Genetic Analysis Of Thermotolerance For Seed-set And Pollen Dispersal Characteristic In Maize

In recent years,with the increasing "greenhouse effect" and the frequent occurrence of extreme high temperature,many areas have suffered from a certain degree of heat damage.High temperature has become one of the main abiotic stress factors in maize production.In Huanghuaihai area,the short-term abnormal high temperatures during summer maize season often caused the observed decrease in maize yield and quality deterioration.As the world’s main food crop,feed crop and industrial raw material,maize plays an important role in China’s food security.Breeding and popularizing heat tolerance varieties are the fundamental ways to protect maize from heat damage.Excavation of heat-tolerant genes and an in-depth understanding of the mechanism of high tolerance are beneficial to accelerate the breeding practice.In this study,linkage analyses combined with genome-wide association analysis（GWAS）were used to explore the heat tolerance loci in maize,and the candidate genes were preliminarily verified.The main results are as follows:1.Established the field heat tolerance identification systems of maize through investigated occurrence regularity and severity in recombinant inbred lines（RIL）population and association population.The heat tolerance of 261 inbred lines derived from CIMMYT,P group,Tangsipingtou,Reid,etc.was evaluated with the seed-setting and pollen dispersal as identification indexes,and finally we have screened 8 heat tolerance inbred lines by reference variety method,such as CNW058,CML162 and CNW063.2.Heat tolerance identification was performed using the RIL population consisting of 237 inbred lines which constructed from the heat susceptible inbred line BT-1 and the heat tolerance inbred line N6.Four QTL on chromosome 5 named qSSR5-1、qSSR5-2、qSSR5-3、qSSR5-4 were identified to be significantly associated with heat tolerance by using the mean level of seed-setting as the identification index.These QTL explained between 10.2%and 11.3%of the variation,of which,qSSR5-4 explained 11.3%of the variation.3.The seed-setting of maize was evaluated by using associated populations containing 261 inbred lines in three environments for 2 years,a total of 42 significant（P<10-4）SNP and 17 heat-related candidate genes were obtained by GWAS.Combined with linkage analysis,we have identified 4 candidate genes:GRMZM2G377194、GRMZM2G026892、GRMZM2G060349 and GRMZM2G122199,they encoding D-type cyclin,cysteine-rich protein（CRP）,DNA mismatch repair proteins（MutS2）and an unknown protein,respectively.Combined with qRT-PCR,it was found that the expression levels of GRMZM2G377194 and GRMZM2G060349 in N6 were significantly up-regulated compared with that in BT-1 after heat stress.By comparing with the sequence of N6,it showed that one amino acid was substituted in the gene GRMZM2G377194 in BT-1,while gene GRMZM2G060349 had an amino acid substitution and an amino acid deletion.4.The heat tolerance of RIL population was evaluated by the average level of pollen dispersal characteristic as identification index in four environments,5 QTL were identified and located on chromosome 3,5,6,8 and 9,respectively,which explained 4.36%to 7.27%of the phenotypic variation.Genome-wide association analysis was performed for the associated population,and there were 17 significant SNP loci and 10 corresponding candidate genes in response to heat.The candidate genes including hydroxyproline-rich glycoprotein（HRGP）,zinc finger protein HIT and actin depolymerization factor,etc..Combined linkage analysis and association analysis,the most significant SNP was S9₁6481394,and it corresponds to a gene that expresses C2H2 zinc finger protein,and the protein is a transcription factor of plants and plays an important role in plant stress response and hormone signal transduction under biological and abiotic stresses.The study is based on understanding the regulation of high temperature occurrence and the period of high tolerance.Establish a scientific high tolerance identification system.Through the evaluation of the tolerance of the material and the genetic research on the high tolerance of maize and the understanding of the genetic mechanism of high tolerance was deepened,which lays a foundation for cloning the high tolerance gene of maize and molecular breeding of heat tolerance varieties.