1、Evaluation of genetic diversity in Lentinula edodes strains usingRAPD,ISSR and SRAP markers Abstract：Random amplified polymorphic DNA(RAPD),inter-simple sequence repeat(ISSR)and sequence-related amplified polymorphism(SRAP)markers were used to evaluate the genetic diversity among 23 elite Lentinula
2、edodes strains in China.A total of 138,77 and 144 bands were detected by 16 RAPD primers,5 ISSR primers and 23 SRAP primer combinations,among which 58.8%,73.5%and 56.3%was polymorphic,respectively.The purpose of this work was using RAPD,ISSR and SRAP markers to investigate genetic diversity among 23
3、 elite strains,and to lay the foundations for directing new cultivars breeding in L.edodes.By UPGMA clustering,a dendrogram was constructed based on each analysis.The three dendrograms showed that 23 L.edodes strains were clustered into three or four groups.The grouping exhibited similar structure a
4、nd was generally consistent with their pedigrees.Twenty-three L.edodes strains shared great similarity indicated that the low level of genetic diversity of L.edodes strains and their relationship between each other.The important source of breeding material,such as wild and exotic types,must be intro
5、duced in order to broaden genetic base and decreases genetic vulnerability of L.edodes.Keywords：Lentinula edodes Random amplified polymorphic DNA(RAPD)Inter simple sequence repeats(ISSR)Sequence-related amplified polymorphism(SRAP)Edible mushroom Strain Genetic diversity Molecular marker.Introductio
6、n:Lentinula edodes.Pegler(Xiang gu or Shiitake mushroom)is the second highest cultivated edible mush-room in total production yield all over the world,and widely cultivated in Eastern Asia,such as China,Japan and Korea because of its flavor,taste,quality and medicinal purposes.Although China is a le
7、ading producer and exporter of L.edodes,the major latent constraint in promoting commercial production of this crop is existing cultivated strains usually derived from a limited number of elite lines,which are often used in the production of many cultivars,resulting in an increasingly narrow genetic
8、 base.The development of new cultivars of edible mushrooms with superior properties such as high fruiting body productivity,disease resistance,and high content of effective constituent that benefits human health to meet changing agronomic and economic requirements in this modern rapid developmental
9、society will be very necessary and important.The purpose of this work was using RAPD,ISSR and SRAP markers to investigate genetic diversity among 23 elite strains,and to lay the foundations for directing new cultivars breeding in L.edodes.Materials and methods:Lentinula edodes strains and culture co
10、nditions Twenty-three L.edodes strains,which planted in many regions in China covering a wide range of ecological conditions from north to south and from mountains to flat irrigated lands,were used in this study.DNA extraction：Extraction of total genomic DNA was done by the sodium dodecyl sulfate-ce
11、tyltrimethy lammonium bromide(SDA-CTAB)method as described by Zeng(2003).The concentration and purity of the DNA were determined using Gene Quant Pro DNA/RNA(GE Healthcare),and extracted DNA was stored at-20after dilution to the required concentration.RAPD,ISSR,SRAP analysis The RAPD,ISSR and SRAP p
12、roducts were all analysed by electrophoresis on 1.5%(w/w)agarose gels which containing 0.5lg ethidium bromide/ml in 19 TAE buffer and then visualized and photographed under ultraviolet light using the JS-380B automatic gel imaging analyzer.The Gene Ruler TM 100bp Ladder Plus was used as a molecular
13、size standard.And the three PCR reactions were all repeated at least twice to confirm the reproducibility of each PCR band.Data analysis Amplified fragments of RAPD,ISSR and SRAP were scored as present(1),and absence(0).Dices similarity coefficients between strains were calculated by the NTSYS-pc 2.
