The linkage maps of Dendrobium species based on RAPD and SRAP markers

Dendrobium plants are used commonly as tonic herbs and health food in many Asian countries,especially in China.Here we report the genetic map construction of two Dendrobium species with a double pseudo-testcross strategy using random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) markers.A F1 mapping population of 90 individuals was developed from a cross between D.officinale and D.hercoglossum.A total of 307 markers,including 209 RAPD and 98 SRAP,were identified and used for genetic linkage group (LG) analysis.The D.officinale linkage map consisted of 11 major linkage groups and 3 doublets,which covered 629.4 cM by a total of 62 markers with an average locus distance of 11.2 cM between two adjacent markers.The D.hercoglossum linkage map contained 112 markers mapped on 15 major and 4 minor linkage groups,spanning a total length of 1,304.6 cM with an average distance of 11.6 cM between two adjacent markers.The maps constructed in this study covered 92.7% and 82.7% of the D.hercoglossum and D.officinale genomes respectively,providing an important basis for the mapping of horticultural and medicinal traits and for the application of marker-assisted selection in Dendrobium breeding program.     

应用RAPD分子标记技术探讨3种石斛属植物的种间关系

采用RAPD分子标记技术,分析了金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的种间关系。10个引物产生的113条DNA扩增片段中,106条（93.81%）具有多态性,利用113个RAPD标记,计算遗传距离,利用非加权组平均法建立聚类图。结果表明,RAPD标记技术较好地从分子水平揭示金钗石斛、铁皮石斛和齿瓣石斛三种石斛属植物的遗传背景、亲缘关系,并为后期在DNA水平上对药用石斛的开发利用提供资料。     

Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers

Combined randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) were used to assess the genetic diversity of Pleurotus ostreatus strains cultivated in China. For the RAPD and SRAP analyses, 479 and 282 polymorphic bands were obtained from 20 P. ostreatus strains using 20 and 13 selected primers or primer pairs, respectively. A combined RAPD/SRAP dendrogram grouped the 20 strains into five clades with a coefficient of 0.690. The comparison of RAPD and SRAP was evaluated in the present study. The combined RAPD/SRAP markers provided reliable information regarding the relationships among the P. ostreatus strains.     

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.     

Evaluation of genetic diversity in Piper spp using RAPD and SRAP markers

Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) analysis were applied to 74 individual plants of Piper spp in Hainan Island. The results showed that the SRAP technique may be more informative and more efficient and effective for studying genetic diversity of Piper spp than the RAPD technique. The overall level of genetic diversity among Piper spp in Hainan was relatively high, with the mean Shannon diversity index being 0.2822 and 0.2909, and the mean Nei's genetic diversity being 0.1880 and 0.1947, calculated with RAPD and SRAP data, respectively. The ranges of the genetic similarity coefficient were 0.486-0.991 and 0.520-1.000 for 74 individual plants of Piper spp (the mean genetic distance was 0.505 and 0.480) and the within-species genetic distance ranged from 0.063 to 0.291 and from 0.096 to 0.234, estimated with RAPD and SRAP data, respectively. These genetic indices indicated that these species are closely related genetically. The dendrogram generated with the RAPD markers was topologically different from the dendrogram based on SRAP markers, but the SRAP technique clearly distinguished all Piper spp from each other. Evaluation of genetic variation levels of six populations showed that the effective number of alleles, Nei's gene diversity and the Shannon information index within Jianfengling and Diaoluoshan populations are higher than those elsewhere; consequently conservation of wild resources of Piper in these two regions should have priority.     

松花型花椰菜主要品种鉴定的分子标记分析

利用RAPD、ISSR和SRAP 3种分子标记对我国南方地区松花型花椰菜主栽品种进行鉴定,分析了品种间的遗传多样性。3种标记共产生370条扩增带,238条为多态性条带,其多态率为64.32%。其中只有SRAP标记的引物m e1/em1可将20个品种全部鉴别。遗传相似系数分析表明,松花型花椰菜品种之间的亲缘关系较近,遗传背景比较狭窄。聚类分析表明品种间的亲缘关系与熟性、地理分布相关。研究表明,分子标记能有效地应用于花椰菜品种鉴定,且综合多种分子标记分析品种间的遗传多样性将更加准确可靠。     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Evaluation of genetic diversity in Lentinula edodes strains using RAPD, ISSR and SRAP markers

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 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. 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 and 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 introduced in order to broaden genetic base and decreases genetic vulnerability of L. edodes.     

