Mapping And Cloning Of Kernel Length And Plant Height Genes In Tetraploid Wheat

Common wheat（Triticum aestivum L.,2n=6x=42,AABBDD）is one of the major food crops for the global population,and China is the world’s largest wheat producer and consumer.The demand of wheat is increasing as the world’s population increases.Therefore,ensuring and improving wheat yield has become even more significant of wheat research.Spike number,kernel number per spike,and kernel size are three key components that determine wheat yield.Among them,the kernel weight is mainly determined by kernel length,kernel width and kernel thickness,indicating that the kernel length can directly affect wheat yield.Meanwhile,plant height is an important trait of wheat,which affects plant lodging,harvest index and wheat yield.Thus,the research on the finding of genes or loci related to wheat kernel length and plant height and their regulation mechanism are of great significance to improve wheat yield and ensure food security.Tetraploid wheat contains a large number of excellent traits and genes related to long kernel and dwarfing,which is an excellent gene source for the genetic improvement of common wheat,but few studies have been done.The objectives of present study are:（1）to investigate and analyze the kernel length and plant height of different tetraploid wheat,and discover excellent genetic resources.Meanwhile,simplified genome sequencing and phylogenetic tree construction of different tetraploid wheat using genotyping-by-sequencing（GBS）are carried out to explore the relationship between these tetraploid wheat.（2）Using the recombinant inbred line（RIL）to detect the QTLs of kernel length and plant height,through genetic and construction of near-isogenic line（NIL）to map,clone and analyze the candidate genes,then using transcriptome sequencing（RNA-seq）to analyze the underlying molecular mechanisms of kernel length and plant height.The main results are as follows:1.Statistics on kernel length and plant height of 37 different tetraploid wheat showed that Triticum polonicum（2n=4x=28,AABB）（except CGN 12291）,T.turanicum（2n=4x=28,AABB）（except PI 352514）and T.ispahanicum（2n=4x=28,AABB）had longer kernels,while T.turgidum AS313（2n=4x=28,AABB,Jianyangailanmai,Ailanmai）had the shortest kernel.The plant height of Xinjiang dwarf Polish wheat AS302（dwarf Polish wheat,DPW）,Japanese dwarf Polish wheat IC 12196 and Ailanmai had shorter,while Xinjiang tall Polish wheat AS304（tall Polish wheat,TPW）was significantly higher than other tetraploid wheat.GBS sequencing and phylogenetic tree analysis showed that T.polonicum except CGN 12191 was closely related to T.durum（2n=4x=28,AABB）and T.turanicum,while T.polonicum CGN 12191 was closely related to T.ispahanicum.T.polonicum CGN12191 has a great difference with other T.polonicum in subgenome A.2.KL-PW,which controls the long kernel of Polish wheat,was detected in the RIL population constructed by DPW and Ailanmai.KL-PW is located on chromosome 7AS.Further genetic mapping and gene sequence analysis confirmed that the candidate gene of KL-PW was VRT-A2^PW,which encoding an MIKC-type MADS-Box protein.The insertion/deletion mutation of the first intron of VRT-A2^PW resulted in alternative splicing,namely,dwarf Polish wheat VRT-A2^PW has two transcripts,VRT-A2^PW1 and VRT-A2^PW2.Gene haplotype analysis found that this mutation type only exists in Polish wheat and Xinjiang rice wheat（T.petopavlovskyi）.Real-time quantitative PCR analysis showed that the expression level of VRT-A2^PW in developing kernels was significantly higher than VRT-A2^Ailanmai.Brassinosteroid（BR）sensitivity detection and BR-related gene expression analysis revealed that VRT-A2^PW might be a positive regulator of BR signaling.Meanwhile,VRT-A2^PW was successfully introduced into hexaploid wheat by hybridization,and significantly increased kernel length of pedigrees.3.Correlation analysis of kernel length and plant height of high generation RIL population（Ailanmai/Ganmai）showed that the short KL gene KL-JY of Ailanmai may be closely linked with the dwarfing gene Rht22.Phenotypic analysis of NIL showed that KL-JY not only led to smaller kernels,but also shorter spike length,increased number of kernels per spike,wider flag leaf,wrinkled leaf,smaller spikelet,shorter awn length,shorter lemma and glume,etc.RNA-seq analysis of NIL revealed that KL-JY regulates pathways related to G protein signaling,mitogen-activated protein kinase signaling,phytohormone and some transcriptional regulators,and then affects the signaling of BR and auxin,and the content of auxin and cytokinin,thereby causing short kernels in Ailanmai.4.Two QTLs controlling plant height,PH-DPW and PH-TPW,were detected in the RIL population constructed by DPW and TPW.PH-DPW is located on chromosome 4BS,which can explain 82.69%of the phenotypic variation,and the candidate gene is Rht-B1b.RNA-seq analysis of NILs showed that Rht-B1b regulates pathways related to hormones,reactive oxygen species,and nitrogen assimilation to modify the cell wall structure,and then limits cell wall loosening and inhibits cell elongation,thereby causing dwarfism in DPW.PH-TPW is located on chromosome 5AL,which can explain 11.45%of the phenotypic variation,and is a new plant height locus.The linkage analysis with newly developed molecular markers further narrowed the candidate region of PH-TPW to522.06-523.88 Mb,and the genetic distance is 3.60 c M.Through the analysis and cloning of candidate genes,we suspected that Traes CS5A02G312000 might be the candidate gene of PH-TPW.