Effect Of Identification And QTL Mapping For Cold Tolerance Of Rice With Exogenous Brassionlides(BRs)

Low temperature chilling is one of the important environmental factors that cause rice to be damaged to varying degrees during each growth period,and its adverse impact on rice yield is also a widespread problem worldwide.Therefore,it is of great significance to study the cold tolerance of rice and screen out the rice varieties with strong cold tolerance,in order to reduce the low-temperature cold damage of rice.Dongxiang wild rice has strong cold tolerance,which provides better materials for the breeding of cold-tolerant rice varieties and cold tolerance gene research.Brassinolide(BRs)can play a better role in resisting plant cold environment.BRs signal transduction pathway is closely related to plant cold tolerance.Taking the cold tolerance identification and population genetic theory under the BRs approach as the research basis,it is of great theoretical and practical value to screen and identify cold-tolerant rice germplasm and to conduct QTL mapping research.In this study,140 backcross recombination inbred lines constructed by Dongxiang Wild Rice and super rice SN265 with outstanding cold tolerance were used as research materials.Under low temperature treatment and low temperature treatment after BRs spraying,cold tolerance identification and QTL mapping and annotation of cold tolerance genes at the bud stage of rice will lay a theoretical foundation for the breeding of cold tolerance germplasm and the discovery of cold tolerance genes.The main results are as follows:(1)The germination rate and seedling rate of low temperature were divided into 1,3,5,7 and 9 grades according to the cold tolerance grade.23 extremely cold resistant materials and 6 extremely cold intolerant materials were screened out with the seedling rate as the reference standard.Cold+BRs treatment was higher than 6 and 17 lines respectively in the first and third grades with strong cold tolerance,while in the seventh grade with weak cold tolerance,Cold+BRs treatment was lower than 10 lines with simple cold tolerance.The Cold+BRs treatment was higher than the simple cold treatment in Grade 1,and the Cold+BRs treatment was lower than the simple cold treatment in Grade 7.In plant height,Although the height and quantity of cold treated and Cold+BRs treated lines were all concentrated around 6cm,the Cold+BRs treatment was more than the cold treated 9 lines.In the first leaf length,the number of cold treated and Cold+BRs treated lines was about 3cm,but the number of Cold+BRs treated lines was more than that of cold treated 4 lines.The results showed that cold treatment after spraying BRs had some effects on low-temperature germination rate,low-temperature seedling rate,plant height and first leaf length.(2)Under the condition of low temperature stress,four cold tolerance indexes of 140 rice backcross lines were measured and analyzed,which were germination rate,seedling rate,plant height and first leaf length.Under the condition of simple cold treatment,there is a very significant positive correlation between germination rate and seedling rate,and between plant height and first leaf length,r = 0.37,r = 0.39,respectively;there is a significant negative correlation between seedling rate and plant height,r =-0.21;but there is no significant correlation between germination rate and first leaf length and plant height,which shows that they are not significant There was no direct relationship between them.Under Cold+BRs treatment,the correlation between the 4 traits is consistent with that of cold treatment alone;In addition,there was a significant positive correlation between the cold and Cold+BRs treatments of the four characters,the correlation was r = 0.73,r = 0.73,r = 0.79,r = 0.73,which indicated that there was a direct relationship between cold and Cold+BRs,and the phenotypic value of Cold+BRs was higher than that of cold alone(3)According to the results of simplified genomic sequencing(GBS),genotyping was carried out with the parameters of deletion rate ≤ 20% and minimum allele frequency ≥ 5%.1145 marker sites and 141 genotypes were selected to construct the genetic linkage map.Its characteristics are as follows: there are 1145 makers on 12 chromosomes,of which 77 are on chromosome 7 with the least maker distribution and 117 are on chromosome 4 with the most maker distribution;the total genetic distance of chromosome is 31883.3c M,the total physical distance is 394.1Mbp,and the genetic distance interval of each marker is between 1.9-146.3c M.(4)In this study,Using ICIM mapping method in Ici Mapping 4.2 software,the LOD threshold is set to 3 for QTL mapping.A total of 73 QTLs were mapping in 4 cold tolerance traits,including 49 QTLs in cold treatment,with the maximum LOD of 5.89 and the maximum phenotype contribution rate of 11.71%;24 QTLs in Cold+BRs treatment,with the maximum LOD of 9.38 and the maximum phenotype contribution rate of 14.69%.The results showed that the LOD and phenotype contribution rate of Cold+BRs treatment were higher than that of cold treatment alone.Among them,there were 1 related to germination rate QTL at low temperature,31 related to seedling rate QTL at low temperature,1 related to plant height QTL,and 16 related to the length of the first leaf QTL;Under Cold+BRs treatment conditions,3 related to low temperature germination rate QTL,12 related to low temperature seedling rate QTL,4 related to plant height QTL,and 5 related to first leaf length QTL,There were more QTLs for the two characters of low temperature seedling rate and first leaf length,but more QTLs for low temperature germination rate and plant height.(5)According to the QTL mapping results of four characters,six cold related genes,DREB1,MYB,WRKY,ERF,BHLH and Os MADS18,were annotated in the physical region of the target region.Among them,DREB1 and Os MADS18 genes are in the coincidence range of seedling rate and germination rate under the condition of simple cold treatment and Cold+ BRs treatment respectively;MYB,WRKY and BHLH genes are in the range of first leaf length,first leaf length and low-temperature seedling rate under the condition of simple cold treatment respectively;ERF gene is in the range of seedling rate under the condition of Cold+ BRs treatment.These results lay a foundation for further study of the specific function and regulatory relationship of cold tolerance genes under BRs pathway.