Genetic diversity and genetic relationships in Hyacinthaceae in India using RAPD and SRAP markers

Genetic diversity and relationship among three genera namely Drimia, Dipcadi and Ledebouria of Hyacinthaceae in India was studied using RAPD and SRAP markers. Twenty one RAPD primers and nine SRAP were used for analyzing 41 accessions. RAPD gave an average 12.6 markers per primer, while SRAP generated 10.1 markers per primer pair. The family emerged very diverged with high polymorphism. The study resolved the three genera into monophyletic groups corresponding to three subfamilies; Urginoideae, Hyacinthoideae and Ornithogaloideae. Drimia wightii emerged a very distinct species and species specific markers were obtained with both marker systems. AMOVA analysis also revealed the genera to be quite well diverged. The two markers showed high correlation (r = 0.932) in Mantel matrix crresspondance test. The combined data also showed a very good correlation with the respective markers individually.     

Genetic diversity and population structure of Butea monosperma (Lam.) Taub.- a potential medicinal legume tree

Three molecular marker systems, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeats (ISSR) and Sequence-Related Amplified Polymorphism (SRAP) were employed to investigate the genetic structure and diversity among the 14 natural populations of Butea monosperma collected from different geographical regions of India. Detected by 17 RAPD, 15 ISSR and 11 SRAP primer combinations, the proportions of polymorphic bands were 84.2 %, 77.2 % and 91.9 %, respectively, and the mean Nei’s genetic distances among the populations were 0.13, 0.10 and 0.13, respectively. Partitioning of genetic variability by Analysis of molecular variance (AMOVA) revealed that the high genetic diversity was distributed within the populations. AMOVA also revealed that the coefficient of gene differentiation among populations based on F_ST was very high irrespective of markers used. The overall gene flow among populations (Nm) was very low. Cophenetic correlation coefficients of Nei’s distance values and clustering pattern by Mental test were statistically significant for all three marker systems used but poor fit for ISSR data than for RAPD, SRAP and combined data set of all three markers. For all markers, a high similarity in dendrogram topologies was obtained, although some differences were observed with ISSR. The dendrogram obtained by RAPD, SRAP and combined data set of all three markers reflect relationship of most of the populations according to their geographic distribution.     

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。     

A doubled haploid rye linkage map with a QTL affecting α-amylase activity

A rye doubled haploid (DH) mapping population (Amilo × Voima) segregating for pre-harvest sprouting (PHS) was generated through anther culture of F₁ plants. A linkage map was constructed using DHs, to our knowledge, for the first time in rye. The map was composed of 289 loci: amplified fragment length polymorphism (AFLP), microsatellite, random amplified polymorphic DNA (RAPD), retrotransposon-microsatellite amplified polymorphism (REMAP), inter-retrotransposon amplified polymorphism (IRAP), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers, and extended altogether 732 cM (one locus in every 2.5 cM). All of the seven rye chromosomes and four unplaced groups were formed. Distorted segregation of markers (P ≤ 0.05) was detected on all chromosomes. One major quantitative trait locus (QTL) affecting α-amylase activity was found, which explained 16.1% of phenotypic variation. The QTL was localized on the long arm of chromosome 5R. Microsatellites SCM74, RMS1115, and SCM77, nearest to the QTL, can be used for marker-assisted selection as a part of a rye breeding program to decrease sprouting damage.     

