The Application On DUS Test Of SRAP And Establishment Of A 2-D Electrophoresis System

The SRAP marker system is a recently developed DNA polymorphism technique with several advantages over other systems: simplicity, reasonable throughput rate, low cost, high reliability, easy isolation of bands for sequencing, which received concerns widely. In the last few years, it has been used in genetic diversity analysis in many plants, like potato,rice,apple,Brassica vegetables and so on. After investigating the factors which influencing the SRAP analysis of lettuce (Lactuca sativa ), including the concentration of Mg²⁺, dNTPs, Tag DNA polymerase, primers, DNA template and annealing temperature in PCR, we set up the optimum reaction system : the 20μl reaction solution contains 50ng DNA, Mg²⁺ 2.0 mM, dNTPs 0.2 mM, Taq DNA polymerase 1U, primer 0.75uM. Reactions were carried out in two steps: the first 5 cycles consist of denature at 94℃, annealing at 35℃and extension at 72℃after pre-denature at 94℃for 5min, the second 30 cycles include the same conditions for denature and extension as in the first step with the changed annealing temperature at 51℃. The last step was extension at 72℃for 7min.18 SRAP markers and 40 morphological traits for DUS test were used for diversity analysis of 50 lettuce (Lactuca sativa) varieties. Average polymorphism information content (PIC) of the SRAP markers was 0.80, ranging from 0.39 to 0.97. Cluster analysis of the SRAP band patterns using UPGMA analysis separated the varieties into three groups, which showed to some extend similarity to the morphological trait-based clustering result. And the correlation coefficient of two markers is 0.5455. SRAP markers coubled be used to complimentary tool for DUS test of new varieties.21 SRAP markers and 54 morphological traits for DUS test were used for diversity analysis of 28 watermelon (Citrullus spp.) varieties. Average polymorphism information content (PIC) of the SRAP markers was 0.72, ranging from 0.525 to 0.892 and the correlation coefficient of vatieties ranging from 0.92 to 0.99. The correlation coefficient ranging from 0.50 to 0.85 in morphological traits test. Cluster analysis of the band patterns (SRAP markers) and morphological traits using UPGMA analysis separated the varieties to four and five groups respectively, which showed some similarity within the two kinds of markers but the correlation coefficient of two markers is only 0.218, indicating little correlation between the two markers. Compared with morphological trait anlysis, SRAP markers show some advantage in variety identification which could be used to as a complimentary tool for DUS test in application for new varieties and varieties protection.Proteome is the total protein encoded by all genome. The Two-dimensional electrophoresis is a common technology used for proteome analysis. Based on IPG-DALT-six system and the 2-DE workflows in model plants such as rice and Arabidopsis, an effective protocol for lettuce proteome analysis was established. At the same time, the distribution of the isoelectric point, molecular weight and relative content of the total proteins of lettuce were investigated. All of these protocols could provide basic tools for further study of lettuce proteomics.