The Study Of Early Heading Mechanism About Foxtail Millet Mutant 110-2-5

What an important process during plant life is from vegetative growth to reproductive growth,especially the stage of earing flowering that mattered.Setaria italica,with the characteristic of high nutrition,drought resistance and barren resistance,is an important cultivation of crops for arid and semi-arid regions.Currently,relative research on earing flowering in millet is rare,and much attention has been paid to increase yield of millet.In this study,we used methyl ethyl methanesulfonate(EMS)to mutagenize Jingu 21 and obtain dwarf and premature mutant 110-2-5,which was carried out in these aspects such as phenotypic observation,agronomic traits investigation and gene function identification.The number of stem segments of 110-2-5 mutant decreased and be thinner,and its plant height,ear number as well as grain weight were significantly decreased.Through 110-2-5 mutant population re-sequencing and mutation site analysis,it was found that 110928 and 1107726 SNP sites were detected in wild-type pool and 110-2-5 mutant pool,respectively.No candidate regions were identified by ED correlation analysis and MutMap+ analysis.The SNP site of wild type and 110-2-5 mutant pool was analyzed by sequencing 110-2-5 transcriptome,selecting Seita.6G095400 as candidate gene that encoding heat shock protein.Researching and analysing on the function and sequence of.Seita.6G095400 gene,we found this protein was closest to Sevir.6G 103400 of Setaria viridis while 79%consistent with chaperone protein ClpB of rice.The results of preliminary phenotypic analysis showed that the rice heat shock protein mutant plants were short,the growth was slow and the tiller was less,which was similar to phenotypic of 110-2-5 mutant of millet.A single nucleotide polymorphism(SNP)of Seita.6G095400 gene was detected in 916 millet,finding 12 SNP loci that means 12 haplotypes were obtained.Finally,the expression of Seita.6G095400 gene was analyzed,finding that the gene was expressed in all tissues of Yugu 1,and it was the highest in leaves.Although the reference genome of foxtail millet has been released,the gene annotation is not optimized.Therefore,we carried out the experiment for the identification of novel genes and the structural optimization of the annotated genes in foxtail millet by RNA sequencing(RNA-Seq)technology.The total RNA wasisolated from the leaves of Jingu 21 and 110-2-5,then used for sequencing library construction.The library was paired-end sequenced using the Illumina HiSeq 2500 sequencing platform and,finally,37072949 and 30356010 high quality clean reads were obtained respectively.To identify novel genes and optimize the annotated gene structure,the clean reads were further aligned with Yugu 1 reference genome.A total of 614 novel genes were identified and 438 genes among them were annotated using COG,GO,KEGG,Swiss-Prot and NR databases.In addition,7175 gene structures were optimized,and 4330 of 5’ends and 5362 of 3’ ends were extendedThrough the transcriptome sequencing analysis,it was found that there were 2405 differentially expressed genes in the precocious mutants 110-2-5,among which 1028 genes were up-regulated and 1377 genes were down-regulated.The expression level of Seita.5G143500 that coding FLOWERING LOCUS T-like(FT)protein was increased by 27.83 times.The Seita.5G143500 gene was cloned into HA-pBA vector to construct an overdose expression vector of 35S-driven Seita.5G143500 gene and transfer it into Arabidopsis by Flora Dip method.At present,the seed of T1 generation has been obtained,and the effect of overexpression of this gene on flowering time of Arabidopsis is under investigation.