Application In Identification Of Common Strains Of Cultivation Mushroom With Molecular Markers

China is the main producer of edible mushroom in the world. However, since mushroom strains can be copied mutually and have a large turnover, strains in the market have been confused. The complexion of that the same strain with different names and different strains with the same name, lead to little protection for the breeder's rights. Because identification of edible mushroom are generally depended on their cultivation characters and isozyme differences, which are easily affected by culture environmental factors, many DNA molecular markers are introduced. In this study, four DNA molecular markers combined with isozyme analysis were employed to differentiate and identify the common strains of cultivation mushroom, to provide alternative molecular approach for the identification and genetic relationship analysis of mushroom strains. The results are as follows:1. The esterase and peroxidase isozyme bands of all the tested strains (Agaricus bitorquis No.3, No.96, No.178, S2, Agaricus bisporus No.1, No.56, U3, No.299, D6, No.1023, No.2796, Agaricus nivesceus S1, Pleurotus ostreatus No.9, Flammulina velutipes No.19, Lentinula edodes No.9015) were clear and polymorphic after cultivated for 20 d on nutritional PDA on 25℃. Cluster results of these two isozyme patterns showed that tested strains were clustered into 2 groups at 60% similarity level. A. bitorquis strains, A. bisporus strains and A. nivesceus S1 were in a group, and P. ostreatus No.9, F. velutipes No.19, L. edodes No.9015 were in another group.2. Restriction fragments from the ITS and IGS amplified products which were digested using 5 restriction enzymes, respectively (AluⅠ,HaeⅢ,HinfⅠ,MspⅠ,RsaⅠ), were used for cluster analysis. Two to four restriction fragments from ITS amplified products were in sizes from 100 bp to 600 bp. Two to seven restriction fragments were generated from IGS amplified products. The restriction fragments from IGS1 were 100～1,000 bp, and IGS2, 200～2,000 bp. The cluster results revealed that tested strains were clustered into 4 groups at 65% similarity level. A. bitorquis strains, A. bisporus strains and A. nivesceus S1 were in a group, and P. ostreatus No.9, F. velutipes No.19, L. edodes No.9015 represented three groups, respectively. 3. Six primers (808~#, 810~#, 811~#, 834~#, 835~#, 842~#) selected from 14 ISSR primers (808~#, 809~#, 810~#, 811~#, 812~#, 822~#, 834~#, 835~#, 841~#, 842~#, 852~#, 853~#, 866~#, 868~#) and 7 pairs of primer (me2em1, me2em2, me2em6, me4em1, me5em2, me5em3, me5em6) screened from 30 pairs of SRAP primer, which were from 5 forward primers (me1, me2, me3, me4, me5) and 6 reverse primers (em1, em2, em3, em4, em5, em6), could amplify DNA fingerprints of all tested strains. Under the optimized ISSR and SRAP reaction conditions, all the DNA fingerprints ampilfied by the above selected primers were used for cluster analysis. In ISSR results, a total of 192 bands were produced by 6 primers, among which 98.4% were polymorphic. Each primer could amplify 28～35 bands in sizes from 250 bp to 2,000 bp. In SRAP results, a total of 211 bands were generated by 7 pairs of primer, among which 94.3% were polymorphic. Each pair of primer could produce 30 bands ranged from 100 bp to 2,000 bp on average. The cluster results of these two techniques, which were almost coincident, were similar with the cluster results of isozyme analysis, ITS and IGS restriction fragments.4. Finally, restriction fragments of ITS and IGS, amplified products of ISSR and SRAP were used for cluster analysis together. Results indicated that when similarity level reached 61%, tested strains were divided into 4 groups. A. bitorquis strains, A. bisporus strains and A. nivesceus S1 were in a group, and P. ostreatus No.9, F. velutipes No.19, L. edodes No.9015 were in three groups, respectively. When similarity level reached 71%, all tested strains were clustered into 5 groups. A. bitorquis No.3, No.96 and No.178 were in groupⅠ, seven A. bisporus strains (No.1, U3, No.56, No.299, D6, No.1023, No.2796) were clustered with A. nivesceus S1 and A. bitorquis S2 in groupⅡ, and P. ostreatus No.9, F. velutipes No.19, L. edodes No.9015 represented groupⅢ, groupⅣand groupⅤ, repectively.