| Maize?Zea mays L.?is one of the most important cereal crops worldwide.The kernel rows number?KRN?of maize is one of important components of the grain yield.The KRN as a quantitative trait is genetically controlled.Considerable efforts have been made to identify quantitative trait loci?QTL?for KRN.However,up to now few genes have been characterized in maize.Therefore,identifying and cloning key genes that control the variation of KRN is useful to illustrate the genetic basis and regulatory network of grain yield,which will provide theoretical guidance and gene resources for high-yield breeding practice.In this study,we performed fine mapping of QTL for KRN and cloning of the candidate gene using the map-based cloning strategy,and analyzed genetic variation and biological function of the candidate genes by resequencing,expression analysis,protein activity detection and genetic transformation.The major results are as follows:1. Construction of near-isogenic lines and QTL confirmationWe screened plants with the heterozygous q KRN5 segment in advanced backcross population derived from NX531 and SIL8 using q KRN5 flanking markers:umc1365and umc2512.One heterozygous plant with a 96.2%of background recovery rate was self-crossed,q KRN5NX531and q KRN5SIL8with homozygous q KRN5 region were used to develop near isogenic lines?NILs?.The width of the ear inflorescence meristem?IM?was significantly different between two NILs(p=3.5×10-7).KRN,kernel number and grain weight per ear were significantly different,while morphological traits and flowering time were the sam between NILs.The results indicated this QTL region controls the size of the IM,KRN and grain yield.2. Dissection of q KRN5Nine individuals with heterozygous recombinant segement in q KRN5 were identified in advanced backcrossed population derived from the two NILs with 12 new developed markers.Combining the phenotype of homozygous progenies from each recombinant individuals with genotypes,q KRN5 was divided into two tightly linked QTL in coupling phase.QTL q KRN5a with 0.34-0.39 additive effect mapped into an approximately 884.5 kb region flanked by SC533 and SC520.QTL q KRN5b with0.93-0.94 additive effect was narrowed into an approximately 1.07 Mb region flanked by bnlg1879 and umc2293.The physical distance between these two QTL was 1.04Mb.3. Fine-mapping and cloning of q KRN5bFive heterozygous recombinant chromosomes were identified in segregation population using 4 new developed markers in q KRN5b.Each recombinant contains5-10 individuals.By progeny test under 4 environments in 2 years,we delineated q KRN5b into a 32.05 kb region flanked by SC36031 and K13.Only one gene was annotated in this region,and was referred to as KRN5b.One SNP at 794 bp of KRN5b coding sequence was identified between q KRN5NX531and q KRN5SIL8,which resulted in an amino acid substitution.In addition,many sequence variations were found in the non-coding regions and promoter of KRN5b between q KRN5NX531and q KRN5SIL8.4. Expression of KRN5b and its protein functionKRN5b was highly expressed in the ear and the tassel,and the relative expression level of KRN5b was 1.5 fold higher in q KRN5NX531than that in q KRN5SIL8.RNA in-situ hybridization showed that expression of KRN5b was enhanced in the vascular,primordia of spikelet pair meristems?SPM?and spikelet meristems?SM?,indicating that KRN5b specifically expressed in reproductive axillary meristems.Purified KRN5b proteins were obtained with a prokaryotic expression system.The phosphatase activity of KRN5b was measured and showed that both PI?4,5?P2and PI?3,4,5?P3were the specific substrates for KRN5b,and the phosphatase activity of KRN5b NX531was higher than that of KRN5b SIL8.5. Biological functions of KRN5bWe created two CRISPR knockout mutant lines KO1 and KO2,which caused premature termination of KRN5b in ZZC01 genetic background.KRN of mutant lines KO1 and KO2 decreased 2.2 and 1.6 rows relative to that of non-transgenic lines NT1and NT2(p=1.1×10-6,1.3×10-4),tassel branch number of KO1 and KO2 decreased2.4 and 3.0 relative to that of NT1 and NT2(p=6.4×10-6,0.004).The spikelet density in tassel also decreased in KOs?p=0.021?.The development of SPMs in the KO ear inflorescence were defected,which showed either developmental arrest,or produced only one SM in the ear inflorescence in KO lines.Percentage of defective SPMs in KO inflorescences was higher than that in NTs(p=5.2×10-4).In tassels,the number of single spikelet?SS?/paired spikelet?PS?increased in KO1 and KO2(p=3.6×10-11,0.003),which led to sparse spikelets at the base of the tassel.The results showed that KRN5b modulates the development of spikelets by controlling the initiation and maintenance of reproductive axillary meristems.6. Evaluation of potential breeding value of q KRN5bWe crossed q KRN5b NX531with the increasing allele to 5 elite inbred lines,and backcrossed to each elite inbred lines,respectively,to improve KRN with marker assisted selection.At present,BC4F1populations with PH6WC,PH4CV,Sheng68 and Sheng62 background and BC3F1population with H21 background were obtained for further studies,respectively.Breeding evaluation of the improved inbred lines is under way. |
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