Analysis of Genetic Diversity Among Chinese Auricularia auricula Cultivars Using Combined ISSR and SRAP Markers

DNA polymorphism among 34 Chinese Auricularia auricula cultivars was analyzed using inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Thirty ISSR primers amplified a total of 129 DNA fragments of which 125 (96.9%) were polymorphic, whereas 11 SRAP primer combinations amplified 154 fragments of which 148 (96.1%) were polymorphic. Both methods were highly effective in discriminating among the test strains. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method distributed the 34 strains into four or five major groups. Clustering analysis based on all the three data sets indicated a high level of genetic diversity among A. auricula, although the combined ISSR/SRAP data were more concordant with the main agronomic characters of strains and their geographical centers of cultivation. Our findings will facilitate future A. auricula breeding programs and the development of bioactive products from this commercially important medicinal mushroom.     

Assessment of genetic diversity of coffee leaf rust pathogen Hemileia vastatrix using SRAP markers

Coffee leaf rust caused by the fungus Hemileia vastatrix (Berk and Br.) is a major disease occurring in coffee plantations. Although the rust fungus exists in different physiological races, the genetic difference between them is meagrely understood. In this study, genetic diversity of 14 identified and two unidentified leaf rust races was determined by sequence‐related amplified polymorphism (SRAP) markers. Of 48 SRAP primer pairs tested, 35 primers are polymorphic and generated 347 distinct scorable fragments. The number of fragments ranged from 4 to 18 with a mean of 9.97 fragments per primer combination. Of the total 347 amplified fragments, 185 fragments (53.31%) are polymorphic with an average of 5.41 fragments per primer combination. The average resolving power (Rp) and the average polymorphism information content (PIC) of the 35 SRAP primer combinations were 13.60 and 0.356, respectively. Of 35 SRAP primer pairs, 15 primer pairs were more informative and generated 25 unique fragments, which are useful for race discrimination. The study demonstrated the existence of genetic variability among various leaf rust races and this information will be helpful in coffee breeding programmes.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

Analysis of genetic diversity among Chinese <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis> Singer cultivars using two molecular marker systems (ISSRs and SRAPs) and morphological traits

To evaluate the genetic diversity of Pleurotus citrinopileatus Singer cultivars in China, 20 P. citrinopileatus strains were analyzed using morphological traits, inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers. Eleven ISSR primers amplified a total of 116 DNA fragments of which 96 (82.91%) were polymorphic, whereas 8 SRAP primer pairs amplified 69 fragments of which 65 (93.47%) were polymorphic. Phylogenetic trees constructed on the basis of ISSR, SRAP, and combined ISSR/SRAP analyses using the Unweighted Pair-group Method with Arithmetic Averages method distributed the 20 strains into three or six major groups. The grouping exhibited great similarity and was generally consistent with their morphological characters and antagonism test, which indicated a high level of genetic diversity among P. citrinopileatus Singer and relationship between each other. Based on the genetic analysis, the primary mini-core strains were constructed with progressive sampling method of the smallest genetic distance. The mini-core germplasm collection included 4 strains (strain 2, 5, 7 and 11). Our findings will provide a scientific fundament for facilitating parent selection for broadening genetic base, accelerating the genetic breeding, identification of cultivated strains and the development of bioactive products from this commercially important medicinal mushroom.     

SRAP技术研究烟粉虱遗传多样性

采用AFLP、SRAP2种标记方法分别对2个烟粉虱Bemisia tabaci Gennadius种群(一品红、甘蓝)的多态性进行分析。结果表明,(1)2种方法平均每对引物组合产生的条带数分别为29.4和21.8。(2)AFLP法每对引物组合产生10～23条多态性带,平均17.20条,多态性带的比例平均为57.93%。SRAP法每对引物组合产生5～18条多态性带,平均13.3条,多态性带的比例平均为60.59%。(3)前者的基因多样性范围为0.1503～0.2838,平均为0.2297;后者的基因多样性范围为0.0977～0.2911,平均为0.2332。证明利用SRAP技术和AFLP技术研究烟粉虱的遗传多样性是有效的。     

Evaluation of Genetic Diversity in Lemons and Some of Their Relatives Based on SRAP and SSR Markers

Genetic diversity was evaluated by sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers among 45 lemons (Citrus limon (L.) Burm. f.), five citrons (Citrus medica L.), four rough lemons (Citrus jambhiri Lush), and two Citrus volkameriana accessions. Twenty-one SRAP primer combinations produced a total of 141 (77%) polymorphic fragments with an average of 6.7 fragments per primer combinations whereas 13 SSR primers produced a total of 26 (76%) polymorphic fragments with an average of 2.0 per primer. The unweighted pair-group method arithmetic average analysis as assessed with combined SRAP and SSR data demonstrated that the accessions had a similarity range from 0.65 to 1.00. Rough lemons and C. volkameriana accessions were relatively closely related. In lemon group, accessions from hybrid origin were distant from the others. We also applied principal components analysis (PCA) for a better presentation of relation among the accessions studies. Using PCA, 88.7% of the total variation in the original dimensions could be represented by just the two dimensions defined by the first two PCs. Although nearly all accessions could be distinguished, there was a low level of genetic diversity detected among lemon cultivars.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

