Genetic Analysis,Gene Mapping And Molecular Basis Study Of The Abnormal Spike Mutant Ptsd1 In Wheat

Wheat（Triticum aestivum L.）spike differentiation determines the grain number per spike,and complete differentiation of the spikes guarantees the final grain yield.At present,the main goal of wheat yield breeding is to increase grain number per spike,because it is difficult to improve the number of spikes per unit area and thousand kernel weight.However,the genetic regulation mechanism of wheat spike differentiation is unclear.One key scientific issue of wheat breeding is how to maintain the spike differentiation status and prolong the spike differentiation stage.Therefore,mining the key genes regulating the wheat spike differentiation process,molecular genetic study,mapping and cloning the important genes are of important significance for wheat breeding.The spike mutants are ideal germplasm resources for molecular genetic studies on wheat spike differentiation.A homozygous wheat prematurely terminated spike differentiation（ptsd1）mutant was obtained from the high yield cultivar‘Guomai 301’treated with ethyl methane sulfonate（EMS）.The differentiation of the upper spikelets terminated prematurely at about double ridge stage,however,a few under spikelets differentiated almost normally.Up to now,we haven’t found such kind of mutants have been reported in wheat.The ptsd1mutant is an ideal material for forward genetic analyses on the regulatory mechanism of wheat spike differentiation.We will fine map and analyze the candidate mutated genes of ptsd1 using the mutant ptsd1,F_2:3segregating family lines and a large segregating population in this project.Meanwhile,we will establish the molecular regulatory mechanism model of abnormal spike differentiation in mutant ptsd1 using the m RNA and miRNA sequencing analysis of‘Guomai 301’and mutant ptsd1.The main research contents and results are as follows:1.The genetic model and gene mapping of mutant ptsd11)The phenotype of mutant ptsd1.Compared with‘Guomai 301’,the upper part of the young spike formed some small spikelet primordia bumps in mutant ptsd1,meristem cells of the upper spikelet primordia didn’t differentiate and the cell arrangement was loose.The upper spikelet primordia of ptsd1 absented their typical meristem characteristics and ultimately degenerated and disappeared.In ptsd1,each spike only formed 6-8 spikelet primordia,the average spike length and fertile spikelet number decreased by 60.0%and77.5%compared to those of‘Guomai 301’.The ptsd1 was a new wheat truncated spike length mutant,prematurely terminated spike differentiation led to the phenotype of truncated spike length and decreased fertile spikelet number.2)The heredity of mutant ptsd1.The karyotype of mutant ptsd1 was normal,and no visible chromosome structural variation was found.The spike length of the F₁plants derived from cross ptsd1 and Zhoumai18 was the same as Zhoumai18,implying that spike length mutation of ptsd1 was a recessive trait.The phenotypes of spike length in the F₂population were segregated into three types designated as normal spike length（NSL,equivalent to Zhoumai18）,medium truncated spike length（MSL,intermediate between NSL and ESL）and extreme truncated spike length（ESL,equivalent to ptsd1）.The segregation ratios of spike length in F₂population didn’t fit the Mendelian ratio of 3:1 or 15:1,which indicated that the spike length trait of ptsd1 wasn’t controlled by a recessive gene or two independent inheritance recessive genes.3)Gene mapping of mutant ptsd1.The 660K SNP array genotyping was performed using bulked-segregant analysis coupled with next-generation sequencing（BSA-seq）for three pairs of the super DNA bulks‘NSL-bulk vs ESL-bulk’from three populations,i.e.the F_2:3and F_2:4lines derived from the cross‘ptsd1×Zhoumai18’,and the F_2:3lines derived from the cross‘ptsd1×CS’.Most of the SNPs linked to spike length were on chromosome5A,the SNPs majorly clustered at two regions,the SNPs on the short