QTL And EQTL Mapping Of Soybean Tolerance To Aluminum Toxicity

Soybean[Glycine max(L.)Merr.]is one of the most important oilseed crops worldwide,which has a wide range of applications in human food,animal feed and industrial production.Aluminum(Al)is the most abundant metal element in the crust,and when the soil pH<5.0,the solid Al changes into a dissolved ionic state.Micro-molar levels of Al3+ can cause poisoning to plants in a matter of minutes,and the lower the pH,the greater the solubility of Al and the greater the toxicity to plants.Previous studies have shown that Al tolerance in soybean is a complex quantitative trait controlled by multiple genes.Therefore,it is of great significance to identify soybean Al tolerance genes by QTL and eQTL mapping and explore the molecular mechanism of soybean tolerance to Al toxicity,which is important to soybean breeding for Al tolerant varieties to improve agricultural production on acidic soil.In this study,we identified two soybean varieties with different Al tolerance,M90-24(Al tolerance)and Pella(Al sensitive),and their derived F2:10 generation of the recombinant inbred lines(RIL,NF2048)were evaluated for Al tolerance,and a high density genetic map was constructed.We used the root growth(RGAl)of soybean primary roots under Al treatment and the relative root growth(RRG)as phenotypic traits for QTL mapping.The differentially expressed genes(DEGs)between Al and control as well as between two parents were analyzed.The results from QTL mapping and DEGs were used to select the Al tolerance candidate genes,and the expression of the candidate gene in the RIL population NF2048 was analyzed by qRT-PCR for eQTL mapping.The main results of this study are as follows:1.QTL mapping of Al tolerance in soybean RIL population NF2048.The RIL population NF2048 was sequenced by RAD-seq,and the genetic map was constructed with a total of 2796 bin markers.The average genetic distance of adjacent bins was 1.491 cM and the average physical distance between adjacent bins was 339 kb.Among the three traits,RGAl and RRG showed a significant positive correlation(correlation coefficient was 0.80 and p<0.0001),while RGCK and RRG were not correlated,which indicated that the higher the RGAl,the greater the RRG,the stronger the tolerance to Al.The Multiple Interval Mapping(MIM)model in WinQTL Cart software was used to map QTL.Under MIM,nine and eight QTL for RGAl and RRG were detected,respectively.The QTL on chromosome 18 explained 16.01%of the phenotypic variation for RGAl and 17.52%of the phenotypic variation for RRG,which is therefore considered as a major QTL,and the additive effect came from the tolerance parent M90-24.The candidate Al tolerance genes could be further explored in this region.2.Comparative transcriptome analysis of the Al tolerant and sensitive parents of the soybean RIL population NF2048.RNA-seq was performed using 16 libraries made from the root tips of M90-24(M)and Pella(P)at 6 h and 12 h Al treatment and control using the Illumina HiSeq 2500 sequencing platform.The differentially expressed genes(DEGs)were selected according to the thresholds of False Discovery Rate(FDR)<0.05 and |log2(Fold Change)|>1.There were 1799 genes showed up-regulated by Al stress specific to M but not P,and 1050 genes were induced by Al in both varieties but with more fold change in M than P.Among these 2849 genes,there were 354 genes also showed higher expression levels in M than P under Al stress,which were enriched in the cellular glucan metabolic process(including cellulose synthase and xyloglucan endo-transglycosylase).The cellulose synthase inhibitor,indaziflam,was used to investigate the role of cellulose synthase in soybean tolerance to Al toxicity.The results confirmed that RRG of M90-24 decreased after adding the cellulose synthase inhibitor.Furthermore,10 samples of extreme Al tolerance and 10 samples of extreme Al sensitive lines from the soybean RIL population NF2048 were selected for qRT-PCR,and the results showed that the relative expression of two cellulose synthase genes was positively correlated with RRG.The results suggest that cellulose synthase might play important roles in soybean tolerance to Al toxicity.3.eQTL mapping of Al tolerance in soybean RIL population NF2048.Based on the above results of QTL mapping and transcriptome analysis,Glyma.18G080600(GmCYP 71)was selected as Al tolerance candidate gene,which is near the major Al tolerance QTL on chromosome 18.The relative expression of GmCYP71 by qRT-PCR was positively correlated with RRG in the RIL population NF2048.The eQTL mapping of GmCYP71 gene was carried out using the genetic map.A total of 29 eQTL were detected by MIM model using WinQTL Cart.The eQTL located on Chr.18 between bin58-59 could explain more than 29%of the variation in the relative expression of GmCYP71,and this eQTL located near the physical position of GmCYP71 which is therefore a cis-eQTL.We also found 27 trans-eQTL for GmCYP71.However,only two trans-eQTL could explain more than 10%of variation in the relative expression of GmCYP71.The results showed that the expression of GmCYP71 gene was controlled by both cis-acting and trans-acting QTL.