Mapping And Functional Analysis Of The Candidate Genes For Zigzag Stem And Vine Growth Habit In Soybean

Breeding of excellent plant types could improve light energy utilization and crop yield.Zigzag stem and stem growth habit are two different plant phenotype of soybean.Zigzag stem soybean has the characteristics of bending main stem,reducing plant height,shortening internode distance,etc..Vine growth and erect growth are two types of stem growth habit in soybean.Vine growth habit is more common in wild soybean,with the characteristics of lengthening main stem,increasing node number,climbing growth.It is beneficial to study the genetic mechanism of zigzag stem and vine growth habit for soybean genetic improvement.Information of genomic sequence provides access to isolate target genes and analyze their genetic mechanism.In this paper,the results of linkage mapping of zigzag stem were verified by near isogenic line re-sequencing,and the candidate genes of vine growth growth were analyzed by population sequencing data.Candidate gene’s function of zigzag stem and vine growth habit of soybean were verified in different methods.The main contents and results are as follows:1.Linkage location results of soybean zigzag stem were verified by near isogenic line re-sequencing.GmCRF4 was important candidate gene for zigzag stem.Near isogenic line re-sequencing can detect different segments in order to verify the results of linkage location.The results showed that 90%of the sites were the same between zigzag and straight stem in near isogenic lines,except in the region 47.5-48.5M of chromosome 14.The separated region was consistent with results of gene linkage analysis as Chr14:47661103-47901399,indicating that this region was the main regulatory region of zigzag stem.Through the re-sequencing and PCR analysis of the candidate genes in the linkage mapping region,it was found that Glyma14g38610 was an important candidate gene,and there were differences between the two near isogenic line of straight stem and zigzag stem materials in sequence and expression levels.This gene belongs to the CRF subfamily of ERF gene family,and it is the homologous gene of Arabidopsis CRF4.This gene changed an amino acid residue in zigzag stem soybeans.2.GmCRF4 regulated soybean’s plant height and growth period by transgenic experiment.Subcellular localization of GmCRF4 was only in the nucleus,consistent with the inference that GmCRF4 belongs to ERF transcription factor gene family.The expression of endogenous GmCRF4 was interfered by BPMV-VIGS.Zigzag stem appeared on 5-8 nodes of main stem 14-21 days after interference,with the expression level of GmCRF4 as much as 6.8%of contrast plants.Over expression of dominant GmCRF4 in Tianlong No.1 resulted in higher plant height and loner growth period,with a significantly higher expression of GmCRF4 as about 8.5 times of the contrast plant.Gene silencing of GmCRF4 resulted in opposite phenotype of over expression,just as lower plant height and shorter growth period.qPCR showed that the expression of GmCRF4 gene in gene silencing plants decreased significantly as only 39%of the contrast plant.GmCRF4 gene silencing did not show a typical zigzag stem,perhaps because of the genetic basis of the transformation receptor.3 QTLs of vine growth habit were detected.Therefore combined with separated sites between vine and erect population,candidate genes of vine growth habit were screened out.The candidate gene VGH1 belongs to the GAox gene family,so the phylogeny and population differentiation of soybean GAox were analyzed.Based on the high-density genetic map of two recombination inbred population(NJRINP,NJRI4P)derived from the cross of two erect soybeans and wild vine type soybeans PI342618B,and combined with the phenotypic data of two years in two growth periods(flowering and maturity),the QTL linkage mapping of vine was carried out.And 10 QTLs including qVGH-18-1,qVGH-18-2,qVGH-19-3 and qVGH-19-4 were detected,among which qVGH-18-2 was detected for 7 times with correlation coefficient R2(12%-23%).Compared with differentiation gene or site between 18 vine and 14 erect soybeans(targeted by FST),many differentiation sites were found in qVGH-18-2 rather than three other main QTLs.It was found that there was a highly differentiated gene VGH1(glyma18g06870)in qVGH-18-2.Compared with the erect type of cultivated soybean,the VGH1 gene of wild soybean PI342618B has three codon deletions at 18N position as an important candidate gene.VGH1 gene belongs to gibberellin 2-oxidase.In addition,GA2ox was also found in the other two important QTLs q VGH-18-1 and qVGH-19-3.Moreover soybean podding habit gene Dt1 was found in q VGH-19-3,which suggested that GAox and Dt1 may be involved in the regulation of soybean vine growth habit.Furthermore the phylogenetic analysis of soybean GAox was carried on.A total of 16 C19-GA2ox,13 C20-GA2ox,12 GA3ox and GA20ox were identified in soybean genome.The differentiation genes of GAox in 18 vine and 14 erect soybeans were analyzed.Finally,many differentiation genes with FST>0.25 were identified in GAox.4.The transgenic experiment showed that VGH1 regulated the number and length of soybean nodes,which affected the vine growth habit of soybean.Over-expressing of VGH1 gene in Tianlong No.1 decreased the number of nodes by 15.7%and increased the internode distance by 23.3%;and knockout plants of VGH1 gene by CRISPR/cas9 increased the number of nodes by 13.8%and decreased the average length of nodes by 38.9%.VGH1 regulated the number and average length of soybean nodes affecting the vine growth habit of soybean.Vine soybean has the characteristics of increasing node number and climbing growth.VGH1 was mutant type in wild vine soybean PI342618B,and wild type in cultivated parents.The absence of VGH1 in wild soybean could lead to increasing node number and shortening node length,which regulated vine growth of soybean.This mutation of VGH1 in wild soybean was helpful to increase the number of nodes leading to vine growth,which could improve the adaptability of wild soybean to the natural environment.