Studies On Genetic Diversity Of Auricularia Auricula-judae Cultivated Germplasm Resources In China

Auricularia auricula-judae, the fourth most cultivated edible mushroom (following Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes) in terms of total world production, is play a very important role in mushroom industry in China. As the cultivated origination and the most productive country of A.auricula-judae in the world, China has abundant cultivated germplasm resources of A. auricula-judae, and the rich gremplasm lays the foundation for further breeding programmer of A.auricula-judae. Accurate identification and objective evaluation on the genetic diversity of A.auricula-judae cultivated germplasm in China, is the basis of cross breeding and new variety exploition, as well as the premise of strengthen the strains management and intellectual property rights protection, which could promote the healthy and continued development of A.auricula-judae industry of China.In this study,32 main cultivars of A.auricula-judae in China were selected as material; four methods (physiological characteristics, TRAP molecular marker, intergenic spacer analysis and somatic incompatibility) were firstly introduced to analyze phylogenetic relationship and genetic diversity of A.auricula-judae cultivated germplasm in China. The aim of the present study was to identify the genetic relationship of A.auricula-judae in China, and evaluate the efficiency of these four different methods in analyzing genetic diversity of A.auricula-judae in China, construct the physiological characteristic information database of main cultivars. The main results of this study were as follows:1. The principal components analysis of 27 physiological characteristic indexes of main cultivars of A.auricula-judae in China demonstrated that, for rapidly and effectively analyze the relationship among different A.auricula-judae strains based on physiological characteristics, the most important factor is the characteristics of mycelium, follow by color and size of fruitbody, and finally, the wrinkle and shape of the fruitbody. According to this method,27 stable and important physiological characteristics could be transformed to fewer and non-related comprehensive indexes, which provided rapidly and accurate information for the classification based on physiological traits.2. The analysis of 27 important physiological indexes demonstrated that distinct difference existed among test strains. Euclidean distance similarity coefficients among the 32 tested strains were ranged from 4.27 up to 11.33, which indicated there was rich genetic diversity in Chinese cultivated germplasm of A.auricula-judae. The co-phenetic correlation between clustering and the data matrix was estimated at 0.77, corresponding to a good fit. UPGMA dendrogram grouped all strains into 3 main clusters at a Euclidean distance index value of 6.73, while H29,186 and C21 was represent a single cluster respectively. The principal coordinate analysis also divided 32 test cultivars into 3 groups; the strains in the same group had more similar physiological traits.3.TRAP molecular marker were applied to analyze the genetic diversity of cultivated strains of A.auricula-judae in China, the PCR primer were designed based on EST sequence and 16 stable primer pairs were selected for demonstrate genetic diversity among 32 test strains. Among 535 amplified DNA fragments,97.9% were polymorphic. Genetic similarities by SM coefficients among the 32 genotypes varied from 0.567 to 0.922, which means a vast genetic diversity was found in Chinese cultivated germplasm of A.auricula-judae. The co-phenetic correlation between the clustering and the data matrices was estimated at 0.92, which means the clustering result reflects the germplasm genetic relationships very well. UPGMA dendrogram grouped the 32 genotypes into 4 main clusters at a similarity index value of 0.67. The principal coordinate analysis also divided 32 test cultivars into 3 groups.4. The complete nuclear rDNA gene complex of AU110 was obtained firstly, and the primer pairs which were more suitable for amplify IGS region of A.auricula-judae were designed. The complete nuclear rDNA length of AU110 was 11209bp, which contains the 18S,5.8S and 28S rDNA gene as well as the ITS and IGS regions. The exact size of each gene was estimated namely 1805 bp for the 18S rDNA,513 bp for ITS sequence,3135 bp for 28S,2334 bp for IGS1,118 bp for 5S and 3304 bp for IGS2 region. IGS1 region have no repeat, and the sequence of IGS2 5'end region was GC-rich (from 8551bp to 8638bp), with (GGGGA)n repeat, which increased the difficulty in complete IGS2 sequencing, and (TTAGG)n repeat existed in the sequence from 8768bp to 8799bp as from 8845bp to 8880bp in IGS2 sequence.5. The IGS1 matrix length of 32 test cultivars of A.auricula-judae was 2312bp, which contain conserves sites 2205bp, variable sites 109bp; The matrix length of IGS2 3'end was 801bp, which contain conserves sites 757bp, variable sites 44bp. Based on the IGS1 complete sequence and IGS2 3'end, the phylogenetic relationship among test cultivars was analyzed by applying the minimum evolution and maximum parsimony methods. The research result demonstrated that the variability of IGS region was high, and a vast genetic diversity in the cultivated germplasm found in China. The 32 test strains were divided into 5 or 6 groups based on IGS1 complete sequence and IGS2 3'end respectively, while divided into 3 groups based on integrated IGS1 and IGS2 3'end region analysis. The integrated sequence analysis contained more variable information than the analysis based on single gene, and can more effectively reflect the phylogenetic relationships among test strains.6. The somatic incompatibility reactions among test cultivars have abundant polymorphism, and obvious difference was found among test strains in three aspects: reaction types, reaction intensity and pigment in the junction of mycelium. The reaction types can be divided into hyphal aggregate, ravine and clear zone. Intensity was scored with compatibility, weak incompatibility and strong incompatibility. Pigment types were indicated by the presence or absence of pigment. There were no correlation among the reaction types, pigment production, and intensity in the SIT phenomenon. UPGMA dendrograms were constructed by setting all the tested strains as samples, and then the types, pigment and intensity respectively as variables. The clustering analysis based on SIT intensity could reflect the relationship among the test strains, while the clustering analysis based on SIT types and pigment failed.7. In the UPGMA clustering analysis based on SIT intensity among test strains, Euclidean distance similarity coefficients was ranged from 3.724 up to 10.633; the co-phenetic correlation between the clustering and the data matrix was estimated at 0.78, which means the clustering result reflects the germplasm genetic relationships very well. The UPGMA dendrogram based on SIT intensity grouped all strains into 6 main clusters at a Euclidean distance index value of 7.26. The principal coordinate analysis also divided 32 test cultivars into 3 groups. The test strains has visible somatic incompatibility reactions were divided into different group in UPGMA and PCO analysis, while those strains compatible with each other were cluster together, the SIT reaction intensity and genetic relationship were positive correlation.As a whole, there was rich genetic diversity in Chinese cultivated germplasm of A.auricula-judae, most strains originated from different cultivated area existed obvious genetic divergence, while the strains originated from same cultivated area have a high similarity genetic background. The 32 tested strains could be divided into three groups corresponding to the Northeast, the South,Central and the Northwest, as well as the North and Southwest regions respectively, which also proved that the cultivated strains were mostly domesticated from the local wild-type strains. The genetic background of cultivated strains in Northeast were similarity, and the strain introduction phenomenon can be found in Central and North region of China, partial strains demonstrated higher similarity coefficients and were suspected to be synonymous. Meanwhile, the result proved all the four analytical methods were reliable for analyze the genetic diversity in Chinese cultivated germplasm of A.auricula-judae. The research conclusion obtained in the present work provides significant information for further cross-breeding and genetic improvement of A. auricula-judae, and providing powerful tools for the development of strains rapidly identification, genetic diversity analysis and strains-special SCAR markers in A.auricula-judae.