Molecular Markers Of Grape Seedless Gene Research

1. The verification of 18 bp specific primer in the grape cultivars. The accuracy of 18 bp specific primer was detected by RAPD technology with 37 cultivars of the seeded grapes and the seedless ones. The size of polymorphism fragment by ainplication was about 590 bp, and the seedless grape cultivars displayed this band, whereas the seeded ones did not. Moreover, this band could distinguish seedless grape cultivars from the seeded ones accurately. This suggested that the 18 bp specific primer, designed and synthesized according to the sequence of UBC-269-484, be capable of the function of detecting the seedless genes in grapes. 2. Cloning, sequencing of RAPD marker and converting into SCAR marker. The specific fragment, amplified by 1 8 bp, was reclaimed from eletrophoresis gel, and cloned in T-easy vector, then sequenced from two directions. The full length of the specific frament was actually 569 bp. According to the sequence, two pairs specific primers, P1+P3 and P2+P3, were designed and synthesized based on the use of the PC-gene software to perform PCR for detecting seedless gene with the seeded grape cultivars and the seedless ones. The sequences of the P1, P2. P3 primers were as follows: P1 CTC ATC TIC TTG ATG GIG Al 3 ; P2 GAC CAG TCT GCA CIT ACA GT 3 P3 AAG TGA AGA ACA TCA TTA AGA AC 3 . By the two-primer amplifications, two fragments, 530bp and 500 bp, could also distinguish the seedless grape cultivars from the seeded ones. This detecting result of the seedless gene was consistent with the RAPD marker. Thus, the RAPD marker was converted into SCAR marker successfully. 3. The application of the molecular markers to the breeding of the seedless grapes. On the fact of the accuracy of 18 bp specific primers and two pairs of sythesized ones, they were used to detect the seedless genes in the cross combinations A, B, C, D and E, whose F1 progenies were 165 that hadn抰 set fruit so far. The RAPD marker amplified by 18 bp and the SCAR marker amplified by two pairs of sythesized primers could distiuguish the seedless plants from seeded ones. The preselection of the seedless trait was successed by the two different markers in young seedlings still in the vegetative state. 4. Southern blot Two restriction enzymes were selected to digest recombinant plasmid on the restriction-site by the sequence analysis of PC-gene software. 310 bp specific fragment was obtained and made into a probe with DIG-labeling for detecting the seedless genes in grapes. The genome DNA of Sultanina, Red Globe and other seeded and seedless grape cultivars were also digested by the Spe I and Hind III for southern blot. The results showed that this probe could be used for distinguishing the seedless grape cultivars from the seeded ones.