29个香蕉基因型遗传多样性的SRAP分析

采用SRAP分子标记技术对29个香蕉品种(系)的多样性进行研究,结果显示,64对SRAP引物中筛选出25个多态性较高的引物组合,共扩增出324条条带;UPGAM聚类图显示所有供试的29个香蕉品种(系)可分为2个类群且与基因型相一致;实验结果与形态、农艺性状标记分类基本一致。研究表明,SRAP技术可有效运用于香蕉基因型的遗传和育种研究。     

Molecular Characterization of Tree Peony Germplasm Using Sequence-Related Amplified Polymorphism Markers

This study examined 63 tree peony specimens, consisting of 3 wild species and 63 cultivars, using sequence-related amplified polymorphism (SRAP) markers for the purpose of detecting genomic polymorphisms. Bulk DNA samples from each specimen were evaluated with 23 SRAP primer pairs. Among the 296 different amplicons, 262 were polymorphic. The maximum parsimony, neighbor-joining, and unweighted pair-group method using arithmetic average trees were largely in congruence. In the three trees, the wild species Paeonia ludlowii and P. delavayi formed separate clusters with strong bootstrap support, and P. ostii was closely related to all cultivars. The cultivars were divided into groups with various corresponding bootstrap values. The genetic similarity among the genotypes ranged from 0.02 to 0.73. These results demonstrate that SRAP markers are effective in detecting genomic polymorphisms in the tree peony and should be useful for linkage map construction and molecular marker assisted selection breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CONSTRUCTION OF A GENETIC LINKAGE MAP FOR PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) BASED ON SEQUENCE‐RELATED AMPLIFIED POLYMORPHISM AND SIMPLE SEQUENCE REPEAT MARKERS1

Molecular markers and molecular genetic maps are prerequisites for molecular breeding in any plant species. A comprehensive genetic linkage map for cultivated Porphyra haitanensis T. J. Chang et B. F. Zheng has not yet been developed. In this study, 157 double haploid (DH) lines [derived from a YSIII (wildtype) × RTPM (red‐type artificial pigmentation mutant) cross] were used as a mapping population in P. haitanensis. A total of 60 pairs of sequence‐related amplified polymorphism (SRAP) primers and 39 pairs of simple sequence repeat (SSR) primers were used to detect polymorphisms between the two parents. Fifteen SRAP and 16 SSR polymorphic primer pairs were selected to analyze the DH population. A linkage genetic map comprising 67 SRAP markers and 20 SSR markers in five linkage groups, with a total length of 830.6 cM and an average of 10.13 cM between markers, was constructed. The markers were distributed evenly in all linkage groups without clustering. The linkage groups comprised 12–23 markers ranging in length from 134.2 to 197.3 cM. The estimated genome length of P. haitanensis was 942.4 cM, with 88.1% coverage. This is the first report of a comprehensive genetic map in P. haitanensis. The map presented here will provide a basis for the development of high‐density genetic linkage maps and lay the foundation for molecular breeding work in P. haitanensis.     

SRAP分子标记分析西瓜遗传多态性

目的:探讨西瓜遗传多样性和遗传基础。方法:采用SRAP分子标记对西瓜品种D1、D2、D3、H1、H2、H3、M1、M2、M3、m1、m2、m3的多态性进行了分析。结果:每对引物组合产生13~25对比较清晰的扩增带.8对引物组合共产生131条扩增带。平均每对引物组合产生16.375条。8对引物组合共产生多态性带37条,每对引物组合产生3~7条,平均4.625条。每对引物组合产生的多态性带的比例为16.666%~38.464%,平均为28.675%。另外,对银染过程进行了优化。结论:SRAP标记多态性还是较高的,可以适于分析西瓜等遗传差异小的作物。     

冬枣×宁梨巨枣的子代SRAP鉴定及遗传多样性分析

应用SRAP分子标记方法对冬枣×宁梨巨枣的子代进行了分子鉴定及遗传多样性分析。采用构建基因池的方法对SRAP分子标记引物进行筛选,从88对引物中筛选出15对多态性好、主带清晰的引物,并对子代进行了真实性鉴定及多态性分析。结果表明:(1)15对引物共产生95个多态性条带,平均每对引物产生6.3个多态性条带,显示了较高的多态性比率。(2)80个子代中44个具有父本特征带,鉴定为真杂种。子代遗传多样性及UPGMA聚类分析表明,子代个体与亲本间的遗传相似系数在0.55~0.98之间,个体差异明显。该研究结果为枣树杂交育种提供了重要的分子证据。     

An improved technique for isolating codominant compound microsatellite markers

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)₆(AG)₅ or (TC)₆(AC)₅ and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.     

