Esteblisment Of Single Chromosome AFLP Molecular Marking System And Library Construction Of Pomelo (Citrus Grandis Osbeck)

Pomelo is an important fruit in China. Beacause of its complex genetic background and long growth cycle, it's hard to construct its accurate physical map., which causes difficulty in the stuys of genomics, molecular marker-assisted breeding and cloning of genes with important horticultural characters.Chromosome microdissection and microcloning technique provides new thoughts of the genetic research of Pomelo. We used the method of random single chromosome microdissection in view of the most small of the single chromosome of Pomelo and hard to recognize. The LA-PCR-AFLP technique was used in our study to classify the chromosomes of Pomelo. And the Southern blot was used to identify the results. The single chromosomes which are homologous were collected together in the end, so as to construct the entire chromosome library of Pomelo.1,Made a study in the chromosome preparation and microdissection of Pomelo, compared two overslip-removing methods between quick-froze by liquid nitrogen and suspension. Optimized the concentration ratio between cellulase and pectinase, and their enzyme digestion time:2% cellulase and 1% pectinase, and digested 1 h; compared two dyeing methods between Carbor fuchsin and Hematoxylin, and finally selected the Carbor fuchsin method; Chose and improved on the glass needle preparing and ways of microdissection. A favorable preparation improved more on the quality and efficiency in microdissection of single chromosome.2,With controlled trials, exogenous DNA contaminations in the LA-PCR-AFLP technical system of the single chromosome in Pcmelo(Citrus grandis Osbeck) were studied using of different types of water, sensitivity test to contaminations of the water between the fore-and-after steps of adding adapter to link and nonspecific bright bands formed in LA-PCR. The results showed that the BT Water(Bioteke, RNase-free and DNase-free Water) was the optimum water among the eight types of test water; The quality of the water had more important influence on the step before adding adapter to link than afterwards; The nonspecific bright bands were primer polymers which had little to do with exogenous contaminations. Exogenous contaminations could be effectively confined by strictly controlling the sources of contamination and strengthening prevention in key steps, and verified by dot blotting. Our research have laid foundation for establishing of high-quality Pomelo single chromosome libraries.3,Got high-quality genomic DNA of Pomelo extracted by SDS-KAC through comparing 3 extracting methods. And made a study to the time of enzyme digestion and the ATP concentration for ligation:a longer digestion time of 4-5 h was used even the digestion times from 1-5 h were all available in order to keep the consistency; and when ligase concentration was more than 5 mM would stop the reaction, and it had less influence even didn't add extra ATP at ligation. The key parameters of LA-PCR-AFLP Technical system were optimized for available PCR protocol, optimum template concentration and better primer pairs on the base of chromosome microdissection and microcloning technique. Results showed that program TD3 which annealing temperature was higher (72℃→65℃30 s (-0.5℃/ cyc)) had better amplified effect; the optimum template amount was diluted with 100 times(10 ng) and 10 primer pairs had better amplified polymorphism. And finally 5 LA-PCR-AFLP fingerprints of different single chromosomes in pomelo were constructed amplified by different groups of primer pairs.4,The high-quality total RNA was obtained and reverse transcribed, then got the amplified products of cDNA by LA-PCR. The products were purified and linked with pMD18-T-vector and then transformed to E.coli and got a hundred of clones, which were purified for following studys.5,Constructed a dot-blot homologous identification system which is suitable for single chromosome of Pomelo:1)Use 2.5μL DIG-High Prime (vial 1) for probes preparation.2)Single chromosomes should be purified for probes preparation, and need not purify when have made.3)Use the first round of PCR products for probes preparation.4)The differential fragments of single chromosomes and the fragments of cDNA clonicons should be purified before used.5)The sample amounts was 1 ng when the single chromosomes of Pomelo and differential segments and cDNA clonicons were used as probe.6,Made a cluster analysis of the polymorphic segments from AFLP-SDS-PAGE maps amplified by 5 prime pairs, and the 55 single chromosomes were classified to 10 groups. Then the 10 groups were hybridized with 134 clonicons from cDNA library, in order to proving the validity of the clusters, and a further Southern blot ensured the validity.A preliminary classification was made and the homologous chromosome libraries were preliminarily constructed, but it is not enough in both integrity and quality, great afforts should be maken in case of construction of high-quality and integrity chromosome library.The results in this study laid the foundation for the construction of accurate physic maps in future, and was very meaningful for subsequent genomic studys in Pomelo. And provided easier, faster and more accurate ways for genetic breeding and the location and cloning of useful genes in Pomelo.