Development Of Specific Markers For Tricholoma Matsuke And Tricholoma Bakamatsutake

Tricholoma matsutake belongs to the Tricholomataceae Tricholoma,is a kind of treasure and endangered wild edible fungus,contains a lot of nutrients in the body and is known as "the king of mushrooms.T.matsutake of Changbai mountain is famous both at home and abroad,is one of the most important area of export of T.matsutake.Tricholoma bakamatsutake also belongs to Tricholoma like T.matsutake,its exterior shape and taste are all similar to the T.matsutake.It is hard to accurately distinguish between T.matsutake and T.bakamatsutake based on the normal morphology and microscopy.In this thesis,T.matsutake and T.bakamatsutake of Changbai mountain were chosen as materials and sequence relevant amplified polymorphism(SRAP)molecular marker technology was used to screen SRAP markers for T.matsutake and T.bakamatsutake.Then,cloning,sequencing and redesigning primers were adopted to develop specific DNA markers for T.matsutake and T.bakamatsutake,so as to quickly and accurately identify them at the molecular level.Forty one primers were screened from 300 pairs using gene pools of T.matsutake and T.bakamatsutake;and four pairs of primers,which were primer ME06 and EM26,ME08 and EM21,ME09 and EM03,and ME 10 and EM06,amplified the 422 bp(SK0626-422)?323 bp(SK0821-323),541 bp(SK0821-541),280 bp(SK0903-280)and 650 bp(SK1006-650)specific bands for T.matsutake,but could not amplify these bands in T.bakamatsutake;and two pairs of primers,which were primer ME01 and EM 10,and ME09 and EM07,amplified the 1168 bp(JSK0110-1168)and 563 bp(JSK0907-563)specific bands for T.bakamtsutake,though could not amplify the two bands in T.matsutake;showing that these SRAP markers are closely related to T.matsutake and T.bakamatsutake and can be used to identify them.According to principle of primer design,one pair of specific primers was designed to SK0626-422,SK0821-323,SK0821-541,SK0903-280,SK1006-650 marks for T matsutake and JSK0110-1168 and JSK0907-563 marks for T bakamatsutake,PCR results indicated that the 243 bp(SK243 from SK0626-422)and 304 bp(SK304 from SK1006-650)specific bands were amplified in all individuals for T.matsutake,but there specific markers were failed to check out for T.bakamatsutake;the 502 bp(JSK502 from JSK0110-1168)and 389 bp(JSK389 from JSK0907-563)specific bands were amplified in all individuals for T.bakamatsutake,however the specific markers were failed to check out for T.matsutake;showing that the specific markers of SK243 and SK304 for T.matsutake and JSK502 and JSK389 for T.bakamatsutake were reliable and repeatable DNA markers to quickly and accurately identify them.By primer combination and optimization of reaction system,a compatible multiplex PCR molecular marker of SK304 and JSK389 combination was obtained,and the specific band SK304 for T.matsutake and the specific band JSK389 for T.bakamatsutake were clearly detected through one time of PCR.In addition,the specific SK243 marker for T matsutake can be expressed at transcriptional level,predicting that it may be a partial gene,this remains to be further identified.