Fine Mapping Of Maize Inflorescence Architecture Traits Pedicellate Spikelet,Sessile Spikelet And Tassel Branch Number

Maize（Zea mays ssp.mays L.）is an important crop in our country,which was used as food,feed and industrial raw materials;it plays a key role in national food security and economic development.Because the increase of corn consumption and the reduction of cultivated land area,we believe that the main subject of maize production and breeding is high yield in the furture.Maize inflorescence archeticture traits were domesticated in the domestication of maize,which also affect yield.Hence,dissection of the genetic basis of inflorescence archeticture traits is help to maize genetic improvement and high yield breeding,and elucidates the molecular mechanism of inflorescence development.In this study,three inflorescence traits were chosen as follow:the pedicellate spikelet aborted reduce kernel number by half（PEDS）,the sessile spikelets develop defect（Sos1）also reduce kernel number by half,the tassel branch number（TBN）affect the yield through nutrition distribution and plant archeticture,to dissect the genetic basis,fine mapping of major loci and candidate gene association analysis.It will help clone the gene and dissect the gene regulator network for inflorescence development,and reveal the domestication mechanism of maize.The main results are as follows:1.For PEDS that double kernel number:（1）maize inbred line SICAU1212 was used as receptor parent and MT1（Zea mays ssp.mexicana）was used as donor parent to develop the dvanced backcross population by“backcross and selfed model”based on the target trait（singe spikelet,PEDS=100%）.（2）Five extreme trait bulks were develop as follow:50 plants with PEDS≥90%were chosen to construct one high extreme phenotype bulk in the BC₃F₂population with 5022 plants in 2014 EEDS environment;100 plants with PEDS≥90%were chosen to marker Y1-Y100,25plants（Y1-Y25）was used as the common plants,to construct three high extreme phenotype bulks with 25 plants（Y26-Y50）,25 plants（Y51-Y75）,25 plants（Y76-Y100）,respectively;50 plants with PEDS=0 was chonse to construct one low extreme phenotype bulk in the BC₃F₂population with 7990 plants in 2014 YN environment;50 plants with PEDS>0 were chosen to construct one high extreme phenotype bulk in the BC₃F₂population with 6720 plants in 2015 YN environment.To perform QTL-seq,we find the SNP-index values located in the chromosome 1L,chromosome 3,chromosome 6S and chromosome 8S were difference between high and low bulks,which may be the regions related to PEDS;Combined with theΔ（SNP-index）,six candidate genomic regions were detected for PEDS（P<0.05）,located on chromosome 1L and chromosome 3 under null hypothesis.（3）To verify the regions detected by QTL-seq,traditional QTL analysis was conducted in the BC₃F₂ populations with 300 and 450 plants under two environments and BC₄F₂population with 700 plants under one environment.As a result,eight QTL related to PEDS were detected on chromosome 1,3,6 and 8,which overlapped to the regions identified by QTL-seq,and the PVE was 1.67%（q15WPEDS1-2）-40.38%（q15WPEDS3-1）.（4）Fine mapping of major QTL（q PEDS3-1）:a total of 1203 plants with PEDS≥20%were chosen from BC₃F₂ and BC₅F₂ populations,which were genotyped by markers chr3-5434 and chr3-1975.There are 240 recombination events at chr3-5434 and 84 recombination events at chr3-1975,so we develop high resolution In Del markers between chr3-5434 and chr3-1975;finally,the q PEDS3-1was narrowed down between PM30 and PM31,the physical position is about 171 kb,and three genes（candidate gene1,candidate gene2 and candidate gene3）locate in the region based on the reference genome sequence（B73-Ref Gen＿v4）.