| Plant height,leaf morphology,grain shape and heading date are crucial agronomic traits of rice(Oryza saliva L.).To better understand the genetic base of rice agronomic traits,we selected two different rice germplasms in phenotypes,Xian80 and Suyunuo(SYN),to construct F2 segregating population for genetics and QTL analysis.Genetis analysis indicated that every trait displayed continuous frequency distributions as quantity traits.Also,high correlations were observed between traits in F2 population.QTL analysis was carried out in F2 population,and twenty five QTLs for six traits were identified,including 4,4,5,2,4,6 QTLs for plant height,leaf length,leaf width,grain length,grain width and heading date,respectively.Interestingly,four QTLs,qPH12,qLW12.2,qLL12,and qGW12.1 were located on the same genome region of chromosome 12 between SSR markers RM519 and RM1103,which explained 50.00%,57.08%,15.41%,and 22.51%phenotypic variation for plant height,leaf width and length,and grain width,respectively.Considering that qPH12 and qLW12.2 had significant genetic effects on plant height and leaf width,the locus was named DNL1(DWARF AND NARROWED LEAF 1).To further narrow down DNL1 locus,the heterozygous individuals of F2 population were selected out,based on the genotype of SSR markers RM519 and RM1103,to develop a segregating population for fine mapping.From this population,a total of 1363 dwarfism and narrowed leaf individuals was selected and screened for recombinants by SSR markers RM519 and RM6947(a marker outboard RM1103),and 141 genetic recombination events was found.High-resolution linkage analysis in the 141 recombination events was performed,using InDel markers within interval of RM519 and RM6947,and DNL1 was narrowed to a 62.39 kb region of InDel markers ID12M28 and HF43.9 Opening Reading Frames(ORFs)exist in the region.Of which,ORF5 encodes a member of CSLD(Cellulose synthase-like D protein)subfamily,named as OsCSLD4,which play important roles in cell wall formation and plant growth.On the basis,ORF5/CSLD4 was determined as the candidate gene of DNL1.To verify that OsCSLD4 is responsible for DNL1 phenotype,nucleotide sequence alignment of OsCSLD4 was performed in 9 rice accessions(including the parents Xian80 and SYN)that well covered genetic diversity.12 sites of single nucleotide polymorphism(SNP1 to SNP12)and one insert and deletion polymorphism(InDel1)were identified.The site of C to T change at nucleotide 2844(C2488T)(SNP8)only occurred in Xian80,which are associated with the dwarf and narrowed leaf phenotype.Consequently,it is crucial for the DNL1 phenotype in Xian80 that the SNP8(C2488T)caused an amino acid residue changes from proline to serine(P746S).DNL1 encodes Cellulose synthase-like D4(OsCSLD4)protein,belonging to glycosyltransferase 2 family(GT2).OsCSLD4 contains a Zf-Ring 4 domain,a Cellulose_synt,two transmembrane domains(TM1 and TM2)toward N terminus,and six transmembrane domains(TM3 to TM8)at C terminus.The Cellulose_synt,which was designated as 'cellulose synthase catalytic subunits',has a GT domain and a 'D,DCD,D,QXXRW' motif between TM2 and TM3.dnll(P746S)happened on the GT domain.The result of multiple sequence alignment displayed that the GT domain was highly conserved in all selected CSLD subfamily members and the SNP8(P746S)was a rare mutation in dnl1.Phylogenetic analysis placed OsCSLD4 and AtCSLD5 in the same clade,suggesting that they may have conserved function in the regulation of plant growth and development cross plant kingdom.In rice,there are five members of OsCSLDs:OsCSLDl to OsCSLD5.Expression analysis showed OsCSLD4 highly expressed in roots,while other CSLD members had comparatively lower expression levels.However,no clear evidence to confirm the truth of altered DNL/CSLD4 expression was associated with its function in Xian80. |
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