Gene Mapping And Expression Of Aroma In Rice Chuanxiang29 And Molecular Assisted Breeding

Aromatic varieties constituting a small but special group of rice have gained greater importance with increase in worldwide demand for super rice.And Aroma is one of the most important characteristics for determining good quality rice.Inheritance and gene fine mapping,molecular assisted selection and gene chip analysis of aroma in rice were worked out in the paper.The main results are as following:1.Genetic analysis and gene fine mapping of aroma in rice1.1 eluting aroma from leaves with dilute KOHAroma in rice is a sensory character,and it is apt to be influenced by environmental condition and other factors.Aroma evaluation with dilute KOH was constructed in different environmental conditions and different sites for many years.The best occasion for identification of aroma is the sun shines again after rain,with the temperature 20～26℃.In this condition,the contrast,aromatic and non-aromatic parents were identified with prediction of more than 99%accuracy;and the behave character of aroma in the plants from segregation population was markedly.1.2 Inheritance of aroma in 29BF₁ grains and plant-leaves from the two crosses of 29B/Lemont and 29B/R2 were non-aromatic.Each F₂ plant was self-crossed to obtain F_2:3 family,F₃ grains were chewed to evaluate the aroma of F_2:3 families.And the genotype of each F₂ plant was inferred from the phenotype of corresponding F_2:3 family.Segregating ratios of F₂ non-aromatic to aromatic plants were 3:1,which indicated the inheritance of a single recessive gene in aromatic rice variety29B.1.3 Aroma gene fine mapping700 simple sequence repeat(SSR) markers well scattered on 12 chromosomes were assessed for polymorphism by comparison of parental alleles.Two hundred sixty-eight polymorphic SSRs between 29B and Le were obtained,and the percentage of polymorphism was 38.29%.The SSR markers RM23097,RM515,RM8264,RM7049, RM7356 and RM7556 were found to be linked with the aroma locus.In addition,two SSR markers Aro1 and Aro7 linked with aroma were developed.The fragrant gene fgr was mapped to the interval between Aro7(0.57 cM) and RM515(0.71 cM) in the F₂ population of 29B/Le.In the 29B/R2 F₂ population,RM23120 and RM3459 were identified to be linked with the scented locus with genetic distance of 0.52 and 1.23 cM.2.Molecular assisted selection for aromatic rice maintainer and restorer2.1 Alleles identification for Markers linked with aromaAccording to the investigations in the past,RM223,SCU-Rice- SSR-1 and RM42 were found to be linked with the aroma locus.However,in this study,RM223 and SCU-Rice- SSR-1 can't reveal the polymorphism between 29B and Le;RM42 was polymorphic between the two parents,but not linked with aroma character.In addition,the markers linked with fragrance identified in the paper were assessed for polymorphism by analysis of parental alleles in 13 scented varieties,and variation alleles were found to distinguish different genotype for each marker.2.2 Genetic background analysis by ISSRTo make use of efficient molecular marker systems to reveal genetic diversity among 23 fragrance and non-fragrance cultivars from different regions,24 ISSR primers selected from 40 ISSR markers were used to analyze the cultivar groups.A total of 208 polymorphic bands were found in 23 varieties with the product from 100 to 2200bp differently.The average number of polymorphism bands per ISSR primer was8.67 with arrange from 5 to16.Genetic similarities among the 23 rice accessions ranged from 0.549 (13 and 22) to 0.887(3 and 11).The clustering analysis based on ISSR data is very identical to that varieties character of maintainer and restorer and climates which the cultivars come from.On the other hand,the polymorphic allele of ISSR marker 812 about 1050bp was assumed to be related with the aroma character.The average number of alleles per SSR locus was4.23 with arrange from 3 to 8.2.3 Marker assisted selection for aromatic maintainer and restorer The fragrance gene fgr was introduced into cultivated rice varieties P18 and Mei B by cross and continuous back crosses,respectively.In this study,we used 29B and 29R as the fgr donors and the elite but without aroma lines P18 and MeiB as recipients to develop aromatic maintainer and restorer lines of hybrid rice.The F₁ of P18/29R and F₂ of MeiB/29B were obtained and fragrance linked markers RM23120 and RM3459 were used to select fgr in MeiB/29B;Arol and RM3459 were used in P18/29R.And marker-assisted selection of rice aroma is feasible.3.The analysis of aroma of rice by gene chip3.1 Genes expressed difference between A1 and N1Gene chip is the new tool for biological research.In this study,cDNA gene chip technique was employed to analyze the leaf RNA expression difference between aromatic and non-aromatic nearly isogenic lines.BC₆F₃ population from 29B/G46 was developed and identified for aroma character and genetic background.The aromatic and non-aromatic plants with identical genetic background were selected and used to do cDNA microarray hybridizing.In the interval of aroma locus mapping on chromosome 8,4 genes were identified at the level of expression fold change larger than or equal to 2.AT3G48170 (gene_id) is relate to Glycine,serine and threonine metabolism,AT4G27070 and AT5G54810 are related to Phenylalanine,tyrosine and tryptophan biosynthesis,and AT3G48170 is related to Valine,leucine and isoleucine degradation.On the other hand, AT3G48170 is the BADH2 gene involved in aroma of rice.Gene ontology was made on 23 sites of different expression patterns,which showed that the function included transcription initiation factor activity,sigma factor activity, DNA-directed RNA polymerase activity and response to stimulus.These sites pathway was involved in the circadian rhythm and sulfur metabolism biological process.Further analysis of RT-PCR was validated the expression difference of aromatic and non-aromatic samples.3.2 The mechanism and biochemistry pathway of aroma in riceAccording to the analysis of the gene chip,previous studies and biological informatics, choline is the important compound in the process of the sk-2 gene controlling aroma character of rice.It is well-known that choline is one of the vitamine B compositions, which is mostly being in the chaff or the surface of rice grain.This is the result of the concentration for aroma character distributing in the surface of rice grains.As the BADH2 gene,aromatic varieties shows a large deletion and three single nucleotide polymorphisms(SNPs) compared to the sequence of non-aromatic varieties, and then terminates prematurely(stop codon TAA) in the exon7 of the gene.Then the function of gene losing that can't encode betaine aldehyde dehydrogenase,and betaine aldehyde is induced into the reaction of Betaine aldehyde + FAD + H20 <=> FADH2 + Betaine with the catalyzation of choline dehydrogenase;this reaction will delete FAD and produce FADH2,which restrained the reaction of proline degradationâ… ;L-proline is then accrued with the action of proline dehydrogenase and finally induce the reaction:L-proline +NAD(P)+=1-pyrroline-2-carboxylate+NAD(P)H+H+,and produce 1-pyrroline-2-carboxylate.The aroma key compound 2-acetyl-1-pyrroline may be from 1-pyrroline-2-carboxylate by methylation as their structural formulas similitude.In addition,sulfide was presumed to be relation with the form or behavior of aroma in rice.