Genetic Diversity Of Wild Auricularia Polytricha In China

Auricularia polytricha is a natural edible and medicinal mushroom; it has a very important economic value. And with the expanding of cultivation area, the commercial name codes of strain were confused in the long term mushroom production, and the phenomenon of the same strain with different names and different strains with the same name was very serious. Meanwhile, aged and degenerate strains were introduced to the production. In addition, strains for cultivation were isolated from limited resources. So the depression is a common problem in cultivated strains. It has an important theoretical and practical significance for the study of genetic diversity, and extensive genetic diversity can improve the adaptation of species viability and the potential for breeding and genetic improvement. However, wild germplasm is a main resource for breeding, it is therefore necessary to evaluate the genetic diversity of Auricularia polytricha among natural populations.ISSR (Inter-simple sequence repeat) and SRAP (Sequence related amplified polymorphism) markers were used to assess the genetic diversity of Auricularia polytricha within and among 27 natural populations, and 145 strains were isolated by dry tissue of basidiocarps from these 27 populations. The purpose of this study was to evaluate the levels of genetic variation and relationships among the strains with different genetic bases, and to provide information for future breeding programs of Auricularia polytricha, for example, the selection of parent strains with high quality of cultivation. The main results of the present study were as follows:The reaction system and amplified procedure of ISSR-PCR suitable for Auricularia polytricha were established. The main elements include,25μL amplification reaction mixture containing 2.5μL 10 x PCR buffer,25 ng template DNA,0.24 mmol/L dNTPs,1 U Taq DNA polymerase,0.9 mmol/L Mg2+ and 2μmol/L primer, ddH2O 12.6μL. Optimal annealing temperature was decided by different primers. Optimal cycling times were thirty-five.12 primers with stable amplification and rich polymorphism for ISSR were selected from 40 ISSR primers.The reaction system of SRAP-PCR suitable for Auricularia polytricha was established. The main elements include 25μL amplification reaction mixture containing 2.5μL 10×PCR buffer,25 ng template DNA,0.48 mmol/L dNTPs,1.5 U Taq DNA polymerase,1.5 mmol/L Mg2+ and 0.4μmol/L primer, ddH2O 14.7μL. Ten pairs of primers with stable amplification and rich polymorphism for SRAP were selected from 130 pairs.At the species level, a total of 509 loci were amplified using eleven ISSR primers at an average of 46 loci per primer, with the percentage of polymorphic loci (P)=100%, Nei's gene diversity (H)= 0.1935, Shannon information index (Ⅰ)=0.3274, and total genetic diversity (Ht)=0.1918. At the population level, P=35.3%,H=0.1277,I=0.1907 and diversity within populations (HS)=0.1135. Take genetic diversity index as evaluating indicator, the samples from Hainan exhibited the highest level of genetic diversity (P=63.85,H=0.1735,I=0.2763), followed by Guangdong (P=61.69%, H =0.1687,I=0.2686), Jiangxi (P=53.63%,H=0.1628,I=0.2535) and Yunnan (P=59.33%,H= 0.1541,I=0.2477). While the least genetic diversity was found in the samples of Jilin, followed by Gansu, Hebei, Zhejiang and Hunan, in which Nei's gene diversity (H) was less than 0.1.At the species level, a total of 478 loci were amplified using nine SRAP primer combinations at an average of 53 loci per primer pair, P=100%,H=0.2323,I=0.3805,Ht,=0.2287. At the population level, P=41.2%,H=0.1501,I=0.2238, Hs=0.1334. The samples from Hainan exhibited the highest level of genetic diversity (P=73.85,H=0.2134,7=0.3347), followed by Jiangxi (P=69.04%,H= 0.2115,I=0.3280), Yunnan (P=74.48%, H=0.2035,I=0.3232) and Guangdong(P=70.08%, H= 0.2005,I=0.3157). While the least genetic diversity was found in the samples of Jilin, followed by Gansu and Hunan, in which Nei's gene diversity (H) was less than 0.1.At the species level, a total of 495 loci were amplified using five ISSR primers and five SRAP primer combinations at an average of 49.5 loci per primer (parirs), P=100%,H=0.2117,I=0.3508, Ht=0.2110. At the population level, P=37.9%, H=0.1384,I=0.2062, Hs=0.1231. The samples from Jiangxi exhibited the highest level of genetic diversity (P=62.42,H=0.1919,I=0.2977), followed by Hainan (P=65.86%,H=0.1843,I=0.2912), Guangdong (P=63.43%,H=0.1797,I=0.2837) and Yunnan (P=65.66%,H=0.1712,I=0.2742). While the least genetic diversity was found in the samples of Jilin, follow by Hunan, Gansu and Zhejiang.A high degree of genetic differentiation (Gst=0.40) among populations was detected on the base of genetic diversity analysis, suggesting that 60% of variation was intrapopulation, but among populations also resided a remarkable genetic differences. Genetic distance analysis also suggested that more genetic differences were found among strains (D:0.320-0.039) than interpopulations (D:0.248-0.025). This genetic structure was mainly due to the combined effects of the limited gene flow (Nm=0.7), geographical isolation, and random genetic drift.Clustering analysis indicated that most of the individuals of a specific population were arranged in population-specific clusters, which was further confirmed by a principal coordinate analysis (PCoA), revealed that a remarkable geographical relatedness was existed among wild strains within population, whereas no distinct geographical pattern occurred in the genetic distance among populations. The first most informative coordinate could analyze the genetic relationship among strains, and accounted for 77%-81% of the variations observed.Comparison of the datas obtained by molecular markers was made by the method of Mantel test. There was a little correlation between ISSR and SRAP marker (r=0.2138), this suggested that there was no disturbing between the two markers. While the correlation between ISSR and ISSR+SRAP (r= 0.5948) was higher than that in SRAP and ISSR+SRAP (r=0.5771).