Cloning And Function Characterization Of A Major QTL KERNEL NUMBER PER ROW6(KNR6) In Zea Mays

Maize(Zea mays L.)is one of the most important crops globally,and improving grain yield is required to meet the rapidly expanding demands for maize-derived food,feed,and fuel.Breeders have worked to improve grain productivity of maize hybrids for more than a century,and have made great successes by pyramiding desirable characteristics for larger ears and wider climate adaptability.However,yield is a complex trait controlled by many of loci with small effects,there is little information on what loci were selected for improving grain productivity,or the molecular basis of beneficial alleles.Kernel number per row(KNR)and Ear length(EL)as important component yield in maize and directly influence grain yield.Through fine-mapping,association mapping,expression analysis and transgenic validation,we establish that KERNEL NUMBER PER ROW6(KNR6),encodes a serine/threonine protein kinase which determines ear length,kernel number per row and flowering time.The major results are as follows:1.Positional Cloning of qKNR6To identify q KNR6,we backcrossed NILq KNR6(Longer ear)to NILqknr6(Shorter ear)and obtained ten recombinants in a selfed-backcross population of ~28,000 individuals.Using recombinant-derived progeny testing,we narrowed q KNR6 into a ~110Kb interval,flanked by markers M6 and M8.We found two predicted genes,Zm00001d036601 and Zm00001d036602,within the 110 Kb interval.Though PCR sequences and expression analysis show that no differences between the QTL parents were found in the coding region of Zm00001d036601,but four polymorphic sites for Zm00001d036602,related to NILqknr6 allele,including a 5054 bp TE absence in 5'-UTR,and three single nucleotide polymorphisms(SNPs)in exons,two of which are non-synonymous,including a C/T SNP in exon1,which leads to the Leucine converted into Phenylalanine(L7F)and a C/G SNP in exon3 which leads to the Leucine converted into Valine(L70V),another G/A SNP in exon3 is a synonymous substitution.Sequence analysis of the 5054 bp TE in KNR6 revealed that it was a Harbinger-like TE,containing a 3bp target-site duplication,and 14 bp terminal inverted repeats(TIRs),with an additional novel TE of 4926 bp inserted within it.2.Expression levels and methylation levels of KNR6 in NIL lines and Expression levels inbred linesHarbinger-like TE lead to the methylation level of theCG,CHG and CHH context in core promoter region of KNR6 is significantly higher in NILqknr6 than that in NILq KNR6.The inserted TE shows high CG methylation level in NILqknr6,these results suggested that the TE insertion leads to an increase in DNA methylation of the 5? region of KNR6,and in turn,a repression of its transcription.Expression levels of KNR6 in 105 inbred lines and NIL lines indicate that KNR6 expression levels were positively correlated with KNR and EL.Compared with the 85 TE-lines lacking the Harbinger-like TE insertion,the 20 TE+ lines had lower expression levels.3.Function characterization of KNR6We generated transgenic maize plants that either silenced KNR6 via RNA interference(KNR6-RNAi)or over-expressed it(KNR6-OE)in inbred line A188.Two independent RNAi transgenic lines(RNAi-1 and RNAi-2)and two over-expression lines(OE3 and OE4)were analyzed.As expected,the RNAi lines had shorter ears(by 1.35 ± 0.11 cm,p = 2.05 × 10-14)with fewer kernels per row(by 2.85 ± 0.22 KNR,p = 1.37 × 10-25),and flowered earlier(by 2.3 ± 0.11 days,p = 1.32× 10-9).In contrast,the over-expression lines produced longer ears(by 1.1 ± 0.26 cm,p = 3.53 × 10-15)with more kernels per row(by 2.5 ± 0.15 KNR,p = 1.85 × 10-28),and later flowering(by 3.1 ± 0.35 days,p = 2.42 × 10-21).4.Association mapping of KNR6We sequenced the locus in a set of 224 diverse maize inbred lines.A total of 93 variants with minor allele frequency(MAF)? 0.05 were detected.Of these,56 variants,including the Harbinger-like transposable element(TE)presence/absence variation(TE-PAV)and one non-synonymous SNP(L7F)were significantly associated with KNR at P = 3.62 × 10-4.The TE-PAV showed the strongest association with KNR(P = 8.81 × 10-5),followed by 50 variants in a strong linkage disequilibrium(LD)block(r2?0.97).5.Genetic effect of the TE-PAV in hybridWe generated 42 hybrids by crosseding 11 TE+ lines and10 TE-s to both NILqknr6 and NILq KNR6,respectively.No matter what genetic background of the inbred lines used,the hybrids crossed from NILqknr6 had significant longer ear with more KNR.These results confirm the value of the KNR6 for genetic improvement of hybrid maize kernel number,and consequently of maize grain yield.we backcrossed the q KNR6 allele,which lacks the TE insertion(TE-),into two TE+ inbred lines widely used in Chinese maize breeding programs zhengdan958 parents lines(Zheng58 and Chang7-2).When the TE+ allele was substituted by the TE allele,the two lines showed an increase in KNR,EL and a delay in flowering time relative to the parental lines.Notably,as a result of improvement of KNR and EL,the grain yield of the improved hybrids was significantly higher than that of the parental Zheng58/Chang7-2 hybrid(13.3T/ha compared to 12.6T/ha,5.6% increase),these results confirm the value of the KNR6 for genetic improvement of hybrid maize kernel number,and consequently of maize grain yield.6.The evolutionary change of the KNR6 locus during maize domestication and improvement.We sequenced KNR6 from 43 teosintes and 275 diverse maize lines,including 214 temperate and 61 tropical inbreds and 24 landraces.The nucleotide diversity analysis suggested that the 5'-UTR intron region of KNR6,around the TE-PAV,was a target of selection during maize domestication,Harbinger-like TE is formed in maize landraces by inserting a novel class of DNA transposon into the MITE,and is further enriched in temperate inbreds.In summary,we cloned a QTL for KNR6(q KNR6)as a gene that encodes a serine/threonine protein kinase which functions as a regulator of KNR,ear length(EL)and flowering time.A Harbinger-like TE-presence/absence variation(TE-PAV)in the 5'-untranslated region(5'-UTR)of KNR6 is a causal variant,with a strong effect on grain yield and weak effect on flowering time.The presence of the Harbinger-like TE repressed KNR6 expression by inducing DNA methylation of flanking sequences.This Harbinger-like TE originated from a MITE in Zea mays ssp.Mexicana,by insertion of the HARBI1 encoding DNA sequence into the MITE in maize landraces and inbreds.