Charcterization And Analysis Of Lax Panicle Mutants In Rice

The panicle architecture is one of key agronomic traits that determines rice yield potential and its molecular mechanism is also a main research area in developmental biology. Thereby, identification of genes related to rice panicle morphology not only contributes theoretical progress in development biology, but also can be used for molecular breeding in rice. Here, we identified13rice lax panicle mutants from a T-DNA insertional mutant library of the Japonica variety Zhonghua11. These mutants show lax panicle features and reduced number of spikelets. In order to determine whether the mutation was due to the fragment insertion or deletion in these two genes, PCR amplification covering the genomic region of LAX1and LAX2is performed in the13lax mutants. The agronomic traits are investigated among these mutants. Genetic analysis for recessive status of these mutants is performed. Determination of the mutation sites and positive assay of T-DNA insertion in lax1-like and lax2-like mutants is also performed. F2mapping population is generated to identify the genes by map-based cloning. The main results of this study are as follows:1. Six allelic lax1mutants are identified from thirteen lax mutants. The mutation sites of6allelic lax1mutants are as follows: lax1-1:the mutant losts the DNA fragment from24kb upstream LAX1start codon to7kb downstream LAX1termination codon; laxl-2is the same as laxl-1; lax1-3:T-DNA is positively inserted at1416bp upstream LAX1start codon and T-DNA is oppositely inserted at1390bp upstream LAX1start codon; laxl-4:Partial T-DNA is oppositely inserted at1581bp upstream LAX1start codon; laxl-5:the mutant losts the DNA fragment from6686bp upstream LAX1start codon to1658bp upstream LAX1start codon; laxl-6:T-DNA inserts at4905bp upstream LAX1start codon in reverse orientation. Analysis of LAX1promoter shows that there are some motifs which function in expression regulation, such as DPBF motif, DOF motif, CACGTG motif and ARF motif. Variation of LAX1expression possibly leads to mutation of6allelic lax1mutants.2. Phenotype analysis of6allelic lax1mutants show that height and panicle length of6allelic lax1mutants are almostly the same as wild type plant. But different degrees of change in number of tillers, primary branches and grains per panicle are observed, lax1-1, laxl-2and laxl-3show serious type of lax1mutant, laxl-4, lax1-5and laxl-6belong to moderate type of lax1mutant. Variation of six different lax1mutants and the corresponding change of phenotye demonstrate potentially that mutation of6lax1allelic mutants is association with the phenotype.3. T-DNA positive assay demonstrates that the mutant morphology in lax-L1, lax-L2, lax-L3, lax-L4, lax-L5and lax-L7are not co-segregate with insertion of T-DNA fragment, the mutant morphology of lax-L6may due to the T-DNA insertion and we have not obtained the corresponding flanking sequence. Genetic analysis of transgenic T2population showed that mutation of these lax-L plants is controlled by a single recessive gene. Comparatively PCR amplification results demonstrate that lax-L1is an allelic mutant of lax2, which is reported in recent pubulication.4. Phenotype analysis of7lax-L mutants show that height and panicle length of these lax-L mutants are almostly same as wild type plant. But the number of tillers of lax-L1, lax-L3, lax-L5and lax-L6is decreased; the number of tillers is increased in lax-L2and lax-L7. The number of primary branches in lax-L2mutant is approximately half of that of wild type plant, and the number of primary branch in other mutants does not change significantly. The secondary branch numbers of these mutants reduce significantly and the number of secondary branches in lax-L2mutant is the least. Grains per panicle of lax-L7mutant are slightly reduced, and several other mutants are almost sterile.5. Three of F2-mapping population by crossing heterozygotes plants of lax-L3, lax-L4and lax-L5and Zhenshan97are generated. Genetic analysis of F2population indicates that mutation of lax-L3, lax-L4and lax-L5is caused by a single recessive gene mutation. Employing genetic mapping method, candidate gene of lax-L3, lax-L4and lax-L5is located between RM16880and RM1205on the long arm of chromosome4, within a physical region about120kb.6. LAX2gene is located in the the regional between RM16880and RM1205. RT-PCR shows LAX2expression is decreased in lax-L3mutant. Sequence analysis showed that lax-L3mutant has the50bp base sequence deletion in first exon of LAX2cDNA. The genomic sequencing showed that lax-L3mutant has lost60bp including50bp in exon and10bp in intron. All these results indicated that the target mutant gene in lax-l3was LAX2.