14、10e.Cluster analysis was performed using the UPGMA algorithm,and a dendrogram was produced based on each simple matching matrix.Because fragments that smaller than100 bp in length gave low reproducibility and could not be accurately performed,DNA fragments which bigger than 100 bases were used for d
15、ata analysis.Fig.1 The extent of RAPD(a),ISSR(b)and SRAP(c)polymorphism observed among 23 L.edodes strains,revealed by primer or prime combination S1028,ISSR2 and Me1-Em16,respectively.Lanes 123,corresponded to the L.edodes strains listed in Table 1;lane M,Molecular ruler(Gene RulerTM 100 bp Ladder
16、Plus The dendrogram based on RAPD data was constructed by UPGMA analysis grouped the 23 strains into three main clusters,with similarity coefficient ranging from 0.64 to 1.00(Fig.2a).Cluster I is the biggest cluster,comprised 17 strains,i.e.,Shenxiang-2,Shenxiang-8,Shenxiang-7,Wuxiang-1,Suxiang,Cr04
17、,Shenxiang-12,Shenxiang-10,L26,Shenxiang-4,L66,Cr02,Shenxiang-6,Shenxiang-9,L03,Hunong-1 and Minfeng-1.Among 17 strains,Shen-xiang-2 and Shenxiang-8,Shenxiang-7 and Wuxiang,Shenxiang-10 and Shenxiang-12,Shenxiang-6 and Shen-xiang-9 appeared to be closer to each other.Cluster II consisted of 241,241-
18、4,Qingke-20,Zhexiang-939-9 and Zhexiang-939-6.Xiangjiu formed a separate OTUs in cluster showing less similarity coefficient(0.64)with other strains studied and appeared to be distinct from all others.ISSR analysis The five primers were screened for their ability to generate ISSR polymorphic DNA ban
19、ds using each strains genomic DNA.In all,77 unambiguous and reproducible amplification products were scored.The five primers amplified fragments across the 23 strains studied,with the number of amplified fragments ranging from twelve(ISSR12)to eighteen(ISSR1)and which varied in size from 150 to 2,00
20、0bp.Of the 77 amplified bands,56 were polymorphic,with an average of 11.2 polymorphic fragments per primer.Percentage polymorphism ranged from 55.6%(ISSR1)to a maximum of 93.3%(ISSR2),with an average of 73.5%polymorphism.3 out of 5 primers showed more than 60%polymorphism.Figure 1b is the representa
21、tive of the extent of polymorphism observed among the L.edodes strains as revealed by ISSR2.Figure 2b shows the dendrogram based on the ISSR data.23 L.edodes strains fell clearly into 4 main clusters,with similarity coefficient ranging from 0.62 to 0.99.The structure of the tree is very similar to t
22、he one from RAPD analysis.The members of the largest group are exactly the same as in RAPD,except Shenxiang-6 and Shenxiang-9,which formed a separate cluster.SRAP analysis A total of 153 different combinations of primers were employed using 9 forward and 17 reverse primers.About two-thirds of them c
23、ould produce clear bands,but considering the polymorphism and reproducibility,23 combinations were chosen in the following study.Among 23L.edodes strains,a total of 144 scorable bands were produced.All the chosen primer combinations amplified fragments across the 23 strains studied,with the number o
24、f amplified fragments ranging from three(Me7-Em2,Me9-Em14)to ten(Me7-Em14)and which varied in size from 150 to 2,000bp.Of the 144 amplified bands,81 were polymorphic,with an average of 3.5 polymorphic fragments per primer combination(Table 3).Percentage polymorphism ranged from 14.3%(Me5-Em16)to a m
25、aximum of 100.0%(Me1-Em5),with an average of 56.3%polymorphism.Only eleven out of 23 primers showed more than 60%polymorphism(Table 3).Figure 1c is the representative of the extent of polymorphism observed among the L.edodes strains as revealed by prime combination Me1-Em16.Figure 2c shows the dendr
26、ogram based on the SRAP data.23 L.edodes strains fell into three main clusters,with similarity coefficient ranging from 0.70 to 1.00.The structure of the tree is very similar to the one from RAPD analysis.Discussion Analysis of genetic diversity is very important in fungi in practical breeding progr
27、ams,and molecular markers offer an approach to unveil the genetic diversity among strains based on nucleic acid polymorphisms.In this study,three makers,RAPD,ISSR and SRAP,were simultaneously used to investigate genetic diversity of 23 elite L.edodes strains in China.These results showed the three m
28、arkers were all suitable for genetic diversity research on L.edodes.The information on the genetic relatedness amongst the strains will also be useful for planning effective breeding programmes which include,for example,the assessment of genetic diversity,the detection of duplicate sample in germpla
29、sm collection and the selection of parent strains to avoid in breeding depression.There were some differences in the positioning of some strains among the three dendrograms.It may reflect the far relationship between the two strains and the other strains,but it also suggests the sensitivity of ISSR.
30、It is reasonable that the principle and virtues of RAPD,ISSR and SRAP are different,and they are aim to amplify a different region of genome.More than one kind of molecular marker was simultaneously used to analysis in genetic diversity maybe received all-sided genetic information.Since genetic fact
31、ors and the selection pressure in distinct agroclimatic zones result in abundant genetic diversity(Sun and Lin 2003),it is not surprising to find a certain extent levels of polymorphism among the 23 strains of L.edodes in RAPD(58.8%),ISSR(73.5%)and SRAP(55.7%)markers in this study,but on the other h
32、and,dendrograms indicated a clear pattern of clustering according to the mushroom breeding institutions by which they were developed,and the 23 L.edodes strains shared great similarity suggested that they were close to each other in genetic relation.These results are accord with the high degree of commonness in their pedigree,primary research in RAPD(Gong et al.2005)and AFLP(Zhuo et al.2006).The important source of breeding material,such as wild and exotic types,must be introduced in order to broaden genetic base and decreases genetic vulnerability of L.edodes.