Genetic diversity across natural populations of <Emphasis Type="Italic">Dendrobium officinale</Emphasis>, the endangered medicinal herb endemic to China,revealed by ISSR and RAPD markers

Dendrobium officinale is a rare and endangered herb with special habitats and endemic to China. Genetic diversity was examined within and among nine natural populations using inter-simple sequence repeat (ISSR) and random amplified polymorphic (RAPD) for conservation. Both molecular markers revealed a high percentage (>89%) of polymorphic bands and ISSR markers detected more diversity than RAPD markers. Analysis of molecular variance (AMOVA) revealed that 78.84% (ISSR) and 78.88% (RAPD) of variability was partitioned among individuals within populations. This genetic structure was probably due to severe genetic drift resulting from habitat fragmentation and human overexploitation since 1950s. Moreover, there is a lack of significant association between genetic and geographic distances (r = 0.276; p > 0.05) in the populations of D. officinale. From the conservation point of view, populations GL, GS and GSD with higher genetic diversity should be protected firstly to maintain the species potential for evolutionary change and population YG with lower diversity but representing a novel evolutionary unit should also be paid more attention to during D. officinale conservation practice. Published in Russian in Genetika, 2009, Vol. 45, No. 3, pp. 375–382. The article is published in the original.     

Molecular Characterization of <Emphasis Type="Italic">Cyclamen</Emphasis> Species Collected from Different Parts of Turkey by RAPD and SRAP Markers

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Ayd?n, Mu?la, ?zmir, Denizli, Kahramanmara?, Osmaniye, Eski?ehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.     

Evaluation of the Use of SCAR Markers for Screening Genetic Diversity of Lentinula edodes Strains

In this study, three molecular marker systems including sequence related amplified polymorphism (SRAP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR) were screened to select polymorphisms of 24 main commercial strains of Lentinula edodes cultivated widely in China. Twenty-nine sequence characterized amplified region (SCAR) markers were developed to set up a dendrogram using UPMGA based on nucleotide sequences of some SRAP, RAPD, and ISSR polymorphic fragments. The grouping showed that the 24 strains were apparently clustered into five groups at a level of 0.68 similarity coefficient, and those that have similar breeding background clustered preferentially into the same subgroup. Results also revealed that the 24 strains had a low level of genetic diversity, and the breeding source of L. edodes should be broadened by exploiting wild types and introducing exotic strains. In addition, the tested strains of L. edodes could be clearly distinguished and identified from others by using different combinations of SCAR primers. Thus, results of this work demonstrated that SCAR was an excellent genetic marker system to characterize and investigate genetic diversity of L. edodes. Furthermore, this provided an alternative method to identify the genetic relationship of different strains of other fungi.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs,SSRs, RAPDs,and SRAPs

Buffalograss [Buchloe dactyloides (Nutt.) Englem.] is the only native grass that is being used extensively as a turfgrass in the Great Plains region. Its low-growth habit, drought resistance, and low-maintenance requirement make it attractive as a turfgrass species. Our objective was to obtain an overview on the genetic relatedness among and within seeded and vegetative biotype buffalograsses using inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPDs), sequence-related amplified polymorphisms (SRAPs), and simple sequence repeats (SSRs) markers that were derived from related species (maize, pearl millet, sorghum, and sugarcane). Twenty individuals per cultivar were genotyped using 30 markers from each marker system. All buffalograss cultivars were uniquely fingerprinted by all four marker systems. Mean genetic similarities were estimated at 0.52, 0.51, 0.62, and 0.57 using SSRs, ISSRs, SRAPs, and RAPDs, respectively. Two main clusters separating the seeded-biotype from the vegetative-biotype cultivars were produced using UPGMA analysis. Further subgroupings were unequivocal. The Mantel test resulted in a very good fit (SRAP=0.92, ISSR=0.90) to good fit (RAPD=0.86, SSR=0.88) of cophenetic values. Comparing the four marker systems to each other, RAPD and SRAP similarity indices were highly correlated (r=0.73), while Spearmans rank correlation coefficient between RAPDs and SSRs was r=0.24 and between ISSRs and SSRs was r=0.66. A genotype-assignment analytical approach might be useful for cultivar identification and property rights protection. Polymorphic SRAPs were abundant and demonstrated genetic diversity among closely related cultivars.Communicated by B. FriebeA contribution of the University of Nebraska Agricultural Research Division, Lincoln, Nebraska 68583. Journal Series No. 14398.     

