Molecular Genetic Position For Walnut(Juglans Regia L.) Precocious Gene

Precocious gene of walnut is rare natural resource. Studying on inheritance of walnut precocious gene and finding DNA molecular markers linked to it are not only significative to walnut breeding and cultivation, but also to revealing flowering regulating genes and their relations with juvenility in woodyplant. Studies of recent years indicated that walnut pecocious characteristic is quantitative characters, and it is controled by multiple genes. In this research, RAPD technique was used to study segregation of a specific DNA marker OPB-08900 in walnut F1 hybrids of cross between precocious parent Lüyuan and later mature parent Lüfeng, and genetic distance to this marker is counted; we cloned and sequencinged this marker, and transformed it to SCAR marker; SRAP marker reaction system in walnut were also established in this research. Main results are followed.①Extraction of walnut genomic DNAThis research indicated that improved CTAB method is a fast, convenient and appropriate way for genomic DNA extraction in walnut. Both the enzyme digestion and agarose gel electrophoresis test indicated that the quality of genomic DNA was high pure, and they were fit for the further studies.②Segregation of RAPD marker OPB-08900 in walnut parents and F1 hybrids of crossA 900bp DNA fragment was amplified by RAPD primer OPB08 in precocious parent Lüyuan, but this fragment did not appear in late mature parent Lüfeng. In 70 precocious hybrids of cross, this fragment appeared in 69 progenies, and in 84 late mature hybrids of cross, this fragment did not appear in 82 progenies.χ2 test indicated that precocious and late mature hybrids of cross accorded with 1:1 segregation proportion, and the marker OP-B08900 segregated with precocious character. In 154 hybrids of cross, there were 3 recombinant types, and the recombinant rate was 1.95%. It indicated that RAPD marker OP-B08900 linked with walnut precocious gene. ③Retrieval, cloning and sequencing of RAPD marker OPB-08900RAPD marker OPB-08900 was retrievaled and cloned into PMD18-T vector in this research, and then transferred into E.coli JM109. White screen was selected from the LB solid culture medium and cultivated in fluid culture medium for 12 hours. Plasmid was extracted from bacteria liquid with MiniBest Plasmid Purification Kit. Sequencing final showed that this DNA fragment was actually 958bp in length. It was submitted to GenBank and the accession number was DQ673614. Sequence analysis carried by software of DNAMAN indicated that there are 36 enzyme digestion sites and it has no gene structure. So it should belong to the DNA fragments which linked to precocious gene of walnut. GeneBank accession number of this marker is DQ673614. Comparability of this fragment was analysed in NCBI, and 26 sequences of other species were found to be homologous to this fragment, but comparability sites are mostly between 27 to 30bp, and E value=0.002-7.0﹥﹥1×10-5. So the homology are very small, and the DQ673614 are unique to the walnut genome.④Transformation of SCAR markerTwo pairs of SCAR primers SEA and SEB were designed according to sequence of OPB08 and its complementary sequence. After identification, pimer pairs SEA are not insensitive to reaction factors at annealing temperature 56℃, and can be used on molecular assistance breeding. SEA amplified a fragment which similar with OPB-08958 on precocious parent, and can not amplify this fragment on late mature parent. Segregation of the SCAR marker in walnut parents and F1 hybrids of cross was in agreement with RAPD marker OPB-08958. This SCAR marker was reclaimed and cloned, and then results of DNA sequencing indicated that it is in agreement with RAPD marker OPB-08958. So this research successfully transformed RAPD marker to SCAR marker.⑤Establishment of SRAP marker reaction system of walnutSRAP marker reaction system of walnut were established in this research. Different primer pairs were used at SRAP-PCR amplification in walnut parents, and a higher polymorphism primer pair, Em10-Me4, was selected and identified in 20 F1 hybrids of across. This primer pair amplified 196 DNA fragments, in which 107 were polymorphism, and diversity rate was 54.6%. So SRAP marker can be used for analyse at genetic diversity of walnut.