Genome-wide Association Analysis Of Wheat Heading Date And Leaf Functional Traits Under Different Nitrogen Levels

Wheat is a crop with a large demand for nitrogen fertilizer.Since the 1950 s,the input of nitrogen fertilizer has played an important role in the increase of wheat yield.However,the largescale application of nitrogen fertilizer has also caused serious ecological pollution and waste of resources.As there are significant genotypic differences in nitrogen efficiency of wheat,and different varieties have high and low responses to nitrogen fertilizers,under the premise of alleviating agricultural environmental risks and ensuring production,we should explore genetic resources related to low nitrogen stress and cultivate low tolerance.New high-yield wheat varieties under nitrogen stress are an effective way to promote green production of wheat.In this study,a natural population composed of 389 wheat varieties was used as the test material.The 660 K SNP chip was used to scan the whole genome,and the mixed linear model（MLM）of the GAPIT software package was used to determine the heading date and normalized vegetation of wheat based on the population structure.Genome-wide association analysis of phenotypic data such as index and leaf functional traits and low-nitrogen tolerance sensitivity index was carried out to clarify the relationship between these traits and low-nitrogen tolerance,and to screen related lownitrogen tolerance candidate genes for selection of low-nitrogen tolerance wheat varieties Provide evidence.The main findings are as follows:1.Use the Plink1.9 software to filter the population genotype data,and eliminate SNPs with a miss rate（miss rate）greater than 20% and a minimum allele frequency（minorallele frequency,MAF）less than 5%,and finally get 397,642 SNPs distributed in 21 On the chromosome,high-quality SNP markers with high polymorphism and good stability are used for subsequent association analysis.2.Structure 2.3.4 software analysis and principal component analysis（PCA）,Neighbor-Joining（NJ）analysis all divide the test materials into 2 subgroups;linkage disequilibrium analysis finds A The attenuation distance of the genome is 0.3Mb,which is similar to 0.27 Mb of the D genome,but much smaller than the 0.79 Mb of the B genome,and the attenuation distance of the whole genome is 0.47 Mb.3.The whole genome association analysis(-log₁₀（P）≥5)of heading date under 8 environments（4 without nitrogen application,4 normal nitrogen）and sensitivity index of low nitrogen tolerance under 4 environments found,The heading date and low-nitrogen tolerance sensitivity index under normal nitrogen and low nitrogen were associated with 53,114,and 33 significantly associated markers,respectively.At least in the two environments,the associated markers were 14,25,and 1,respectively;Balance（Linkage disequilibrium,LD）attenuation distance found that these markers formed a total of 11 Quantitative trait locus（QTL）,of which q HD-2D-2 and q HD-7D-3 were repeated in 6 and 4 environments,respectively Detected and consistent with the results of previous studies.It is worth noting that the QTL related to nitrogen tolerance（q HDSSI-2D-2）was also detected at the same locus q HD-2D-2;located on the 1BS chromosome q HD-1B-1,q HD-1B-2,q HD-1B-3 and q HD-2B-1 on the 2BL chromosomes have been repeatedly detected in at least 4environments,and there is no report that may be a new locus,the remaining 4 loci q HD-2D-1,q HD-7D-1,q HD-7D-2 were only detected in 2 environments without nitrogen application,and q HD-1D-1 was only repeatedly detected in 3 environments with normal nitrogen;A total of 10 excellent allelic variants were screened using the most significant markers among the above QTLs.Among them,AX-111956072-T can not only advance the heading date but also improve the tolerance to low nitrogen;extract genes in the candidate interval based on the flanking positions of 11 QTLs Ten candidate genes that may be related to wheat heading date and low nitrogen tolerance index under low nitrogen stress were screened.Two of them have been proved to be related to heading date in previous studies(Traes CS2D02G079600（Ppd-D1）,Traes CS7D02G111600（Vrn-D3）,7 may play a role in the process of plants resisting abiotic stress,and 1 may be related to the synthesis and response of plant hormones.4.Genome-wide association analysis(-log₁₀（P）≥ 4)was performed on the normalized vegetation index（NDVI）and low-nitrogen tolerance sensitivity index of natural populations at different growth periods.For NDVI（Shanxi Shenfeng 2019）,it will survive the winter without applying nitrogen.30,16,94,53,55 and 20 significant association markers were detected in 6environments,namely,normal nitrogen overwintering period,low nitrogen overwintering period,low nitrogen jointing period,normal nitrogen jointing period,low nitrogen filling period,and normal nitrogen filling period.Among them,14 SNP markers were repeatedly detected in at least 2environments.Associated markers,of which 14 SNP markers have been repeatedly detected in at least 2 environments.According to the LD attenuation distance,these markers form a total of 3QTLs.Among them,q NDVI-5D-3 contains 12 stable associated SNPs,which have not been reported.They may be new locus,and the remaining two all contain only one significant associated marker;Further correlation of the NDVI tolerance to low nitrogen sensitivity index（SSI）of each growth period,it was found that the correlation markers of the NDVI tolerance to low nitrogen sensitivity index（SSI）in the overwintering stage,jointing stage,and filling stage were 5,217,and 2,respectively,of which 25 A SNP was also repeatedly detected in the non-nitrogen environment.According to the LD attenuation distance,a total of 4 QTLs are formed,among which q NDVISSI-1B-1,q NDVISSI-1B-2,and q NDVISSI-1B-3 on chromosome 1B overlap with the positions of QNDVI.ubo-1B.2 previously studied.The QTL on 1D has not been reported;the most significant markers of the above QTLs were used to screen out 7 excellent allelic variants,which accounted for a relatively high proportion in the tested population.According to the flanking position of QTL,the genes in the candidate interval were extracted,and 15 candidate genes that may be related to the normalized vegetation index of wheat and the low-nitrogen tolerance sensitivity index were screened,which are respectively related to cytochrome metabolism,nitrate,ammonium salt nitrogen uptake and transport,Transcription factors（WRKY,MYB,HTH）,ethylene metabolism related.5.Perform genome-wide association analysis on flag leaf-related traits of natural populations and their low-nitrogen tolerance sensitivity index.A total of 1390 associated markers were detected,which are widely distributed on 21 chromosomes,including chlorophyll content,leaf length,leaf width,and senescence parameters The number of SNPs detected were 186（125 low nitrogen,61 with normal nitrogen）,33（8 low nitrogen,25 with normal nitrogen）,283（33 low nitrogen,250 with normal nitrogen）),595（49 low nitrogen,546 with normal nitrogen）,of which 16 SNP markers are repeatedly associated with wheat flag leaf-related traits,including physiological traits（9 chlorophyll content）,morphological traits（leaf length 6）,senescence parameters（1 at the beginning of senescence）,a total of 9 QTLs are formed according to the LD attenuation distance,of which q SPAD-1B-2 on chromosome 1B and q Ll-6B-2 on chromosome 6B contain multiple significant SNP markers,And there is no report,it may be a new locus that controls chlorophyll content and leaf length.The remaining 7 QTLs contain only one significant SNP;5 SNPs have one-cause pleiotropy and are distributed on chromosomes 2B and 4D.It is related to aging parameters and nitrogen tolerance;using the most significant markers of these QTLs to select 7 excellent allelic variants,of which AX-111578849-A has a lower proportion in the tested materials,and the remaining 6 are in the tested population Accounted for relatively high.According to the flanking positions of the above QTLs,the genes in the candidate range were extracted,including 10 candidate genes that may be related to wheat flag leaf-related traits and low nitrogen tolerance sensitivity index,of which 3 are related to chlorophyll metabolism and 1 is related to the initiation of senescence.Six are related to nitrogen metabolism pathways.