Genome-wide Association Studies And Important Gene Mining Of Maize Stalk Strength Related Traits

Maize lodging seriously affects yield and mechanized harvesting.The occurrence of lodging will reduce the number of kernels per ear and 1 000 grain weight,resulting in yield reduction.It also leads to an increase in the number of dropped ears,the occurrence of moldy or germination of grains,an increase in crop harvesting difficulty,and a decrease in crop quality.Maize stalk strength is closely related to stalk lodging.However,the mechanism of maize stalk strength formation is still unclear.In this study,301 maize inbred lines were used as materials to investigate the stalk strength-related traits,and the traits were analyzed by genome-wide association analysis.Maize inbred lines with extremely strong and weak stalk strength were selected for stem anatomical analysis.The F2 population and F2:3 families with stiff-stalk line HB08F1 and non-stiff-stalk line SJ201304 were used for BSA-seq analysis.The mechanism of maize stalk strength formation was further analyzed by transcriptome analysis.Combined with transcriptome analysis,the differentially expressed genes in the candidate interval of significantly SNP loci with stalk strength in GWAS were mined.The main results were as follows:1.The rind penetrometer resistance at 20 days after flowering(F20RPR)and maturity(MRPR)stages and three-point bending test(TPBT)of the inbred line population in 2016 in Tai'an(TA16)and 2017 in Tai'an(TA17)and Wenkou(WK17)were measured,and then the BLUE values were calculated respectively.There was a wide range of variations in stalk strength.The variation coefficient of F20RPR is 18.82%–21.23%,MRPR is 18.72%–24.56%,and TPBT is 40.54%–54.68%.2.The results of the correlation analysis show that F20RPR,MRPR,and TPBT are significantly positive correlations.Internode length was positively correlated with RPR(p<0.001),while stem diameter was negatively correlated with RPR(p<0.001).The TPBT was a positively correlated with internode length only in TA17 and WK17(p<0.05),and the correlation between stem diameters and TPBT was not significant.The dry weight,dry weight per unit length,and the weight of hemicellulose,cellulose,and lignin per unit length were significantly positively correlated with the stalk strength(p<0.001).Ten stiff-stalk lines and11 non-stiff-stalk lines were selected by cluster analysis.It was found that the difference in stalk strength between stiff-stalk lines and non-stiff-stalk lines was related to the thickness of the rind and the cell wall thickness of the rind and vascular bundle sheath cells.There was no significant difference in the percentage of hemicellulose,cellulose,and lignin in stiff-stalk lines and non-stiff-stalk lines.However,compared with non-stiff-stalk lines,stiff-stalk lines contain more hemicellulose,cellulose,and lignin per unit length of the internode.3.Genome-wide association studies(GWAS)were performed on RPR and TPBT combined with 41074 high-quality SNPs.At P?2.43×10^-5(-log₁₀P=4.61),11,6,and 7significant SNPs were detected in F20RPR,MRPR,and TPBT,respectively.At P?2.43×10^-4(-log₁₀P=3.61),F20RPR,MRPR,and TPBT detected 75,37,and 29 significant SNPs,respectively.Based on the GWAS results of F20RPR,MRPR,and TPBT,the significant SNPs detected in at least two traits were speculated to be associated with stalk strength.Twenty SNPs significantly associated with stalk strength were screened,which were located on chromosomes1,3,4,7,9,and 10.GWAS analysis of HWUL and CWUL,at P?2.43×10^-5(-log₁₀P=4.61),both HWUL and CWUL detected two significant SNPs.At P?2.43×10^-4(-log₁₀P=3.61),HWUL and CWUL detected 23 and 14 significant SNPs,respectively.4.BSA-seq analysis of the F2 population and F2:3 population constructed by stiff-stalk line HB08F1 and non-stiff-stalk line SJ201304,two association regions of maize stalk strength were identified,which were 239.1 Mb–272.0 Mb on chromosome 1 and 157.1 Mb–192.9 Mb on chromosome 3,respectively.The significant SNPs PZE-103090393,PZE-103103090,and PZD00008.3 in the GWAS overlapped 157.1 MB–165.0 MB with the association region of chromosome 3 of BSA-seq,which is the common interval between GWAS and BSA-seq.5.Transcriptome analysis was performed using stiff-stalk lines HB08F1 and A801,and non-stiff-stalk line SJ201304.Transcriptome analysis of maize stalk at different growth stages showed the involvement of hydrolyzing O-glycosyl compounds and cytoskeleton organization-related genes in internode development at the ninth leaf stage.Cell wall metabolism-related genes are implicated in the internode development of inbred lines with different stalk strengths at the tasseling stage,and the GO term significantly enriched in the biological process group is a microtubule-based process.Therefore,cell wall metabolism and cytoskeleton related genes might play important roles in internode development.6.The candidate intervals of SNPs significantly associated with stalk strength in GWAS were screened by transcriptome data from stiff-stalk lines HB08F1 and A801,and non-stiff-stalk line SJ201304,and 26 differentially expressed genes were obtained.SYN3989,a SNP significantly associated with stalk strength on chromosome 1,had a maximum-log₁₀P of 5.16in TPBT-Blue,and the phenotypic variation rate R² was 8.63%.SYN3989 is located in the gene Zm00001d029343,which is predicted to encode an F-actin capping protein?subunit,which is a homologous gene of At CPB in Arabidopsis so it is named Zm CPB.Meanwhile,Zm CPB was also enriched in cytoskeleton organization(GO:0007010,p=1.00×10^-2)in RNA-seq analysis.In the preliminary investigation of the RPR of the Mu mutant cpb of Zm CPB,the results showed that the MRPR of cpb was significantly lower than that of B73(p?0.01).At present,no study has proven that the F-actin capping protein can affect the development of the thick-walled cell wall of maize stems.Therefore,mining the F-actin capping protein?subunit gene Zm CPB is innovative and feasible as an important gene of maize stalk strength.7.The NAC and MYB transcription factors related to secondary cell wall synthesis in maize stems were screened,using the transcriptome of different parts of the basal third internode that are developing at the ninth leaf stage in the stiff-stalk lines HB08F1 and A801,and the non-stiff-stalk line SJ201304.The results showed that Zm NAC87,Zm MYB109,Zm MYB 91,Zm MYB 76,Zm MYB 142,Zm MYB 106,and Zm MYB 37 may be related to maize stalk strength.Because Zm NAC87 is differentially expressed between stiff-stalk lines and non-stiff-stalk line,q RT-PCR analysis found that Zm NAC87 is differentially expressed between multiple stiff-stalk lines and non-stiff-stalk lines.Therefore,it is speculated that Zm NAC87 regulates the development of secondary cell walls in maize internodes.