QTL Mapping Of Drought Related Traits And Isolation And Functional Analysis Of VrERF1 Gene In Mungbean

The rising drought stress caused by climate change has brought severe challenges to the global food security.It is very important for food security to breed varietiy with drought tolerance and stable yield in various crops.Mungbean?Vigna radiata L.?is one of the most important food legume crops.However its genetic research is relatively backward than other major crops,and the researches on QTL mapping of drought resistance traits and gene cloning are scarce up to date.In this study,for the purpose to improve the molecular breeding of drought tolerance and accelerate drought resistant breeding process in mungbean,on the one hand,we developed a full-coverage genetic linkage map based on simple sequence repeat?SSR?markers using a recombinant inbred line?RIL?population derived from an intra-specific cross between two different drought-resistant varieties,and we subsequently detected QTLs for plant height?PH?,the maximum leaf area?MLA?,biomass?BM?,the relative water content?RWC?and seed yield?Yield?in irrigated and drought environments at two locations;on the other hand,after differential expression gene screening under PEG simulated drought stress,we cloned a AP2/ERF transcription factor gene from mungbean,and analyzed its gene function by genetic transformation of Arabidopsis thaliana.Our results are mainly as follows:1.A RIL population consisting of 256 lines was obtained from intra-species hybridization of two different drought-resistant mungbean varieties.The population was developed through single-seed descent from the F₂ to F₆ generations.We measured PH,MLA,BM,RWC,and Yield of this RIL population and its parents under well-watered and drought stress treatments at two locations?Shijiazhuang and Beijing?.There were significant differences between the parental lines for all traits,except for RWC in the open field experiment at Shijiazhuang location.In comparison,only MLA was significantly different between the parental lines in the pot culture experiment at Beijing location.The RIL population showed a high degree of morphological variation with a continuous distribution and exhibited quantitative inheritance and bidirectional transgressive in segregation was observed for all of the traits measured at each location and under each water treatment.The correlation analysis showed that PH,MLA,BM,and Yield were positively correlated with each other.2.A total of 3,690 SSR markers were used to screen for polymorphisms between the two parental genotypes,which included two main classes.Among which,1,695 SSR primer pairs were newly designed from the mungbean genome databases,and the other 1,995 SSR primer pairs were from mungbean transcriptomes and related references.After screening,we obtained 423 polymorphic SSR markers between the two parental genotypes.3.Finally,a total of 312 co-dominant SSR markers and one phenotypic marker?young stem color?were employed for the construction of a genetic linkage map.The final map was anchored with 313markers,and the total map length was 1010.18 cM across 11 linkage groups,covering the entire genome of mungbean with a saturation of one marker every 3.23 cM.There was no gap greater than 20 cM on this map.Comparative analyses were performed between the linkage map and a former linkage map of mungbean.The 24 similar markers between our linkage map and the former linkage map showed high conservation of orders and positions.In addition,the BLAST analysis demonstrated that all 11 linkage groups in both linkage maps were consistently mapped to the same chromosomes,which implies a high quality of the present linkage map,and it could be useful in comparative analysis and QTL mapping.4.We detected QTLs for PH,MLA,BM,RWC and Yield in irrigated and drought environments at two locations?Shijiazhuang and Beijing?by using the novel genetic linkage map.A total of 50 QTLs were detected based on single-environment analysis,with the loci accounting for 2.86%to 29.45%of the total phenotypic variation.Thirty-four of these QTLs were consistently detected two or more times at similar linkage positions.Notably,qPH5A and qMLA2A were consistently identified in the marker intervals from GMES5773 to MUS128 in LG05 and from Mchr11-34 to the HAAS_VR₁812 region in LG02 at four environments,contributing 6.40-20.06%and 6.97-7.94%of the observed phenotypic variation,respectively.The results of these analyses will facilitate the isolation of drought-related genes and help to clarify the mechanism of drought tolerance in mungbean.5.Several AP2/ERF transcription factors were isolated from Unigenes of mungbean transcriptomes previously,and were screened for differential expression under PEG simulated drought stress in this study.Among which,VrERF1 was found to be differentially expressed at different stages of the treatment.Therefore,we amplified it from the cDNA and genome DNA of mungbean.After gene cloning and sequencing of VrERF1,we found there was no intron in its nucleotide sequence.It encodes an ERF transcription factor protein which includes 167 amino acid.And there is a typical AP2/ERF domain of the 95¹55 amino acid sequence.A pGFPGUSPlus-VrERF1 expression vector was constructed for sub-cellular localization.The VrERF1 gene was expressed in the aerial root induced by Agrobacterium rhizogenes K599 which harboring the pGFPGUSPlus-VrERF1 expression vector.The result observed by a fluorescence confocal microscopy indicated the protein of VrERF1 was located at the nucleus.The VrERF1 gene was transformed into wild-type Arabidopsis thaliana by Agrobacterium tumefaciens GV3101 for over expression.The sensitivity of transgenic Arabidopsis plants to salt stress at germination stage was higher than that of wild type;however,the salt tolerance at seedling stage was significantly higher than that in wild type,suggesting that VrERF1 gene was involved in the response to salt stress.In addition,a drought stress trial showed significantly survival rate in transgenic Arabidopsis plants than in wild type plants,suggesting that VrERF1 gene was also involved in the response to drought stress.