Study Of RAPD Marker And It's Application Of The Wild Grapes Native To China

The use of the RAPD technique was investigated on a set of 73 accessions of 18 wild grape species native to China,and one interspecific hybrid,7 Vitis Vinifera cultivars,one rootstock cultivar and one strain of V.riparia. RAPD fingerprints of 83 grape materials were obtained. An inheritance distance matrix was constructed on the basis of the presence or absense of 191 DNA bands generated by 20 Operon primers, and cluster analysis was performed. Genetic diversity among these grapes was investigated using RAPD assay. Genetic linkage maps were created for two parents 83-4-96(V.quinquanglaris) and Muscat Rose (V. vinifera) using RAPD marker data from 60 F1 progenies (83-4-96×Muscat Rose). We used the RAPD technique to identify molecular genetic markers linked to grape downy resistant and downy susceptible genes OPO06-1500 and OPO10-835. The two RAPD markers were cloned in T-vector easy and sequenced. According to the sequences, two pairs of specific primers were designed to perform PCR. The RAPD markers were converted to SCAR markers successfully. The main content of this paper was as follows: 1. Improving of CTAB method adapting to grape genomic DNA isolation and optimization of RAPD reaction system. An improved DNA extraction protocol with CTAB was developed. The DNA was extracted directly from young woody canes tips within vigorous growth peoriod. An economic and effective RAPD system was obtained which adapted to grape RAPD analysis. The optimized system inclued 10ng template DNA, 150μM dNTPs, 1.5 mM Mg²⁺ concentration, 1U Taq DNA polymerase, 0.5μl Operon random primer (4pM) with 25μl of the total volume. This was the basis of large scale research of many grape materials′RAPD analysis. 2. The RAPD fingerprints of the wild grapes native to China and their molecular taxonomy. The screening of 280 decamer oligonucleotides allowed the selection of 20 primers used for the analysis. They revealed a high level of polymorphism among the materials. One hundred and ninty one bands,intense and easy to score,were chosen as markers. Identification of 83 materials was obtained with only one of two primers (OPQ04 and OPJ01) combined with one of five primers(OPH19, OPP02,OPA15, OPJ07 and OPU16). The average bands number was 3～7 each sample/primer. The rate of polymorphic bands was 16.7～90.9%, and the size of bands ranged from 100bp to 3000 bp. The simmilarity coefficents (s) and genetic distances (D=-Lns) were calculated using Nei′s coefficient of genetic distances. Relationships among the eighty three grape accessions based on their genetic distances, were clustered using unweighted pair group average cluster method (UPGMA) in a dendrogram. Twenty two clusters which fortunetly adapted to 22 grape species level were clearly resolved on the dendrogram. Relationships represented in the dendrogram were consistent with the available pedigree information. The largest distance was found between V. riparia, V. vinifera, V. larbrusca and the wild grapes native to China. V. hancockii was the oldest species and V. qinlingensis was the next to V. hancockii in the Chinese wild Vitis species based on their genetic distances. The seventeen wild grape species native to China were grouped into nine subclusters. Genetic distances among different flower type accessions of the same species were larger than the same flower type accessions in spite of coming from different geographical conditions. Relationships among the different accessions of one species with same flower type were consistant with their geographical distribution. 3. RAPD markers of grape downy mildew resistant (MR) and downy mildew susceptible (FS) genes and their location. The resistance to grape downy mildew is important genetic trait in disease resistance modification for grape cultivars. The objective of the research was to seek molecular markers with MR and FS genes by RAPD analysis. Two RAPD markers OPO06-1500 and OPO10-800 were tightly linked to MR and FS respectively. Using 88-110 cross F1, three F2 populations and 88-84 cross V. quinquangularis (Xun3) ×V. vinifera (Ugni Blanc). The two markers were verified in the wild grapes. Based on Mapmarker software analysis, the map distances of MR and OPO06-1500, FS and OPO10-800 were 1.7 cM and 0 cM respectively. OPO06-1500 was localized on P1-5 linkage group. OPO10-800 was localized on P-2-2 linkage group with the same position as FS. 4.Cloning and sequencing of OPO06-1500 and OPO10-800 which were converted into SCAR markers. OPO06-1500 and OPO-800 fragments were cut from electrophoresis gel andcloned in T-vector easy, then sequenced from two sides. 586bp and 452bp of OPO06-1500 were sequenced from two directions. The actual length of OPO10-800 which was sequenced was 835 bp. Then OPO10-800 was marked into OPO10-835. According to the sequences, two pairs of specific primers were designed to perform PCR. The RAPD markers were tranformed into SCAR markers successfully. 5.RAPD based genetic linkage maps of two grape accessions. A set of 280 random primers was screened for RAPD fragments within a sample of 60 interspecific F1 hybrids to generate RAPD markers and construct the molecular maps for V. quinquangularis (83-4-96) and V. vinifera (Muscat Rose). The maps consisted primarily of RAPD markers, but also contained four morphological markers (MR, grape downy mildew resistance; FS, grape downy mildew susceptibility; MFT,female flower ♀;FFT,perfect flower). Maps were constructed using a double pseudotest cross mapping format and Mapmarker version 3.0 software. A 3.0 linklod parameter and a maximum map distance of 37.3cM were the minimum criteria used to define linkage groups. Map distances were computed using the kosambi mapping function and default maplod threshold of 0.05. 58 repeatable RAPD markers and 4 morphological markers were assigned to 14 differen linkage growps including 8 linkage groups of 83-4-96 and 6 of Muscat Rose. The 83-4-96 linkage maps covered a total genetic distance of over 687.5cM (centimorgan), the average distance between markers was 17.6cM. The Muscat Rose′s maps covered a total genetic distance of over 654.6cM, The average distance between markers was 19.3cM. The sixth linkage groups of both parents (P1-6,P2-6)were homologous to linkage groups. There were 33 unlinked markers. It supplied a linkage framework for a more saturated linkage map of grape. 6.The segregation patterns of RAPD marker in an interspecific F1 hybrids of 88-110 cross between V.quinquangularis (83-4-96) and V.vinifera (Muscat Rose). The segregation patterns of RAPD marker in an interspecific F1 hybrids between V.quinquangularis (83-4-96) and V. vinifera (Muscat Rose) were studied. According to the segregation patterns, RAPD markers were grouped into three types:(1)normal Mendelian inheritance with segregation ratio of nearly 1∶1,3∶1or 1 ∶0(non segregation);(2)deviation from mendelian segregation ratios; (3)abnormal segregation in low frequency, including none parental markers, absent in both parents but present in progenies. Basing on statistics of RAPD markers, there exist 83.5% of 1∶1 and 3∶1 mendelian segregation marker, 44.9% of none segregation marker, 2.1% of abnormal segregation merker. RAPD markers presenting in both parents were the main none segregation markers, markers presenting in only one parent mainly showed mendelian segregation. There werefive non parential marker. Markers of presenting 1∶1 and 3∶1 segregation were used for constructing RAPD molecular linkage map. The fact that abnormal segregation was resulted from the exchange between chromosomes was proved by RAPD assay.