（5）Candidate gene 2 was selected for candidate gene association analysis according to the expression datas in different tissues of maize and homologous gene analysis.First,the full-length of candidate gene 2 allels between parents were cloned by PCR,and a 12bp insertion deletion site（cg-In Del4-1）was indentified in exon 4.The cg-In Del4-1was clone in 114 maize inbred lines（Tropical,NSS and SS is 47,24 and 43,respectively）and 89 teosintes,combined with the PEDS phenotype,to perferm the candidate gene association analysis,as a result,cg-In Del4-1 is significantly associated with PEDS.2.For Sos1:（1）the mutant Sos1 was used as the common parent,crossed to B73,Mo17,RP125 and SICAU1212,to develop segregating populations.The phenotype of F₁ suggested that Sos1 was controlled by dominant gene in Mo17 and SICAU1212genetic background,and was controlled by semi-dominant gene in B73 and RP125genetic background.The ratio of Sos1 phenotype in four F₂ populations indicated that Sos1 was controlled by a single gene.（2）Sos1 was mapped on chromosome 4S,flanked by markers chr4-6475 and chr4-10247,and the PVE near to 100%in four F₂populations.（3）To fine mapping of Sos1,endosperm of 18463 seeds were genotyped by markers chr4-5415 and chr4-10247 that contain Sos1 region;combined with the Sos1 phenotype in the field and high resolution In Del markers,Sos1 was mapped between chr4-SM8 and chr4-SM3,the physical position is about 63 kb;then four key recomibation plants were genotyped by GBS,as a result,Sos1 was narrow down 27kb,no gene was annotated in this region based on the reference genome sequence（B73-Ref Gen＿v4）.3 For TBN:（1）The BC₄F₂ population was developed by“backcross and selfed model”based on the target trait（TBN）derived from maize inbred line 18-599 with TBN=9-11 and 3237 with<1 TBN.（2）Two extreme bulks were constructed by chose 24 plants with TBN≤3 and TBN≥13 from BC₄F₂ population with 399plants in the 16WJ environment.Thirteen candidage genomic regions（P<0.05）were identified under null hypothesis by QTL-seq,located on chromosome 2 and chromosome 5.（3）To verified the candidage genomic regions detected by QTL-seq,BC₄F₂ population with 227 plants in the 16WJ environment and BC₄F₂ population with 192 plants in the 16JH environment were selected to perform QTL analysis;a total,three QTL（q16TBN2-1,q16TBN2-2 and q16TBN5-1）were indentified,and the PVE was 6.13%-18.79%.（4）For each major QTL（q16TBN2-2 and q16TBN5-1）,the plants that QTL region is heterozygous and the other regions are homozygous were chosen and selfed to develop RHLs.In the RHLs,two QTL（q16TBN2-2.1 and q16TBN2-2.2）were mapped in the q16TBN2-2 region,and the PVE were 6.96%and21.57%,respectively;one QTL（q17WTBN5-1）was detected that is same as q16TBN5-1,and the PVE was 30.75%.（5）The major QTL（q TBN2-2）was chosen to fine mapping:the endosperm of 7968 seeds from BC₄F₄ families was genotyped by markers In Del3 and In Del6 to screen recombination events,and 494 recombination events were detected.To exclude the effect of q16TBN2-2.1 flanked by In Del3 and In Del4,the plants that q16TBN2-2.1 was 18-599 genotype and the recombination events happened between markers In Del5 and In Del6 were chosen.Recombinant homozygotes were developed by selfed and MAS,and TBN were investigated in the recombinant homozygote families and T-test were performed;finally,q TBN2-2 was mapped between TM5 and TM2,the physical position is about 39 kb,only one gene located in this region,name TBN2-2.2.（6）For candidate gene association analysis,105 maize inbred lines（Tropical,NSS,SS is 42,22,41,respectively）were chosen to PCR amplification the sequence of TBN2-2.2,600 bp 5’-upstream of TBN2-2.2 and100 bp 3’-downstream of TBN2-2.2,a total about 2200 bp;combining with the TBN phenotype of 105 inbred line under three environments,two significant loci were identified association with TBN in the exon and 3’-UTR of TBN2-2.2,which consist of 4 haplotypes.Haplotype 1 increased TBN and haplotype 2 reduced TBN,the proportion of Haplotype 1 decreased significantly and Haplotype 2 incresed significantly in the improvement of from tropical maize to temperate maize.We found TBN2-2.2 had undergone strong reduction in nucleotide diversity from teosintes to maizes withπmaize/πteosinte=0.19,indicating that TBN2-2.2 was under selection during domestication from teosinte to maize.