Gene Cloning The Super Early Maturity Mutant Xiaomi In Foxtail Millet And Establishment Of A Model Plant System

Foxtail millet,as a nurturing crop of the Chinese nation,has the advantages of drought resistance,barren tolerance and high C₄ light efficiency.The optimal heading and flowering time of foxtail millet determines to a large extent its adaptation to the local environment and the expansion of the cultivation area,which is very important for foxtail millet production.Arabidopsis and rice play an important role in leading the major scientific and technological discovery and functional research as model plants,while Arabidopsis and rice are C₃ plants,which can not solve the basic problems such as high light efficiency of C₄ plants.Foxtail millet has a small genome and is self-pollinated for diploids.Plants are diverse and have the potential to develop into C₄ model plants.The famous millet Jingu 21 was mutated by EMS,and super early maturity mutant of foxtail millet was screened and named xiaomi.Use the late maturing G1（♀）×early maturing xiaomi（♂）F₂ population for genetic analysis and gene cloning.By constructing a high-quality xiaomi reference genome,gene expression profile and an efficient and stable genetic transformation system mediated by Agrobacterium,xiaomi has become an ideal model system for studying C₄ plant functions.1.Screening and phenotypic analysis of super early maturity mini foxtail millet mutant xiaomiJingu 21,a high-quality and high-yield variety in Shanxi Province,was subjected to EMS mutagenesis,and a super early maturity mini mutant was selected from the M₂ generation population of about 20,000 mutant lines and named it xiaomi.xiaomi.has a short growth cycle,with earing in about 39 days after sowing in field conditions,and maturity in about 70 days;the plant is short,only about 67 cm.The seed setting rate is 12.83%higher than that of its wild-type Jingu 21,but the thousand-grain weight and seed size are not significantly different from those of Jingu 21.Under long-day conditions（16 h light/ 8 h dark,28℃/ 22℃）,the heading date of xiaomi was further advanced and the plant height decreased to about 30 cm.Foxtail millet is a typical short-day crop,but the early heading phenotype of xiaomi is long-day dependent.Under short-day conditions,the heading is about one month later than that under long-day conditions.2.Genetic analysis and gene cloningIn order to clone the XIAOMI gene,the xiaomi mutant was crossed with the late-maturing variety G1 to construct an F₂ population.Genetic analysis showed that the ratio of late heading to early heading in the xiaomi×G1 hybrid F₂ population was 3:1（χ²=0.318<χ²0.05,1=3.841）,indicating that the early heading date of xiaomi was caused by a recessive single-gene mutation.Using map-based cloning and Super-BSA high-throughput whole-genome sequencing technology to locate the XIAOMI gene in the 212 kb interval of chromosome 9,there is only one base mutation in this region: Phytochrome C（PHYC,Si9g09200）gene 2469 G changed to T.PHYC encodes the photoreceptors necessary for photoperiod flowering.3.Abnormal photoperiodic pathway leads to early heading phenotype of xiaomiIn order to understand how XIAOMI gene functional site mutations affect flowering period under long-day conditions,transcriptome sequencing was performed on the last two leaves of Jingu 21 and xiaomi 30 days after sowing.A total of 1281 differentially expressed genes were screened in xiaomi,of which 711 were up-regulated genes and 570 were down-regulated genes.Further analysis showed that the expression of PRRS,GI,COL/Ghd7,EHD1,FT/ Hd3 a and AP1/ MADS in xiaomi were significantly affected.The above results indicate that mutations in the PHYC gene cause abnormal photoperiod pathways.4.Establishment of C₄ cereal model plant system based on xiaomiIn order to develop xiaomi into C₄ cereal model plant,we using third-generation single-molecule sequencing technology,second-generation Hi-Seq sequencing technology and Hi-C technology,a high-quality xiaomi genome was assembled with a genome size of 429.94 Mb and an N50 length of 42.41 Mb.Using three-generation full-length transcriptome and small RNA sequencing data,combined with ab initio prediction and homology search,annotated 34,436 protein-coding genes,919 rRNA genes,3516 tRNA genes,2631 pseudogenes,340 microRNA（miRNA）precursors,28260 long non-coding RNA（lncRNA）precursors and 1318 circularRNA（circRNA）precursors.To construct a gene dynamic expression map for the whole growth period of xiaomi,RNA-seq was used to detect gene expression in 11 tissues and organs of xiaomi,including seeds germinating for 3 days,seedlings at 2weeks old,roots,stems,the last leaf of seedlings at 2 weeks old,the second leaf,flag leaf and penlast fourth leaf of seedlings at 30 days old,young panicle,panicle at pollination stage and panicle at grain filling stage.In order to make these expression data convenient use,developed digital xiaomi gene expression through（http://sky.sxau.edu.cn/MDSi.htm）.In the browser,gene expression data can be presented as a heat map.To solve the problem of foxtail millet genetic transformation suffering,the efficient and stable Agrobacterium-mediated genetic transformation system of foxtail millet was established.The transgenic system only needed 2-3 months from Agrobacterium-mediated co-culture to the transgenic seeds,and the transformation efficiency was as high as 23.28%.The problem of low transformation efficiency of millet as model plant was fundamentally solved.