Mapping And Cloning Of Rice Few-tillering Candidate Gene And Rice Bacterial Blight Resistance Breeding By Molecular Marker-assisted

Tillering is an important agricultural trait of rice yield, and it is a model system for studying on molecular mechanism of branching of endogen. So, it is necessary to create mutant associated with tillering and clone its controlling genes. In this study, a few-tillering rice mutant was identified and the genetic analysis, fine mapping and cand i date gene cloning were carried out and two transformation vectors for verifying the function of the candidate gene were constructed. The main results obtained in this study are following:1. A few-tillering rice mutant, G069, was identified from the anther culture progeny of an indica-japonnica hybrid combination, Gui630/02428. And the NILs, G069-fti, between G069 and 02428 was established by using 02428 as back crossing parent to continuously back-cross with G069-fti and the few-tillering plants of F₂ populations. Several mutant traits expressed in the mutant NIL, such as few-tillering, delayed tillering development, yellowing apex and margin of early leaves, enhancing the ability of re-generation, dwarf, and so on.2. The statistics analysis results of frequency of tillering distribution in F₂ under two d i f f erent f ertility levels and planting densities indicated that the distribution of tillering are display two peak distribution on both conditions, and the peak values are similar with G069-ft1 and 02428, respectively. Furthermore, the segregating proportion correspond 3:1 (X²_c=0.52872₀.05,1,=3.84 and X²_c=0.72652₀.05,1=3.84, respectively), which suggested that the few-tillering trait was controlled by a recessive gene.3. Fine mapping of the few-tillering gene was completed. The gene was successfully mapped near RFLP marker, S13984, which is locates between RM263 and C424 on the No. 2 chromosome of rice. The linkage map and complete sequence of the genomic among the few-tillering gene were obtained from NCBI and the BAC sequence of Nipponbare on the foundation of molecular marker mapping of ft1, and the fine mapping of ft1 was carried out by using the new markers designed based on the linkage map and complete sequence of the genomic amongthe few-t i I ler ing gene. Two markers, RGD219 and RGD224, were co-segregate with ft1 and the distance between the two markers are 5.5kb.4. There are 9 putative genes in 40kb genomic sequence among the two co-segregate markers by predicting genes on Internet. 4 candidate genes of Ft\, F;t1H\ Ff\-g, Fti-h and Ff\-\, were chosen out by BLAS and function analysis on NCBI.5. RT-PCR analysis of the 4 putative genes in 4 leavies stage showed that the other three putative genes except Ffl-f can transcript in this stage.6. Cloned and sequenced Ft1-g> Ffl-h and Ft1-i. The results of sequence analysis of these three genes showed that only Fti-g display difference on genomic sequence and ami no acid sequence. So it was selected out as candidate gene of Ft).7. The results of gene structure and similar analysis of Ff\-g indicated that it is consist of 6 exons and can transcript an 1188bp mRNA and encode 395 amino acids. Its cDNA have 99% similarity with a japonica rice cDNA (XM66964.1) among 900bp.8. The results of structure and function predict of Ft\ coding protein showed that it is a receptor protein kinase and have Serine/Threonine protein kinases conserve catalytic domain and have 88% similarity with a rice Ser ine/Threonine protein kinases among 338 amino acid. It infer that Ft\-g coding a receptor protein kinase of signal conducting pathway.9. Comparing the gene and protein structure of Ft\-g among indica, japonica and the mutant, we can come to the conclusion that Ft\-g displayed dual-morphism allele mutant character during indica-japonica evolution because of the deletion of a fragment of 26 amino acids and 15 amino acids in the protein of Ft\-g in indica and japonica, respectively. But it seemed that this kind of variation couldn't change the function of Ff\-g. But, when we cross indica with japonica in this study, an intragene homologous recombination happened and led both of the two amino acids fragment to be deleted. It made Fti-g cann't form 3D-structure correctly and lost its function.10. The sense gene and anti-sense gene transformation vectors for verifying the function of Ft\-g were constructed successfully, respectively.