Genetic Diversity Evaluation And Identification Of Gene Resources Involved In Drought Avoidance In Dongxiang Wild Rice

Dongxiang wild rice（DXWR）is the northernmost common wild rice in China and even across the globe that is recognized as precious germplasm resources in terms of a well-developed root system and strong drought resistance due to its evolution in the special environments.Evaluating the genetic diversity of the existing Dongxiang wild rice and proposing appropriate protection strategies are the prerequisite for exploration and utilization of its superior genes.However,its genetic diversity has not been thoroughly investigated,and there is lacking necessary understanding of the genetic basis of its superior characters of drought resistance.In this study,mitochondrial markers and SNPs from nuclear genome were used to analyze the genetic diversity of DXWR,supporting the proposed protection strategies.Meanwhile,the discovered lines with strong drought resistance were further assessed,and used in development of segregating population for quantitative trait loci（QTLs）mapping and candidate gene identification.The main results are presented as follows:1.A total of 220 DXWR accessions from previously described 9 natural populations could be divided into 4 subpopulations based on 12-pair mitochondrial polymorphic markers,and into 2 subpopulations based on single nucleotide polymorphisms（SNPs）of the nuclear genomes.The varied results of the population structures based on different data are probably attributed to the complex evolutionary history of DXWR.The current classification of 9 natural populations failed to reflect the real population structure of DXWR.The results will serve as an important basis for formulating protection and research strategies of DXWR.2.As embodied in private SNP and Indel variations of DXWR population,DXWR possessed many specific genetic resources in comparison to cultivated rice and common wild rice from other sources.The two subpopulations based on nuclear SNPs varied in drought resistance,especially in the deep-rooting mediated drought escape.Moreover,a lot of DXWR-specific drought responsive metabolites were identified by analyzing the root system metabolomes of a typical drought-resistant line（DY80）.In addition,the auxin metabolism mediated by tryptophan biosynthesis was found to be a potential pathway underlying the drought response of DY80 roots.Above results hinted that DXWR is of great values for exploring drought escape and for rice breeding.3.Mapping populations were constructed using drought resistant line DY80 and the early indica restorer line R974.There are a total of 234 lines in the BC₂F₇population.A linkage map containing 1,977 bin markers was established via dd RADSeq.Through two-year,two-location phenotypic experiments,a total of 31 QTLs were mapped for 4root system characters（deep root number,shallow root number,total root number and deep root ratio）,among that enhancing alleles of 15 QTLs were derived from DXWR,including 6 key regions.Within a 70 kb region on chromosome 5,two QTLs（named q DR5.1 and q TR5.2）that simultaneously influenced deep root number and total root number were repeatedly detected in two years.Wherein,q DR5.1 explained 13.35%and12.01%,and q TR5.2 explained 10.88%and 10.93%of the phenotypic variations,respectively.Enhancing alleles of both QTL came from DY80.The cumulative effect analysis of QTLs revealed that the phenotypic value can be increased by pyramiding multiple QTLs for deep root number and deep root ratio.4.A natural population containing 175 local varieties was used to analyze the association between SNPs in target QTL intervals and root phenotypes.We found that 23SNPs within 10 candidate genes were identified to be associated with deep root traits.According to the response of candidate genes in the target intervals to different hormones（ABA,IAA,CTK,GA3 and ET）,a total of 17 candidate genes that might participate in responses to different hormones were screened out from those candidate genes within the target region.Two candidate genes（LOC＿Os10g30610 and LOC＿Os10g33310）were repeatedly detected in the two methods.Based on the quantitative polymerase chain reaction（q PCR）test for 25 candidate genes within the target regions,expressions of five genes showed significant differences between two parents（DY80 and R974）.Expressions of LOC＿Os02g01355 and LOC＿Os02g04160 were significantly higher in DY80 than thoes in R974 under normal condition（CK）.Expressions of LOC＿Os05g02140 and LOC＿Os07g03000 were significantly higher in DY80 than thoes in R974 under both CK and osmotic stress.The expression of LOC＿Os10g30390 in DY80 was significantly higher than that in R974 under the osmotic stress.We further detected four DXWR-specific SNPs within 3 candidate genes（LOC＿Os02g01355,LOC＿Os07g03000and LOC＿Os10g30390）in DY80.The DXWR-specific SNPs could be used to identify candidate genes controlling drought resistance in DXWR and differentiate it from other cultivated rice varieties.5.According to the root system proteomics results from lines with different introgressed segments of deep root ratio in DXWR,all up-regulated differentially expressed proteins（DEPs）could be significantly enriched in pathways of phenylpropane biosynthesis,glycometabolism and amino acid metabolism,while down-regulated DEPs could be evidently enriched in pathwaysof secondary metabolite biosynthesis,purine metabolism and amino acid synthesis.By combining the results of proteome and QTL localization,it was discovered that 8 DEPs may be related to the deep rooting of DY80.In the follow-up study,the functional mechanism of these candidate genes and their application to rice breeding will be further explored.In this study,the genetic diversities of 220 DXWR resources in ex situ DXWR conservation plots were systematically analyzed,expecting to render a reference basis for protecting and investigating DXWR germplasm resources.In the meanwhile,backcross recombinant inbred line populations were constructed using drought-resisting line DY80and early indica restorer line R974 of DXWR.Next,QTL localization and candidate gene analysis were performed for root system characters of DXWR,including deep root number,shallow root number,total root number and deep root ratio.On this basis,important candidate genes related to such root system characters were acquired,thus laying a foundation for mining and applying drought-evading genes of DXWR.