arm of chromosome5A covered a 100 Mb（from 20 to 120 Mb）physical region;the SNPs on the long arm of chromosome 5A covered a 80 Mb（from 380 to 460 Mb）physical region.The results indicated that there were two mutation genes in mutant ptsd1,which were named as ptsd1-5AS and ptsd1-5AL respectively.The linked SSR marker analysis was performed in two super DNA bulks‘NSL-bulk vs ESL-bulk’from two F_2:3populations derived from the crosses‘ptsd1×Zhoumai18’and‘ptsd1×CS’.Among the 210 SSR markers,we identified17 linked SSR markers in the F_2:3population derived from the cross‘ptsd1×Zhoumai18’and 10 linked SSR markers in the F_2:3population derived from the cross‘ptsd1×CS’.The results of linkage analysis using SSR markers were consistent with the results of 660K SNP array analysis,the gene ptsd1-5AS was located between SSR marker Xbarc56 and Xbarc358covered a 38 Mb（from 71 to 109 Mb）physical region,and the gene ptsd1-5AL was located between SSR marker TC84150 and Xgpw4093 covered an 16 Mb（from 427 to 443 Mb）physical region.4)Fine mapping of ptsd1-5AS.Twelve kompetitive allele-specific PCR（KASP）markers were identified in the candidate region for ptsd1-5AS on chromosome 5A after analyzing polymorphism between parents and bulks.They were used to genotype the F_2:3population derived from the cross‘ptsd1×Zhoumai18’for fine mapping ptsd1-5AS.Finally,the ptsd1-5AS locus was narrowed down to a 0.77 c M interval flanked by KASP markers AX-111485371 and AX-108768318 covered a 6 Mb（from 90 to 96 Mb）physical region.There were 29 genes in the candidate region of ptsd1-5AS referred to wheat reference genome Ref Seq-v1.0.2.Gene regulation mechanism of mutant ptsd11)The m RNA and miRNA sequencing analysis of the young spikes in‘Guomai 301’and mutant ptsd1 indicated that 3532 differentially expressed genes（DEGs）were highly expressed,but 907 DEGs were lowly expressed in the young spike of ptsd1.The WRKY and NAC families were the top two largest families having the most DEGs,and most of them were highly expressed in the young spike of ptsd1.The homeotic genes Ta SEP5,Ta SEP6 and Ta FL,which were associated with spikelet formation and determine the spike differentiation,were lowly expressed in ptsd1 during the young spike differentiation stages.Cytokinin signal transduction was weakened and ethylene signal transduction was enhanced in ptsd1.The reactive oxygen species（ROS）signal transduction related genes were highly expressed in ptsd1,and the concentration of H₂O₂in ptsd1 was significantly increased by31%compared to‘Guomai 301’.DNA degradation was widespread in the undifferentiated upper spikelets of the ptsd1.The tae-miR164 and novel-miR49 were extensively involved in the gene regulatory network of ptsd1,and they had 11 and 16 targets respectively.Overall,spike differentiation related biological processes were depressed,and senescence related biological processes such as catabolism and programmed cell death（PCD）were activated.The ROS accumulation caused oxidative damage and cell death,and ultimately resulted in young spike degenerated phenotype of ptsd1.2)The physiological metabolism of mutant ptsd1.The content of protein granules was lower in undifferentiated upper spikelets of ptsd1 compared to‘Guomai 301’indicated that protein was degraded in young spikes of ptsd1,and the protein content decreased by 25.0%.In ptsd1,the content of nitriate nitrogen was significantly decreased by 43.3%,while the content of NH₄⁺was significantly increased by 14.7%.The glutamine synthetase（GS）activity was 3.5-fold higher in ptsd1 compared to‘Guomai 301’.Ammonium assimilation occurred via GS and ultimately formed glutamine.Meanwhile,the content of lignin was significantly increased by 48.2%in ptsd1.A regulatory model was put forward to elucidate the response mechanism of prematurely terminated spike differentiation based on the metabolism related genes,and provided a theoretical basis for a more comprehensive understanding of wheat spike differentiation metabolic mechanism.