Cucumber Female Rapd Markers And The Acc Synthase Gene Cloning

Cucumber (Cucumis sativus L.) is an important vegetable crop in Cucurbitaceae. Its sex phenotype is complicated and various. Cucumber is one classic material in research of sex differentiation and sex expression patterns in plants. The development stage and distributing state of female flower that is the sexual representation are closely related to maturation stage, yield and quality etc. Presently, gynoecy or sub-gynoecy is important approach in cucumber heterosis breeding. Sexual representation of cucumber is controlled not only by genetic factors, influenced but also by environmental factors etc. So the selection efficiency of gynoecious gene based on its phenotype through traditional method is lower. Recently, the molecular marker-assisted selection of cucumber has more and more gotten attention by researchers. This technique can make up the traditional breeding method, improve exactness of selection and quicken the step of breeding. The study on molecular marker of the related gene is the basis of molecular marker-assisted selection, isolation and cloning of genes from cucumber.Genetic analyses in cucumber showed that the different sex types were determined by three major genes: F, m, a. The F gene in the dominant condition regulates the female expression in cucumber. In several hormones of plants, IAA and ethylene can promote femaleness. The ethylene production within the apices of stem is tremendously correlated with the differentiation of female flower in plants. As the ACC synthase is a key enzyme which plays a role in the endogenous ethylene biosynthesis, the function of ACC synthase gene in the biological process of sex expression in higher plants has been drawn attention. The postulation that the ACC synthase gene is of close linkage with the F locus determining femaleness of cucumber has come to an agreement.The aims in this paper are to screen the molecular marker correlated to gynoecy of cucumber by the technique of random amplified polymorphic DNA. At the same time, we have converted the RAPD markers into SCAR marker, which is better than RAPD markers in specificity and stability. The research can provide the basis for sex identification of seedling and marker-assisted breeding of the character of gynoecy in cucumber. In addition, by cloning and sequencing the ACC synthase gene, we tried to find out the gene difference among various lines of cucumber, compare the relationship between the gene and diverse sex types of cucumber and probe into the role of ACC synthase gene in the process of sex determination in cucumber. The consequences are as follows:(1) The genomic DNA was extracted from cucumber by CTAB method. RAPD technique was employed to study the molecular markers related to gynoecious gene of cucumber. In this paper, 39 randomly selected 10-bp primers was elementarily screened, of which 12 primers showed polymorphism in cucumber tested. These polymorphic primers were used as candidates to further screen and obtain gynoecy-specific molecular marker by the RAPD technique.(2) The polymorphic primers were used to identify all the gynoecious cucumber by RAPD technique. The results showed that two specific bands of about 2000 bp and 500 bp, scored as S10-2 kb and S435-0.5 kb, could be amplified stably and clearly in cucumber of gynoecious lines with the primer of S10 and S435 respectively, while the band was not found in the monoecious individuals. So we consider that the two molecular markers may be related to cucumber sex.(3) Two fragments of 2003 bp and 470 bp in full length were obtained by sequencing the recovered S10-2 kb and S435-0.5 kb markers. Then the sequences were analyzed by B1AST on NCBI. Sequence analysis showed that the S10-2 kb marker had very low homology with issued sequences of cucumber in database and maybe a new one harboring a largest ORF coding a short peptide of 62-amino-acid residues. We had successfully submitted to the GenBank and obtained an accession number of EF 057801. The blast analysis of S435-0.5 kb revealed that there was no homology compared with the conserned sequences of Cucurbitaceae. This sequence, whose accession number is EF 062502, seemed to be a new one of cucumber harboring a largest ORF encoding a short peptide of 68-amino-acid residues. The functions of these short peptides specified by two RAPD markers were not determined by now and their biological significance remains to be further studied.(4) According to the sequence segment of 2003 bp, we designed two pairs of specific primers scored CS1 and CS2 to convert RAPD marker into SCAR marker. The amplified results showed that the polymorphism vanished in the pair of primers CS1. While a band of expected size correlation to gynoecy of cucumber can be amplified by primer CS2, and not found in monoecious cucumber. These results showed that the RAPD marker had been converted into SCAR marker successfully. So the pair of primer CS2 could be used as a pair of specific primers to identify sex of seedling stage in cucumber.(5) Based on the published sequence of ACC synthase gene, we designed a pair of specific primers and tried to amplify the ACC synthase gene in the lines of gynoecious, sub-gynoecious and monoecious cucumber. As a result, ACC synthase gene in expected size exists only in the lines of gynoecious and sub-gynoecious cucumber, while it is not found in monoecious individuals. A full sequence of 1042 bp in length we obtained by sequencing the clone, was one bp longer than the previous sequence, and the sequence of amino acid encoded by a longest ORF is extremely coincident with the previous result. Then the sequence was compared with the database of NCBI by the BLSATn. Sequencing analysis revealed that the sequence was especially similar to CS-ACS1 and CS-ACS1G with homology of 85% or so. So we elementally infer that the fragment may be ACC synthase gene called ACSG. In view of the relationship between ACSG gene and the phenotype of gynoecious lines, ACSG gene maybe a molecular marker which can be used to identify the line of gynoecious. In addition, the sequence had moderate homology with the ACC synthase genes of other crops of Cucurbitaceae and the plants of Rosaceae, and had lower homology with the ACC synthase genes of the plants of Cruciferae, Leguminosae and Solanaceae etc.Conclusion: It is feasible in search of the molecular marker correlated to the character of gynoecy in cucumber by the RAPD technique. In fact, the accurateness and the selected efficiency of marker-assisted selection can be distinctly improved after converting the RAPD markers into SCAR marker. In this paper, a pair of SCAR primer, i.e. CS2, can be used to distinguish the sex of cucumber seedling; the fragment of ACC synthase gene which exists in both the lines of gynoecious and subgynoecious cucumber is associated with the sexual phenotype of cucumber, and maybe female line-specific. We have cloned and obtained the full sequence in length of ACC synthase gene (ACSG) in gynoecious cucumber. In view of a close connection between ACC synthase gene and the femininity in cucumber, ACSG could be as a molecular marker to identify the female lines.