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Cloning and Sequence Measuring of the Marker Linked to Seed Trait in Soft-seeded Pomegranate

Author: LuLiJuan
Tutor: ZhuLiWu
School: Anhui Agricultural University
Course: Pomology
Keywords: Pomegranate RAPD analysis Target fragment Clone SCAR marker
CLC: Q943.2
Type: Master's thesis
Year: 2006
Downloads: 111
Quote: 3
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Abstract


White jade seed bud mutation of three white pomegranate varieties, Dialogue jade seed and three the white pomegranate main economic traits comparative study showed that both mature before harvest fruit cracking, no significant differences in storability and resistance. Fuzzy comprehensive evaluation results, the white jade seed economic traits significantly better than three white pomegranate. White jade seed variation characteristics of the outstanding performance for the kernel weight increase of 84.3%, seed hardness soften. Preliminary pilot study found that pomegranate Grain hardness bimodal curve, there may be a major gene in gene control pomegranate grain hardness. Test for the study of three white pomegranate and white jade seed material to explore the differences between genomic DNA to find genetic markers associated with pomegranate soft seed traits, and cloned and sequenced into SCAR (Sequence characterized amplified region ) mark. Soft pomegranate seeds breeding the basis of molecular biology. Using the modified CTAB method to extract pomegranate leaves gene set of DNA, RAPD reaction Taq enzyme concentration, template DNA concentration, the concentration of Mg2, concentration of dNTPs and random primers concentration carried out the by-optimized, established pomegranate genome of DNA RAPD-PCR analysis of the appropriate reaction system : total reaction volume of 25μl, wherein 1.0U of Taq polymerase, primers 0.3μmol / L, and dNTPs 0.2mmol / L of MgCl2 2.0mmol / L, and a template DNA 40ng / μl. Screened from 113 random primers could amplify a specific band of nine primers amplified a total of 11 specific bands. Said to understand the jade seed presents a greater range of variation at the DNA level. Repeat amplification nine primers screened, choose a better stability, clear bands of three specific bands were isolated and purified. Purified target fragment connected to PMT18-T vector, and then transferred into E. coli for amplification, then colony PCR testing; fragment was sequenced to an overnight bacterial culture. According to the sequencing results using Primer Premier 5.0 software to analyze the design size 18 ~ 22bp specific primers, SCAR-PCR amplification of to white jade seed and three white pomegranate genomic DNA as a template. The results show that the target fragment separation and purification of three, two are present in the the white jade seed and three white pomegranate, its molecular size 562bp and 804bp SCAR marker RAPD two specific bands is not real; the amplified molecular weight in the white jade seed for 537bp specific band, three white pomegranate does not appear that understand jade seed with three white pomegranate presence of this mutation sites. The test is successful white jade seed variation RAPD markers into SCAR markers. The test results for pomegranate RAPD markers into SCAR marker technology system; RAPD markers for the future use of the pomegranate fruit traits to control the gene location mapping and the use of maps clone (Map-based cloning) fruit trait loci DNA evidence; pomegranate soft early identification of seed varieties or seed breeding provide a reference.

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