QTL Mapping Of Ear-kernel Related Traits In Maize(Zea Mays L.)

Ear-kernel related traits are the key effect that determine the yield per ear and thus the yield per plant.Breeders have studied the genetic developmental characteristics of ear-kernel related traits and increased the yield of maize hybrids and meet the growing human demands for maize derivatives.Therefore,the study of maize ear-kernel related trait loci（QTL）,and subsequent discovery of related genes,not only provided a theoretical basis for improving maize yield,but also laid the foundation for molecular improvement and genetic mutation mechanisms in maize breeding.In the present study,a distinctive line,EHel（extremely low Ear Height,derived from Dan299×Pioneer hybrid）was crossed with B73 to construct a segregating populations of F₂ and F_2:3.Phenotypic characterization of seven ear-kernel related traits,i.e.ear length（EL）,ear diameter（ED）,kernel row number（KRN）,ear weight（EW）,ear-kernel weight（EKW）,50-kernel weight（50KW）,and kernel ratio（KR）,was performed for parental lines of EHel and B73,F₂,and F_2:3.Besides,SNP chipset consisted of～10000 SNPs was used to genotype EHel,B73,and all F₂ individuals for QTL detection of seven ear-kernel related trais.Based on the QTL mapping results,the annotated genes within the QTL regions were selected for further verification.The main findings are as follows:1.Phenotypic characterizationPhenotyping results showed that EHel possessed statistical lower ear length（EL）in both 2018（P=0.0002）and 2019（P=0.0047）than that of B73.While no significant differences were observed on both ear diameter（ED）and kernel ratio（KR）between EHel and B73 in both years.In addition,EHel and B73 exhibited non-statistical difference on both kernel row number（KRN）and 50-kernel weight（50KW）in 2018,while in 2019,extremely significances were obserbed on both traits of KRN（P=0.008）and 50KW（P=0.0000）.Contrary trends were obsereved on the other pairs of traits,i.e.ear weight（EW）and ear-kernel weight（EKW）.EHel presented significantly lower EW（P=0.002）and EKW（P=0.011）than those of B73 in 2018,while non-statistical differences were detected on both traits between EHel and B73 in 2019（P=0.38 for EW,and 0.73 for EKW）.Pearson correlation analysis results of 7 target traits via F_2:3:3 population showed that pairs of 5 traits,i.e.EL,ED,KRN,EW,and EKW,possessed significant positive correlation between each other,and the correlation coefficient ranged from 0.41（EL vs KRN）to 0.99（EW vs EKW）.Besides,positive correlation was also observed among trait pairs of KR and the other 3 traits,i.e.KRN,EW,and EKW,and the correlation coefficient ranged from 0.45（KR vs KRN）to 0.62（KR vs EKW）.Trait pairs of 50KW vs the other6,i.e.EL,ED,KRN,EW,EKW,and KR,exhibited weak correlations,and the coefficients were less than 0.1,except pairs of 50KW vs EL（-0.17）and 50KW vs EKW（0.10）.Non-significant positive correlations were also observed on both pairs of KR vs EL,and KR vs ED.2.Construction of genetic map and QTL mappingFrom the about 10,000 SNPs integrated by 10K chip,2108 SNPS with stable polymorphism between EHel and B73 were screened out to construct the linkage map.The total length of the linkage map constructed by 2108 polymorphic SNPS was 3155.30cM,and the average spacing of markers was 1.50 cM.Among 10 chromosomes（Chr）（or10 linked groups）,five chromosomes,i.e.Chr1-Chr4,and Chr8 captured more than 200SNPS each.Considering the average length of pairs of SNPs,six chromosomes,i.e.Chr1（1.09 cM）,Chr2（1.20 cM）,Chr4（1.11 cM）,Chr6（1.32 cM）,Chr8（1.46 cM）,and Chr10（1.14 cM）possessed smaller average length than the total average（1.50 cM）of the whole genome.The results of QTL mapping showed that a total of 54 QTLs controlling six target traits were detected among all 10 chromosomes.Among these 54 QTLs,Ten located on Chr1,controlling 4 traits of ED,EW,50KW,and KR.Three QTLs located on Chr2,one for EL,thr other two for KR.Four corresponding QTLs separately located on Chr3,Chr4,Chr7,and Chr10,controlling EL,ED,50KW,and KR.Additionally,QTLs controlling ED and KR on Chr10 located in the same maker interval.QTLs for KRN only located on Chr6（qKRN6-1 and qKRN6-2）and Chr9（qKRN9）.Besides,QTLs for EL,ED,50KW,and KR were also detected to locate on Chr6.7 QTLs located on Chr8,among which,2for EL and KR,respectively,and 3 for 50KW.Aong 10 chromosomes,Chr1 and Chr5 captured 10 and 15 QTLs,respectively,higher than the other chromosomes.Chr9 captured the lowest QTL,only one for KRN.The genome-wide distribution of detected QTLs suggested unequal characteristics of the candidate regions,and possibile genes as well,for all target traits along the whole genome of maize.In addition,some of QTLs responsing for different traits were detected to locate at the same chromosomal region,reflecting the characteristics of one gene for multiple effects.3.SSR maker selection and verification based on BSABSA（Bulked Segregation Analysis）strategy based on F₂ individuals was used to carry out retesting of target traits.The retesting traits selected were EL and 50KW,for both traits presented statistical differneces in 2019 between parental lines of EHel and B73,and the PVE of detected QTLs for both traits were larger than 10%.The results of BSA analysis indicated a homogous segment（EHel/EHel）among selected F₂ individuals with lowest performance of EL and 50KW between SNPs of AX-86326358 and AX-90635232（7.07 Mb）on Chr5.The left border of this homogous segment neighbored to the detected QTLs for EL and 50KD.Polymorphic SSRs located within this segment were screened out to shorten the candidate region,and finally the possible targed region for EL and 50KW werer fixed to the interval of umc1072 and umc1792.Within this region,the annotated gene,Zm0001d018208 presented the highest expression level in maize ear than the others,and was selected as the possible candidate gene for EL and 50KW in the present study.4.Sequence alignment and expression analysis of Zm0001d018208Through the method of Escherichia coli monocloning,Zm00001d018208 was sequenced via EHel and B73 according to the reference genome sequence（B73RefGen＿v4,Reference）.Allignment results showed that sequence cloned from pollen parent of B73 matched 100%to that of the Reference,exhibiting no polymorphism.While nine polymorphic sites were observed between sequence pairs of EHel and B73,in cluding eight single nucleotides changings,and a 3 bp deletion,resulting five amino acids changing and one amino acid deletion between the predicted coding products.Real time quantitative PCR（QPCR）was used to reveal the relative expression of Zm00001d018208 among four tissues,i.e.developing ear,root,stem and leaf,of EHel and B73.Results showed that Zm0001d018208 exhibited statistical higher relative expression level in all four issues of EHel than those of B73,respectively.Besides,Zm0001d018208 presented the highest expression level in developing ear of EHel（>20）,more than 4 times to that of B73,suggesting that this gene might serve as a negative regulator for the performance of both EL and 50KW in maize.