was utilized to identify the 23 key factors for fertility transformation in a wheat photo-thermosensitive genic male sterile 24 line. 25 Authors' contributions 26 JB supervised the experiments, wrote manuscript. Zl: observed cytological events and 27 wrote manuscript. YW: edited the manuscript and completed the writing. LG: analyzed the 28 RAN-seq data. HG: performed the RT-PCR and qPCR. ZT and WD: performed ultrathin 29 sections. SY: managed the plants. YL, JY and TL: collected the samples. FZ: supervised 30 the experiments. CZ: conceived the original screening and research plans. LZ: conceived 31 the research plan, serves as the author responsible for contact and ensures communication. 32 All authors carefully checked and approved this version of the manuscript. 33 Abstract: 42 Wheat photo-thermosensitive genic male sterile (PTGMS) line is a vital material in the 43 two-line hybrid wheat breeding system in which functional pollen production is highly 44 associated with temperature during early developmental stage. Understanding the potential 45 mechanism of pollen infertility induced by low temperature in PTGMS wheat is crucial for 46 the effective utilization of genetic resources to guide wheat breeding. Herein, we combined 47 full-length single-molecular sequencing and Illumina short reads sequencing data to obtain 48 the high-resolution spatio-temporal transcriptome map of pollen under low temperature 49 stress at mother cell, dyad and tetrad stages in PTGMS line BS366. Cytological descriptions 50 and whole transcriptome analysis revealed a global landscape of low temperature altered 51 pollen fertility transformation via regulating the transcriptional patterns of cytoskeleton-52 related lncRNAs and their target genes, which involved in the calcium signaling and vesicle 53 trafficking pathways on cytoskeleton homeostasis at different stages of meiosis. Overall, 54 our results provided the transcriptional and cytological evidences for understanding the low 55 temperature-induced pollen sterility deficiency in PTGMS wheat line.56