Development Of RSAP-A Novel DNA Marker Technique And Its Application In Pepper Genetics And Breeding

Capsicum spp., Originated from tropic region of America, now is cultured in many regions of the world and especially most widely grown in China. Of these, long-fruit hot pepper (Capsicum annuum var. longum) is one of the main variety types in China. High-yield breeding is main breeding objectives of pepper. Based on quantitative trait loci (QTL) analysis, Marker assisted selection for yield-related traits can be an efficient way to this ends. Hitherto, all of DNA makers based on restriction site require digestion with restriction enzyme, which make the procedure complexity and sometimes result in pseudo-polymorphism.Based on restriction site, a new marker technique called restriction site amplified polymorphism (RSAP) was designed in this paper. With special designed primer in RSAP, the polymorphism in restriction sites can be detected in a simple PCR without digestion. RSAP were mainly tested and refined with pepper in this paper, then tested in rice and balsam pear for its widely application. To further evaluate the efficiency and application of RSAP, the genetic difference among 10 elite pepper inbred lines were analysis by RSAP and SRAP and SSR, the genetic analysis ability of RSAP was compared to the results of RSAP and SSR. Cluster of ten pepper inbred lines based on RSAP were also contrast with SRAP and SSR. Using RSAP and SRAP techniques, a pepper genetic linkage map was constructed based on 93 F2 plants from two elite breeding inbred lines cross. Based on the linkage map and phenotype values of 15 yield-related traits, QTL analysis were performed by multiple interval mapping. Dynamic QTLs affecting plant height were mapped and analyzed by the combination of multiple interval mapping and the conditional analysis method. The main results of this study were as follows:1. A new marker technique called restriction site amplified polymorphism (RSAP) was developed. This technique adopted two primers of 18 nucleotides, starting at the 5' end of each primer are 12-14 bases of arbitary sequence, followed restriction site sequences (4-6 bases). The restriction sites of two primers are difference. PCR amplification is run for the first 5 cycles with an annealing temperature of 35℃, followed by 35 cycles with an annealing temperature of 48℃. Amplified DNA fragments can be separated by denaturing acrylamide gels and detected by silver staining. The 25μl PCR reaction of RSAP includes 20ng DNA templates, 2.5mmol/L of Mg2+, 0.2mmol/L of dNTP, 1.5U of Taq DNA polymerase, 600 nmol/L of each primer. Without digestion, RSAP is simpler than other DNA marker technique based on restriction site. RSAP also has moderate throughput ratio and reliability and can be well amplified in Oryza sativa L. and Momordica charntia L.2. RSAP and SRAP and SSR were adopted to analyse the genetic difference among 10 elite inbred lines. The results showed that RSAP had the higher number of total loci (51) and polymorphic bands (13) per assay ratio, being 1.5-fold and 1.3-fold higher than SRAP and 17-fold and 6.5-fold higher than SSR respectively. The correlations between pairwise genetic distances generated by RSAP maker system with SRAP (0.5324) and SSR (0.5429) were high. Ten long-fruit hot pepper varieties based on SRAP were classified into three groups that were in agreement with practice of pepper heterosis breeding and pepper fruit shape type.3. Based on 251 polymorphic markers produced by RSAP and SRAP, a pepper intra-specific linkage map was constructed on JoinMap3.0 software. The map was composed of 12 bigger linkage groups and 3 smalls, with 113 markers including 35 RSAP markers and 78 SRAP markers. The map covered genome 995.73 cM. An average distance between markers was 8.82cM. In the construction of intra-specific map, RSAP showed power ability for polymorphism detection and high efficiency.4 A total of 49 QTLs were detected for 15 yield-related traits in pepper. The number of QTLs per trait ranged from one to six. Most of the QTLs were found in LG1, LG2, LG8, LG9 and LG10, in which similar QTL positions were identified for many traits, suggesting gene linkage or pleiotropy are existed. Some single QTLs explained more than 40% variance, was detected for plant height, fruit shape, pericarp thickness, fruit number, early yield and total yield.5. Eleven unconditional QTLs were located at different regions of seven linkage groups for plant height at six development stages. The results showed that only one QTL detected at all of six stages and effects of QTLs on plant height were varied at different developmental stages, suggesting that the expression of each QTL was dissimilar at different stages. With net growth value, six conditional QTLs for plant height were detected, indicating that QTLs for plant height are selected to express at different development stages that should be considered when marker assistant selection.