Characteristics and a comparison of three classes of microsatellite-based markers and their application in plants

Microsatellites (SSR--simple sequence repeats, STR--short tandem repeats, SSLP--simple sequence length polymorphism, VNTR--variable number of tandem repeats) are the class of repetitive DNA sequences present in all living organisms. Particular characteristics of microsatellites, such as their presence in the genomes of all living organisms, high level of allelic variation, co-dominant mode of inheritance and potential for automated analysis make them an excellent tool for a number of approaches like genotyping, mapping and positional cloning of genes. The three most popular types of markers containing microsatellite sequences that are presently used are: (1) SSR (simple sequence repeats), generated by amplifying in a PCR reaction with the use of primers complementary to flanking regions; (2) ISSR (inter-simple sequence repeats), based on the amplification of regions between inversely oriented closely spaced microsatellites; and (3) SAMPL (selective amplification of microsatellite polymorphic loci), which utilises AFLP (amplified fragment-length polymorphism) methodology, with one exception--for the second amplification, one of the starters is complementary to the microsatellite sequence. The usefulness of the three above-mentioned markers for numerous purposes has been well documented for plants.     

ISSR分子标记及其在植物遗传学研究中的应用

ISSR分子标记是在SSR标记基础上发展起来的一种新技术,其基本原理是在SSR的5′或3′端加锚1~4个嘌呤或嘧啶碱基,然后以此为引物,对两侧具有反向排列SSR的一段基因组DNA序列进行扩增。重复序列和锚定碱基是随机选择的,扩增产物经聚丙烯酰胺或琼脂糖凝胶电泳分离后,每个引物可以产生比RAPD方法更多的扩增片段,因此,ISSR标记是一种快速、可靠、可以提供有关基因组丰富信息的DNA指纹技术。ISSR标记呈孟德尔式遗传,在多数物种中是显性的,目前已广泛用于植物品种鉴定、遗传作图、基因定位、遗传多样性、进化及分子生态学研究中。 ISSR Markers and Their Applications in Plant Genetics WANG Jian-bo Key Laboratory of MOE for Plant Developmental Biology,Wuhan University,Wuhan 430072,China Abstract:Recently,inter-simple sequence repeat (ISSR) markers have emerged as an alternative system with reliability and advantages of microsatellites (SSR).The technique involves amplification of genomic segments flanked by inversely oriented and closely spaced microsatellite sequences by a single primer or a pair of primers based on SSRs anchored 5′ or 3′ with 1-4 purine or pyramidine residues.The sequences of repeats and anchor nucleates are arbitrarily selected.Coupled with the separation of amplification products on a polyacrylamide or agarose gels,ISSR amplification can reveal a much larger number of fragments per primer than RAPD.It is concluded that ISSR technique provides a quick,reliable and highly informative system for DNA fingerprinting.ISSR markers are inherited in Mendelin mode and segregated as dominant markers.This technique has been widely used in the studies of cultivar identification,genetic mapping,gene tagging,genetic diversity,evolution and molecular ecology. Key words：molecular markers; ISSR; plant;applications     

Nonanchored Inter Simple Sequence Repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting

Many molecular marker techniques are available today. PCR-based approaches are in demand because of their simplicity and requirement for only small quantities of sample DNA. Nonanchored inter simple sequence repeats (ISSRs) are arbitrary multiloci markers produced by PCR amplification with a microsatellite primer. They are advantageous because no prior genomic information is required for their use. We found the technique stable across a wide range of PCR parameters. Polymorphisms were abundant among 7 dicot species tested with 2 tri-nucleotide and 2 tetra-nucleotide primers. Thus, nonanchored ISSR markers are a good choice for DNA fingerprinting.     

Assessment of genetic diversity in dent and popcorn (Zea mays L.) inbred lines using inter-simple sequence repeat (ISSR) amplification

Popcorn (Zea mays L.) hybrids grown in the United States are derived from narrow-based germplasm, and standard RFLP analysis detects relatively little polymorphism. Inter-simple sequence repeat (ISSR) amplification, a novel technique based on PCR amplification of inter-microsatellite sequences to target multiple loci in the genome, was employed to investigate its potential for detection of polymorphism among nineteen popcorn and eight dent corn inbred lines. ISSR yielded an average of 54 bands/primer/inbred line, with over 98% of the bands repeatable across DNA extractions and separate PCR runs. Ten primers based on di- and tri-nucleotide tandem repeats revealed 73% and 87% polymorphism among popcorn and dent corn lines, respectively, with an overall 95% polymorphism rate. Principal component and cluster analyses resulted in grouping of dent and popcorn lines corresponding to their heterotic breeding pools. ISSR amplification, in addition to being both simple and cost and time efficient, provides for rapid production of highly polymorphic markers which appear to correspond to known pedigree information. Therefore, the ISSR technique may have great potential for identifying polymorphism in species with narrow-based germplasm, and for use in DNA marker-assisted breeding approaches.     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

花椰菜细胞质雄性不育系及保持系中特异序列的克隆、分析

以细胞质雄性不育花椰菜ogura-A和相应的保持系ogura-B为材料进行ISSR及DDRTPCR分析.选用30条ISSR引物,经扩增共产生306条清晰可辨的条带.每条引物可产生6-12条带,其中引物ISSR3在两系中呈现多态性扩增,在保持系中特异扩增出一条1100 bp的片段,序列分析表明该片段与油菜、拟南芥线粒体基因组的部分序列高度相似.推测其可能来源于花椰菜线粒体基因组.在DDRT-PCR分析中,选用3条锚定引物、15条随机引物进行组合,最终共获得1 122条大小在1 000-50 bp的带.经反向Northern杂交验证只有4条带特异的存在于两系中,分别命名为ogura-A205、ogura-A383、ogura-B307、ogura-B352.其中ogura-A205、ogura-A 383只在细胞质雄性不育系中表达,而ogura-B307、ogura-B352在保持系中呈现特异性表达,分析表明四个差异序列均为首次报道.其中ogura-A205、ogura-B307至今尚未发现与之相似的序列,有待于进一步研究;ogura-A383、ogura-352与报道的拟南芥、大白菜等的叶绿体基因的一些片段具较高相似性,推测ogura-A33、ogura-B352搅可能来源于花椰菜叶绿体基因组.以上结果为进一步阐明花椰菜细胞质雄性不育及育性保持的分子机制提供了新线索.     