Musa Genetic Diversity Revealed by SRAP and AFLP

The sequence-related amplified polymorphism (SRAP) technique, aimed for the amplification of open reading frames (ORFs), vis-â-vis that of the amplified fragment length polymorphisms (AFLP) were used to analyze the genetic variation and relationships among forty Musa accessions; which include commercial cultivars and wild species of interest for the genetic enhancement of Musa. A total of 403 SRAP and 837 AFLP amplicons were generated by 10 SRAP and 15 AFLP primer combinations, of which 353 and 787 bands were polymorphic, respectively. Both cluster analysis of unweighted pair-grouping method with arithmetic averages (UPGMA) and principal coordinate (PCO) analysis separated the forty accessions into their recognized sections (Eumusa, Australimusa, Callimusa and Rhodochlamys) and species. The percentage of polymorphism amongst sections and species and the relationships within Eumusa species and subspecies varied between the two marker systems. In addition to its practical simplicity, SRAP exhibited approximately threefold more specific and unique bands than AFLP, 37 and 13%, respectively. SRAP markers are demonstrated here to be proficient tools for discriminating amongst M. acuminata, M. balbisiana and M. schizocarpa in the Eumusa section, as well as between plantains and cooking bananas within triploid cultivars.     

Genetic variability and relationships between and within grape cultivated varieties and wild species based on SRAP markers

Sequence-related amplified polymorphism (SRAP) markers were used to assess genetic relationships among 76 grape genotypes including Chinese indigenous and newly bred varieties, representatives of foreign grape varieties, and wild Vitis species. Nineteen informative primers were selected from 100 SRAP primer pairs due to their ability to produce clearly and repeatedly polymorphic and unambiguous bands among the varieties. A total of 228 bands were produced; 78.63% of them were polymorphic; the average polymorphism information content (PIC) is 0.76. Genetic relationships were obtained using Nei and Li similarity coefficients. Cluster analysis of SRAP markers through the unweighted pair-group method of arithmetic averages (UPGMA) analysis and principal coordinate analysis (PCoA) were largely consistent. The definition of clusters in the dendrogram and PCoA plot is the same and some degree of grouping by types of grape, ecogeographical origin, and taxonomic status of the varieties was revealed. Three main groups were found after cluster analysis, i.e., table grape of Vitis vinifera; table grape of Euro-America hybrid and wine grape of V. vinifera; wild Vitis species. Groupings indicated a divergence between the table and wine-type varieties of V. vinifera. The results showed that the wild Vitis species that originated from America and China could be clearly differentiated and Vitis hancockii is the most distant from the others of Asian Vitis species. The results also indicated that SRAP markers are informative and could distinguish bud sports of grape. The present analysis revealed that Chinese cultivated and wild grape germplasm are highly variable and have abundant genetic diversity.     

Molecular Phylogeny Analysis and Species Identification of Dendrobium (Orchidaceae) in China

Dendrobium plants are important commercial herbs in China, widely used in traditional medicine and ornamental horticulture. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to molecular phylogeny analysis and species identification of 31 Chinese Dendrobium species. Fourteen SRAP primer pairs produced 727 loci, 97% of which (706) showed polymorphism. Average polymorphism information content of the SRAP pairs was 0.987 (0.982–0.991), showing that plenty of genetic diversity exists at the interspecies level of Chinese Dendrobium. The molecular phylogeny analysis (UPGMA) grouped the 31 Dendrobium species into six clusters. We obtained 18 species-specific markers, which can be used to identify 10 of the 31 species. Our results indicate the SRAP marker system is informative and would facilitate further application in germplasm appraisal, evolution, and genetic diversity studies in the genus Dendrobium.     

Characterization and mapping of NBS-LRR resistance gene analogs in apricot (Prunus armeniaca L.)

Genomic DNA sequences sharing homology with the NBS-LRR (nucleotide binding site-leucine-rich repeat) resistance genes were isolated and cloned from apricot (Prunus armeniaca L.) using a PCR approach with degenerate primers designed from conserved regions of the NBS domain. Restriction digestion and sequence analyses of the amplified fragments led to the identification of 43 unique amino acid sequences grouped into six families of resistance gene analogs (RGAs). All of the RGAs identified belong to the Toll-Interleukin receptor (TIR) group of the plant disease resistance genes (R-genes). RGA-specific primers based on non-conserved regions of the NBS domain were developed from the consensus sequences of each RGA family. These primers were used to develop amplified fragment length polymorphism (AFLP)-RGA markers by means of an AFLP-modified procedure where one standard primer is substituted by an RGA-specific primer. Using this method, 27 polymorphic markers, six of which shared homology with the TIR class of the NBS-LRR R-genes, were obtained from 17 different primer combinations. Of these 27 markers, 16 mapped in an apricot genetic map previously constructed from the self-pollination of the cultivar Lito. The development of AFLP-RGA markers may prove to be useful for marker-assisted selection and map-based cloning of R-genes in apricot.     

Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.