Genetic Localization Of The Pur09 On Purple Trait In Purple-caital

Purple-Caitai(brassica rapa.L),is a kind vegetables of flower stalk as the main edible parts for a vegetable of Brassica,originating in China,mainly in Hubei,Hunan and other places widely cultivated.There is Higher anthocyanins of purple Caitai,so,it is high nutritional value for people.This experiment use the purple-caitai as female and white-caitai as male hybrid,building F2 Segregating population,through the genetic population in extreme plant mixed pool sequencing and analyzing the consequence of the DNA-sequence screening of chromosome 9 associated with the purple-caitai color gene.It will provide theoretical and practical basis for follow-up experiments and applications.1.Genetic analysis and field investigation of purple-Caitai purple traitThe Purple-caitai(female parent)and white-caitai(male parent)cross to F1 generation,F1 self-cross to F2 generation.F2 generation is planted in Wuhan area which number is 1001,until commercial trait maturity,color trait of P1?P2?F1?F2generation plants are investigated,trait investigation results show:F1 showed red,BC1(P1)showed red,F2 generation show character separation,character level can be divided into four types,0(green)?1(pale red)?3(red)?5(Deep red),generally similar with the normal distribution.The number of purple trait for 884 strains,the number of white characters of 117 strains,the proportion is 7.56:1,which can launch control characters red purple-caitai is composed of multiple genetic loci control,and not a single site simple control,the trait is a quantitative trait.2.Screening of linked markers of purple trait related genes and identifying the genotype of F2 isolate populationAccording to the analysis of previous sequencing results,more than 400 pairs molecular markers primers are designed on in the peak area(on the rapa chromosomeA07 and A09).then,the designed primers were first screened by the purple-aitai parent and white-caitai parent and two extreme pools to screened molecular markers primers of different bands which are high repeatability and stability,and then,the polymorphism molecular markers primers were screened in F2 segregating groups.ultimately,nine pairs polymorphism molecular markers associated with purple-caitai traits were Screened,respectively.A09-75,A09-15,A09-16,A09-31,A07-8,A07-42,A07-58,A07-12 and A09-3.35 F2 plants were selected which were used to screen the candidate loci on the A09 chromosome,and the candidate loci pure on the other chromosome.3.Construction of the F2:3 populations and Screening of candidate genes for single gene segregating populationsThe 35 F2 plants in the F2 isolate population are selected which were used to screen the candidate loci on the A09 chromosome,and the candidate loci pure on the other chromosome,and then,The 35 F2 plants are self-pollination to get F2:3groups(Purple-caitai is self-incompatibility,and need to bud self-cross).Each group planted 100 plant,combined with molecular markers and field characters of the survey results,identify candidate groups.According to the band pattern amplified by molecular markers in the population,the ratio of the statistical bands was selected,and some better isolated groups were screened out.Two single gene segregating populations,F2:3(12-1)and F2:3(8-1),were screened in combination with field phenotype and molecular marker banding patterns.4.Screening of markers linked to target genes on the rapa chromosome A09Pass through the investigation of the field traits and the the genotype of molecular markers,determining the target gene linkage CAPS markers A09-75 and A09-15 on the A09 chromosome,located near 7.5M and 15.7M respectively,the corresponding Single gene isolation candidate groups are F2:3(12-1)and F2:3(8-1).5.Changing of anthocyanin content under stress,and the expression analysis of anthocyanin biosythesis-related gene of A09 chromosomeUnder simulated drought stress conditions,the content of anthocyanin content with the prolonging of treatment time is increased,the increase in processing after 12 hours to 24 hours after large,slow growth;in the low temperature conditions,the anthocyanin content appeared significant growth in 24 hours 48 hours,96 hours to reach the maximum value in the condition of light stress;under 1ight Stress,the anthocyanin content slowly increased from 12 hours to 72 hours after growthslowed.The anthocyanin content of cabbage sprouts did not change significantly.In the light of stress under the condition of 9 A09 on anthocyanin synthesis related gene Q-PCR analysis of purple-caitai parent and white-caitai parent,results showed that the expression of Bra027457 purple-caitai gene and Bra036828 gene in white-caitai was significantly higher than that of moss,expression of Bra036828 gene in red cabbage cabbage was 7.23 times of the moss,the expression of Bra027457 genes in red cabbage was 3.62 times of the cabbage moss had no obvious difference,the expression of other 7 genes.The synthesis pathway of these two genes Bra027457 and Bra036828 may be involved in anthocyanin purple-caitai,located at 10.9M and27.0M.Under other stress conditions,there were no significant differences in the expression of 9 genes between 2 parents.