Application Of New Molecular Markers To Evaluate The Genetic Diversity In Germplasm Of Lentinula Edodes In China

Lentinula edodes is the second most cultivated edible mushroom (following Agaricus bisporus) in terms of total world production, and play a very important role in mushroom industry in China. China stretches across a vast area, with complex geographic environment and climatic conditions, thus forming abundant wild germplasm of L. edodes. The rich germplasm lays the groundwork for further breeding improved variety of L. edodes. Preservation and utilization of shiitake germplasm are based on accurate identification and objective evaluation of these germplasm. Compared to morphological markers and biochemical markers that are commonly used, DNA-based molecular markers are stable, accurate, and independent of environmental impacts, which is powerful tools for all research field of genetics for edible mushroom. Each molecular marker technique would uncover different genetic information, because each technique has its own unique technological principle and system. So, it would be complementary among genetic information revealed by different molecular marker techniques. Currently, molecular marker techniques on the basis of universal primer PCR were widely used in genetics for mushroom. However, those techniques based on species-special primer PCR, such as SSR markers based on the DNA sequences of both sides of microsatellites, IRAP and REMAP markers based on sequences of retrotransposons, and TRAP markers based on sequences of special genes, were seldom used for mushroom research.In this study, it was the first time in L. edodes to develop special primers of above four new molecular markers, and to optimize their PCR technological systems. These four new molecular markers were then used to uncover genetic diversity and phylogenetic relationship of wild and cultivated germplasm of L. edodes in China. The aim of the present study was to assess the efficacy of these marker techniques in analyzing genetic diversity and phylogenetic relationship of L. edodes in China, and establish several new research techniques for future research. The second aim was to identify the genetic relationship of shiitake germplasm in China, thus providing scientific basis for rational preservation and utilization of these germplasm. The main results of this study were as follows:1. Two methods, database search and ISSR-suppression PCR, were used to isolate microsatellite markers from L. edodes. Six wild strains and two cultivated strains were used to assess polymorphism of developed SSR markers.21 primer pairs were designed by database search,11 out of them with polymorphism. The number of alleles produced by these 11 markers was 3.3 per locus in average. As to the method of ISSR-suppression PCR, eight primer pairs were developed and five of them had polymorphism. There were three alleles per locus in average produced by these five markers. Results showed that two methods were effective and applicable to develop SSR from L. edodes.2. Orthogonal design and gradient PCR were used to optimize SSR technological system. SSR markers were then utilized to reveal the genetic diversity of 55 wild strains and a cultivated strain of L. edodes grown in China. A total of 224 DNA bands were detected through 25 primer pairs, of which,223 bands (99.6%) were polymorphic between two or more strains. The pairwise genetic similarity coefficient ranged from 0.692 to 0.987, with an average of 0.803. The variation in SSR DNA band patterns, average genetic similarity, and Shannon's Information Index among the wild-type strains of L. edodes obtained from the same region uncovered a vast genetic diversity in the wild germplasm found in China. Compared with L. edodes strains originating from other areas, the genetic diversity of those from the Yunnan Plateau, Hengduanshan mountains, Taiwan, and South China was significantly greater.Based on cluster analysis and PCoA analysis, the results indicated that all L. edodes strains were divided into three major groups. These results effectively displayed the differences between the strains from north and south China, and those from the same or adjoining regions could cluster preferentially into small groups in most cases, suggesting the positive correlation between the clustering results and the geographical origin for the wild germplasm of Chinese L. edodes.3. Single-factor cycle screening was used to optimize TRAP technological system. TRAP technique was also used to evaluate genetic diversity of above 56 strains of L. edodes in China.932 DNA fragments were amplified using 12 primer combinations, of which,929 fragments (99.68%) were polymorphic between two or more strains. The average pairwise genetic similarity coefficient was 0.696, with a range from 0.503 to 0.947. Results of average genetic similarity and Shannon's Information Index revealed a vast genetic diversity in the wild germplasm found in China, and the genetic diversity of strains from the Yunnan Plateau, Hengduanshan mountains, Taiwan, South and Northeast China was much higher than that of other regions. UPGMA cluster and PCoA analysis separated the tested strains into two major groups, and Group A was further divided into seven subgroups. Those strains from the same or adjoining regions could cluster preferentially into small groups in most cases. Results effectively displayed that genetic relationships among wild strains of L. edodes were highly associated with geographic distribution.4. Both results of SSR and TRAP indicated that these two highly polymorphic marker techniques were particularly useful for study on L. edodes'wild germplasm in China. There was rich genetic diversity in Chinese wild germplasm of L. edodes, and significant genetic differences exited in eight natural populations of shiitake mushroom in China. Results well reflected geographical distribution of tested wild strains of L. edodes in this study. Populations of L. edodes from Yunnan Plateau and Northeast China were separated independently to other ones among the eight populations. Data from both SSR and TRAP analysis were combined to reveal genetic diversity in Chinese wild germplasm of L. edodes. Results of combined analysis were highly similar to those from single TRAP analysis (Relative coefficient was 0.99 between them), however, were little similar to those from SSR analysis (Relative coefficient was 0.68 between them). UPGMA analysis based on average genetic similarity between two different populations also preferably displayed geographical distribution among the eight populations.5. Single-factor cycle screening and gradient PCR were used to optimize IRAP technological system. Based on the optimized PCR system,19 pairs of polymorphic primer combinations of IRAP and REMAP were utilized to demonstrate genetic diversity among 44 cultivated strains of L. edodes in China. Among 281 amplified DNA fragments, 273 (99.6%) out of them were polymorphic between two or more strains. The pairwise genetic similarity coefficient ranged from 0.495 to 0.975, with an average of 0.668. Both results from UPGMA cluster and PCoA showed that all the strains of L. edodes were divided into four groups, according to differences in cultivated substrate, temperature and growth period of L. edodes. Group A1 was mainly composed of L. edodes strains for sawdust cultivation, fruiting in high or broad temperature. Group A2 consisted of strains for sawdust cultivation, fruiting in medium or low-medium temperature with medium-short growth period. Group A3 included strains for sawdust cultivation, fruiting in medium or low-medium temperature with medium-long growth period. Group B mainly contained L. edodes strains for wood log cultivation. Some strains with close cluster relation indicated some similar agronomic characters.6. SCAR markers were developed based on IRAP and REMAP analysis of cultivated stains of Chinese shiitake mushroom. Two specific amplified DNA fragments from strain 9608 (1712bp) and L135 (2549bp) were excised from agarose gels, cloned and sequenced. Results of BLAST search displayed that the sequence of specific fragment from strain 9608 showed homology with transposase of Phakopsora pachyrhizi. According to the sequence of the strain-specific fragments, two pairs of SCAR primers was designed to diagnose strain 9608 and L135 respectively, using on-line Primer 3 program. This study provided a new developing method for SCAR markers, and SCAR markers exploited here were useful for precisely and rapidly distinguishing strains of L. edodes.As a whole, all of the four new molecular markers are highly polymorphic, and suitable for analyzing genetic diversity of L. edodes in China. Genetic information of L. edodes germplasm in China revealed by the four markers was consistent with those of previous studies. Therefore, the technological systems of four new molecular markers optimized and established in present study were practical and efficient, thus providing powerful tools for development of strains-special SCAR markers, cross breeding, construction of genetic map and mapping QTL in L. edodes.