The Application Of Mating Test And Degenerate PCR In Genetic Analysis Of Auricularia Auricula

The molecular fingerprints and genetic similarities of 34 Auricularia auricula strains cultivated mainly in China were studied by their esteraseisozyme,ISSR and SRAP markers in our laboratory in the early stage, which showed that partial strains have high similarity and may be synonym such as Xinke 1 and Xinke 5, strain 139 and strain 8129. Therefore, mating test and degenerate PCR were applified for genetic analysis of partial parent monokaryons and F₁ sporulated monokaryons in order to study identification techniques of cultivated strains for A. auricula, in the paper.The cultivated strains were divided into 3 groups: Xinke 1 and Xinke 5 (Group I), 139 and 8129 (Group II), Xinke 5 and Hei 29 (GroupIII). The parent monokaryons and F₁ sporulated monokaryons were developed from every strain by protoplast monokaryonization and single-spore isolation technique, respectively. Mating tests were carried out between parents with F₁ sporulated monokaryons from same or different strains in same group. Based on affinity analysis between two sets of parents and F₁ sporulated monokaryons in same group, genetic similarities index were calculated for two sets of parents. The result revealed that: (Group I) Mating type genes of Xinke 1 and Xinke 5 have high similarity coefficient matrix, which were 0.96 for XK1-S70 and XK5-P55, and 0.75 for XK1-P11 and XK5-P32. (Group II) By the same token, the similarity coefficient matrix for mating type genes was 0.93 for 139-S46 and 8129-S22, and 0.86 for 139-S13 and 8129-S8 respectively. (GroupIII) Affinity ratio between a set of monokaryons from Hei 29 with monokaryons of Xinke 5 were all 100%. Comparision with parents monokaryons of Xinke 5, F₁ monokaryons showed genetic recombination on mating genes after crossover for chromosome of basidia during meiosis period. The result showed that there were differences for allele genes among different strains to some extent. There were significant similarity in Group I and Group II, low similarity in GroupIII. Mating type marker should be used for detection of A. auricula strains.ISSR marker was applied for study on genetic analysis of monokaryotic strains from A. auricula. Seventy-three ISSR primers were screened and Thirteen which could differentiate parents T₁ and T₂ effectively were selected to amplify strain XinKe 5 and F₁ sporulated monokaryons. A total of seventy fragments were amplified, among which sixty-three fragments(90.0%) were polymorphic. According to amplification results, genetic similarity coefficient matrix among thirty-six strains ranged from 0.25 for the lowest similarity coefficient between parent monokaryotic strains T₁ and T₂ to 0.83 for the greatest extent of similarity. The ISSR dendrogram based on the similarity coefficient matrix was constructed by using NTsys 2.10e software. Tested strains could be classified into 2 major groups: T₁ and twenty-four of F₁ sporulated monokaryons fell into same group, while T₂ and the rest of F₁ sporulated monokaryons fell into the other group. Monokaryons were not classified strictly based on mating typing. It was showed that chromosome crossing-over in each basidia of fruitbody was complex and polymorphic during meiosis, and the most of F₁ sporulated monokaryons resembled parental monokaryon T₁. It revealed that mating type factor could be formed with exchangeable subunits in A. auricula.To screen the molecular markers related to mating type genes of A. auricula, degenerate primers br1-F and br1-R were employed to amplify monokaryons from different strains. About 775bp DNA fragments were obtained. Sequencing analysis revealed that they belong to pheromone receptor gene. The result showed that pheromone receptor genes of monokaryons from different strains were significant alignments. Also, by means of degenerate PCR we obtained partial DNA fragment (2591bp) whose back 850bp encoded short-chain dehydrogenase by BLASTX. Sequencing analysis revealed that the amino acid sequence was of a membrane protein which have 2 transmembrane helices. At the same time, we could find the DNA sequence according to introns distribution rule in pheromone gene. It indicated that perhaps the DNA fragment contained downstream sequences of pheromone gene of A. auricula.