用Langdon二体代换系统建立小麦染色体RAPD标记

以一套Ｌａｎｇｄｏｎ硬粒小麦二体代换系及其亲本Ｌａｎｇｄｏｎ、中国春和中国春双端体为材料,研究适于硬粒小麦和普通小麦的理想ＲＡＰＤ分析条件,进行小麦Ａ、Ｂ和Ｄ染色体组各个染色体的ＲＡＰＤ分析。结果表明,ＡｍｐｌｉＴａｑＳｔｏｆｆｅｌｆｒａｇｍｅｎｔ比ＴａｑＤＮＡＰｏｌｙｍｅｒａｓｅ优越。所用１２个随机引物中,７个引物扩增出的１３个特异产物,可确定在硬粒小麦ＬａｎｇｄｏｎＡ、Ｂ染色体组和中国春Ｄ染色体组中的１０个个别染色体上。４个标记进一步定位在相应的４个染色体臂上。结果还表明,用Ｌａｎｇｄｏｎ二体代换系统、中国春双端体为材料,容易得到重复性高、特异性强的ＲＡＰＤ标记。     

青藏高原早熟甘蓝型春油菜遗传资源研究

利用SSR和SRAP 2种分子标记研究了69份试验材料的遗传差异及其亲缘关系.29对SSR标记共扩增出118条多态性带,多态性位点占总扩增位点的97.5%,27对SRAP引物扩增出123条多态性带,多态性比率为70.3%.两种标记聚类结果表明.在相似系数0.566处所有材料可以分为A、B 2个大类群;B类在相似系数0.620处又可分为7个亚类,10个天然双低早熟甘蓝型品系、2个甘蓝型亲本和4个新型品系聚在第1亚类中,其余的51个新型甘蓝型油菜品系分别聚在其他6个亚类中.对55份新型品系进行遗传成分分析,结果表明,每个品系都合有4种带型,各品系所舍不同带所占比率不同.对各品系中含有白菜型亲本带所占比率分别与其对应的两亲本之间的遗传距离进行相关分析,结果表明新型甘蓝型油菜品系中白菜型亲本带所占比率与白菜型素本间的遗传距离为负相关(-0.52),且达到极显著水平;与甘蓝型亲本间的遗传距离为正相关(0.31),且达到显著水平.对试验材料之间的遗传距离及其来源进行分析(除与2个白菜型亲本间),遗传距离排名前20位的都来自新型品系之间或天然品系与新型之间,最大为0.544.     

Use of a subset of doubled-haploid lines for RAPD interval mapping in barley.

Molecular markers have been used in barley to locate genes and quantitative trait loci. Only a few RAPD markers have been located on barley marker maps. The objectives of this study were (i) to place RAPD markers in specific intervals on the barley linkage map developed from the cross Steptoe (S) x Morex (M), (ii) to examine the distribution of RAPD markers, and (iii) to compare markers amplified by Taq DNA polymerase with those amplified by the Stoffel fragment of Taq DNA polymerase. Screening of DNA from S and M with 362 decamer primers identified 85 that amplified 127 reliable RAPDs. A subset of 15 doubled-haploid (DH) lines from the 150 DH line mapping population was used to place these RAPD markers in intervals on the SM map. This subset can be used for rapid placement of any new markers on the SM linkage map. Most of the RAPD markers were dominant but four codominant RAPDs were identified. The RAPDs were not evenly distributed, with many clustered around the centromeric region of each chromosome. Two of these clusters were located in intervals larger than 15 cM. Testing of 38 to 42 additional DH lines provided more precise placement of eight of the markers in these clusters. Reliable RAPDs were detected with 44% of the primers tested with the Stoffel fragment, but with only 17% of the primers tested with Taq DNA polymerase. These RAPDs provide additional markers for use in barley improvement.