RAPD Analysis Of Loquat Genetic Resources

In this experiment loquat genetic resources including 11 main species or types in Eriobotrya, 65 accessions of loquat germplasm as well as 12 accessions of 'jiefangzhong' materials from 12 different production areas were used as materials to establish the method of leaf DNA extraction, to optimize the RAPD reaction system, and to conduct RAPD analyses to discuss the genetic relationship and genetic diversity of loquat germplasm combined with clustering analysis. The results were as follows:1. The available method of DNA extraction from loquat leaves. Loquats were rich in the secondary metabolites such as polyphenols, polysaccharides and pigments, which makes it rather difficult to obtain high quality genomic DNA from their tissues. The improved SDS method was suitable to extract pure and high-quality genomic DNA from loquat leaves for RAPD amplification. But some attentions should be paid in this method. The first, the fresh and tender leaves should be used as the materials; the second, 3% PVP should be added into to the extraction buffer; and the third, the steps (adding 500μl 20% SDS and 1500μl 5M KAc) should be duplicated one more time after the first 25000g high speed centrifugation.2. Optimization of RAPD-PCR system in loquat . Based on the common RAPD reaction program and adjusting experiments, the optimal amplification program for loquat RAPD-PCR was described as follows: 94℃ for 240 s; 45 cycles at 94℃ for 60s, 38℃ for 90s and 72℃ for 120s;72℃ for 600s. Effect of the content of Mg2+, dNTP, DNA templates, primers andDNA polymerase on experimental results were tested and the optimal reaction system of RAPD for loquat germplasm was determined as follows: 0.6mmol-L"' Mg2+, 500 umol-L'1 dNTP, 300n mol-L'1 primer,50 ng DNA and 1.25 unit Taq DNA polymerse in total 25uL reaction volume.3. RAPD analysis for 11 main species (cultivars) in Eriobotrya, 11 accessions of loquat genetic resources including Eriobotrya japonica (cvs. Jiefangzhong, Moriowase, Zaozhong No.6, Wuqi, Baili, Luoyangqing, Yantangpipa, Hubeiliuer) E. prinoides, E. deflexa, Guizhouyesheng,etc. were analyzed by RAPD with 14 arbitrary 10-mer primers. A total of 130 DNA bands were amplified, among which 3 bands were non-polymorphic. The genetic diversity degree was 97.69%. By UPGMA (unweighted pair group with mathematic averages), as Dl=0.599, 11 accessions of loquat genetic resources were divided into 2 groups(cultivated group and non-cultivated group); as D2=0.649, Eriobotrya japonica were divided into two groups (white color pulp group and red color pulp group). Therefore, loquat pulp color was proved to be a taxonomic index on DNA molecular level. RAPD provides a new path for identification and classification of loquat germplasm.4> RAPD analysis of 65 accessions of loquat germplasm. The genetic relationship and classification of 65 accessions of loquat germplasm was analysised by RAPD. 16 lObp-primers were screened from Sangon arbitrary primers to amplify the genomic DNA of 65 accessions of loquat. A total of 210 bands were amplified, among which 210 bands(100%) were polymorphic. The results showed polymorphism of loquat germplasm was high and nuclear germplasms identified primarily were Duanbingbianhe^ Tiancaozaosheng^ E. prinoides, E. deflexa^ Jiefangzhong^ Xiangzhong No. 11.The genetic distance among 65 accessions ranged from 0.045 to 1. Based on UPGMA cluster analysis of genetic distance, a DNA molecular dendrogram was established for 65 accessions of loquat, which divided 65 accessions into 2 groups as D=0.847. One group including No.7 (￡. prinoides, E. deflexa)^ No. 1 (Guizhouyesheng ),No.5( Taiwanpipa) f P No.8 (Hainanyesheng) was non-cultivated type, the others was cultivated type. The classification was consistent with those of the previous studies, but the classification of sub-groups of cultivated type according to cluster analysis is somewhat different from those of previous studies.5. RAPD analysis of genetic relationship among Zaozhong No. 6, Jiefangzhong and Moriowase in loquat .Genomic DNA from three loquat cultivars (Zaozhong No.6, Jiefangzhong and Moriowase) was extracted for RAPD analysis with 43 random primers .The results showed that 255 bands were amplified from the 3 cultivars, among them 82 bands were common with the monomorphic degree of 32.16%, which suggested that the genetic relationship be close among the three loquat cultivar. Parents'chiracteristic RAPDs emerged in filial generations by comparison with parent's common bands. 7 amplifying bands were detected in the 3 cultivars by RAPD analysis with S7) primer. Among them , 780 bp and 1500 bp bands were female parent (Jiefangzhong) and male parent (Moriowase) characteristic bands, respectively. Both the two characteristic bands occurred stably in filial generation (Zaozhong No. 6). The above results confirmed that Zaozhong No. 6 was the hybrid between Jiefangzhong and Moriowase by experiment in the DNA level.6. RAPD analysis for Jiefangzhong materials from 12 different spots.12 accessions of Jiefangzhong materials from 12 spots were investigated by RAPD analysis. 13 selected Sangon 10 bp primers generated64 bands, in which different primer amplified band number ranged from l(S60) to 8(S80), average band number amplified by each primer is 4.85. Genomic DNAs from 12 spots amplified by eleven primers generated same DNA bands. But Genomic DNAs of sample NO. 3 amplified by S66 deleted one DNA band at 850 bp and NO. 9 by S80 deleted one DNA band at 250 bp. The results indicated that genetic stability was high in Jiefangzhong, but some genetic variations occurred with the changes of environments and the passage of time.