Cross Breeding Of Grifola Frondosa And Study On Function Of Its Phenylalanine Ammonia Lyase Gene

Grifola frondosa(Dicks.)S.F.Gray is a rare edible and medicinal fungus.Its market demand is increasing day by day for its unique taste flavor and efficient health care effect.At present,the cultivars of G.frondosa in China are mainly from wild domestication or foreign introduction,in which there are some deficiencies such as poor adaptability,low polysaccharide content and low biological efficiency.More excellent varieties are needed to improve the yield and quality of G.frondosa.The production of G.frondosa in China is mainly based on the traditional agricultural cultivation mode,and high temperature could lead to reduced or even no production.In this study,the genetic diversity of 42 G.frondosa strains was analyzed by intergrating the mycelial antagonistic reaction,phylogenetic analysis based on ITS sequences and ISSR and SRAP molecular markers.Combining the results ofvariety comparison test,the parent strains with far genetic relationship and complementary comprehensive agronomic traits were selected for mon-mon hybridization to obtain excellent hybrids.The GfPAL were also cloned,and the expression of GfPAL under different high temperature treatments was analyzed to explore the biological function of GfPAL in response to heat stress,which could provide scientific basis for breeding thermo-tolerant varieties.The results are summarized as follows:(1)Forty-two G.frondosa strains were divided into 17 groups by the antagonistic reaction.The phylogenetic tree based on ITS sequence could distinguish G.frondosa strains from Asia and America,which indicated that there was a certain correlation between the geographic distance and genetic distance of G.frondosa.The clustering analysis resultbased on integration of ISSR and SRAP showed that the 42 strains were clustered into 6 groups with a genetic similarity coefficient of 0.779,indicating a rich genetic diversity across the G.frondosa strains.The 6 strains of Jinxianghuishuhua、Hui3、Hui4、HuishuhuaGF54、Qinghui151、Qinghui152 with good agronomic traits were screened out by variety comparison test.Combining the above results,the wild strain Tihui NO.1 and the cultivar Qinghui 151 were selected as parent strains.(2)One hundred hybrid combinations were conducted using 10 spore monokaryons of each parent strain,and 65 real hybrids were obtained after observing clamp connection and antagonistic reaction with their parents.Twenty-three hybrids with fast growth rate and strong growth vigour were selected for factory pilot cultivation experiment.Seven hybrids with excellent agronomic traits were screened out,among which zj86 had the best comprehensive traits.The strain zj86 has big fruiting body cluster.Its fruiting body is dark with short tubule.The average yield was 291.30 g per bag that was 74.50%(P<0.01)and 20.53%(P<0.01)higher than the parent strain Tihui NO.1 and Qinghui151,separately.The biological efficiency was more than 50%.The polysaccharide content in fruiting bodies of zj86 was 4.74% that was 33.52%(P<0.01)and 11.52%(P<0.05)higher than the parent strain Tihui NO.1 and Qinghui151,separately.ISSR and SRAP molecular markers were used to identify the selected 7 hybrids.The results showed that there were different degrees of genetic variation between the hybrids and parents.So the hybrids were truly new strains.(3)The 42 G.frondosa strains and 65 hybrids were treated with high temperature to screen thermo-tolerant strains.After being treated at 37 ℃ for 48 h,8 thermo-tolerant strains and 4 thermo-sensitive strains were screened out.The selected 8 thermo-tolerant strains were treated at 39 ℃ for 48 h,and the 4 thermo-sensitive strains were treated at 35 ℃ for 48 h for secondary screening.Thermo-tolerant strain CGMCC5.404 and thermo-sensitive strain Luqian NO.3 were screened out according to recovery growth rate and growth vigour of mycelium after heat stress.(4)The cDNA and genome sequences of GfPAL were cloned.The expression of GfPAL in thermo-tolerant strain CGMCC5.404,thermo-sensitive strain Luqian NO.3 and cultivar Qinghui 151 treated at 37 ℃ for 0 h,8 h,24 h,36 h,42 h,48 h were analyzed by qRT-PCR.The results showed that the expression of GfPAL in three strains showed a trend of increasing first,then decreasing and then increasing,and all of them peaked at 8 h.The relative expression of GfPAL in CGMCC5.404 was 24.15 times that of Luqian NO.3 at 8h.During the whole process of heat stress,the expression of GfPAL in the hypha of Luqian NO.3 was low and changed little,while the relative expression and the amplitude of variation in CGMCC5.404 were all higher than that in Luqian NO.3.The results showed that heat stress caused significant changes in the expression of GfPAL,which indicated that GfPAL was involved in the response of G.frondosa to heat stress and closely correlated with the heat tolerance of the strains.