Map-based Cloning And Functional Conifrmation Of Narrow And Rolled Leaf1&Genetic Mechanism Of Hybrid Weakness In Rice

Leaf is a main organ of photosynthesis and contributes to ideal plant type in rice. Therefore, leafshape has become one of hot spots by molecular geneticist and breeder. In order to furtherunderstanding of the regulatory mechanism of leaf development, we isolated three allelic mutants ofnrl1, nrl1-1and nrl1-2from an M2population of the japonica rice cultivar Nipponbare, nrl1-3from thejaponica rice cultivar Wuyunjing7after EMS mutagenesis. Three mutants showed phenotypes ofdwarfism with different degree, reduced leaf width, semi-rolled leaves and slim grain. Microscopicanalysis indicated that the NRL1involved in development of different cells and tissues, i.e. the bulliformcells, longitudinal veins, sclerenchyma cells and parenchyma cells were changed in size or number.Among them, smaller bulliform cells may explain the semi-rolled leaves and fewer leaf veins result inthe narrowed leaves. The content of cellulose, hemicellulose and lignin also increased in mutants,suggesting NRL1likely affect their biosynthesis.The NRL1gene was fine mapped to an interval of8.71kb, where one open reading frame (ORF)located on chromosome12via map-based cloning strategy. To define the mutation locus, the DNAfragments of wild-type and three allelic mutants were amplified and sequenced. Single base-pairsubstitutions were found at different loci in each of the three allelic mutants. To confirm the biologicalfunction of the NRL1gene, we constructed complementation vector and over-expression vector. Thephenotype of mutant was completely suppressed while Nipponbare not changed in complementation testand over-expression analysis which confirming NRL1without dosage effect. NRL1exhibitedconstitutive expression at transcription level at the heading stage, and decreased in the mutants, alteredlittle in complementary transgenic plants and increased significantly in over-expression plants.Sequence analysis of cDNA revealed that the NRL1gene is composed of two exons and one intron,which encodes a protein of1215amino acids in the KOME database. BLAST analysis indicated that theNRL1protein is the cellulose synthase-like protein D4(OsCslD4), which contains the D, D, D,QXXRW motif and eight trans-membrane segments characterized in Csl family.As the phenomenon of postzygotic reproduction isolation, hybrid weakness is common in plants.Genetic study of hybrid weakness will contribute to understanding the molecular mechanism ofreproduction isolation and its application. In this study, the F1plants from the cross between Peruvianvariety Jamaica and japonica varieties exhibited phenotypes of dwarfism, single tiller and notablyshortened roots. The F1seeds were normal in embryonic development and germination, but hybridweakness exhibited weak growth potential after germination, while growth was inhibited in root earlierthan in shoot. The plants will die under conventional field management, but flowering and seed settingwas achieved under favorable conditions without survival competition. Genetic analysis indicatedhybrid weakness was controlled by a pair of dominant complementary genes. The genotype of Jamaica,japonica and indica rice is Hwc1Hwc1hwc2hwc2, hwc1hwc1Hwc2Hwc2and hwc1hwc1hwc2hwc2,respectively. When the locus of Hwc1and Hwc2are dominant homozygous or dominant heterozygous,the plant appears hybrid weakness and it is normal if one or two loci is recessive homozygous. The hybrid weakness gene Hwc1was fine mapped into a region of22.9kb on chromosome1,which had only one ORF by PCR rapid screening and test cross of recombinant. Hwc2was also finemapped in24.6kb on chromosome4, where total four genes were predicted. Complementation vectorsfor both genes have constructed and transgenic experiment is being carried out. In order to apply thehybrid weakness gene in rice production, we introduced Hwc1into a transgenic plant with Bt, Roundupand Bar gene. Now, herbicide and pest resistant plants with Hwc1have obtained by gene pyramiding toestablish a new technology of preventing the exogenous gene escape. The Hwc1also has beenintroduced into maintainer line Wuyunjing7and restorer line C Bao by backcrosses and have gainedNILs of BC6and BC7respectively, which provides basis for production apllication in hybrid rice.