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.     

铁皮石斛微卫星SSR设计与应用

通过Websat对来源于NCBI公共数据库的2 447条石斛属(Dendrobium)核苷酸序列进行简单重复序列SSR的搜索,剔除冗余序列后,找到124个SSR位点。利用primer3.0软件设计引物75对,并通过改良的方法提取铁皮石斛DNA作为模板,对铁皮石斛(Dendrobium officinaleKimuraetMigo)的SSR引物进行筛选,选出21对有较清晰且稳定的目标扩增产物的引物,对8个种源的铁皮石斛进行多态性分析和聚类分析,得到8个种源的铁皮石斛进行遗传多样性和亲缘关系。     

A computer program for selection of oligonucleotide primers for polymerase chain reactions.

We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

R-ISSR-for fingerprinting, mapping and identification of new genomic loci in rye (Secale cereale L.)

The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Ot1-3), hybrid F₁ and two bulks (tolerant and susceptible) formed from recombinant inbred lines—RILs (F₉) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons—R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low T _m were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer—repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to r _AB.C = 0.870.     

荔枝SSR标记的研究

以无核荔枝A4号为实验材料,应用选择性扩增微卫星（SAM）法分离、克隆了100个简单序列重复（SSR）序列,其中88个非重复,可用。加上搜索数据库所获得的1个SSR序列,一共89个序列用于特异引物的设计。仅从71个序列的82个基因座设计出特异引物。合成41条特异引物(与5′锚定简并引物配对,个别相互配对),对其中的39个基因座进行检测。其中15对引物扩增出相应大小的片段,另外11对引物扩增出非预期片段。最后,以37个荔枝种质的基因组DNA为模板,从26对出带的引物中,筛选出多态性引物21对,获得了22个荔枝基因座特异性SSR标记。     

Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

Evaluation of genetic homogeneity of in vitro-raised plants of <Emphasis Type="Italic">Tecomella undulata</Emphasis> (Sm.) Seem. using molecular markers

Tecomella undulata (Sm.) Seem (family Bignoniaceae) is an economically and pharmaceutically important timber tree of arid regions of India. Overexploitation of natural stands coupled with minimal conservation and reforestation efforts has led to its incorporation in list of endangered species. This monotypic genus can be propagated only through seeds as no methods are available for its vegetative propagation. Therefore, protocol for multiplication of T. undulata via direct regeneration using nodal segments from mature trees has been standardized. Authentication of genetic homogeneity of these in vitro-raised plants is necessary for commercial-scale application of the developed micropropagation protocol. PCR-based molecular markers which have emerged as simple, fast, reliable, and labor-effective tools for testing the genetic homogeneity of in vitro-raised plants were used in the present study. Arbitrary (random amplified polymorphic DNA, RAPD), semi-arbitrary (inter-simple sequence repeat, ISSR; start codon targeted (SCoT) polymorphism), and sequence-based (simple sequence repeat, SSR) markers were used. DNA samples of shoots maintained in vitro for 2 years collected after every 4 subculture cycles (of 3 weeks each) and field-transferred plantlets were compared with the mother tree DNA using 131 primers (25 each of RAPD, ISSR, SCoT and 56 SSR). Scorable unambiguous and reproducible DNA fragments were produced by 77 (21 RAPD, 20 ISSR, 22 SCoT and 14 SSR) primers. A total of 71, 93, 94, and 42 distinct and scorable DNA fragments were produced by RAPD, ISSR, SCoT, and SSR primers respectively with an average of 3.38, 4.65, 4.27, and 3.0 DNA fragments per primer. The true-to-type nature of the in vitro-raised plants of T. undulata undergoing up to 32 subculture passages over a period of approximately 2 years was authenticated by monomorphic DNA fragments amplified with all primer combinations. Therefore, the developed micropropagation protocol can be safely used on a commercial scale for multiplying T. undulata plants.     

Genetic diversity in Arabidopsis thaliana L. Heynh. investigated by cleaved amplified polymorphic sequence (CAPS) and inter-simple sequence repeat (ISSR) markers

In this study, we investigated genetic diversity among 37 accessions in Arabidopsis thaliana from Eurasia, North Africa and North America using morphological traits and two polymerase chain reaction (PCR)-based marker systems: cleaved amplified polymorphic sequences (CAPS) and inter-simple sequence repeats (ISSR). Cluster analysis based on genetic similarities calculated from CAPS data grouped the accessions roughly according to their geographical origin: one large group contained accessions from Western, Northern and Southern Europe as well as North Africa, a second group consisted of Eastern European and Asian continental accessions. North American accessions were interspersed into these groups. Contrary to the CAPS analysis, the dendrogram obtained from the ISSR data did not reflect the geographical origin of the accessions, and the calculated genetic distances did not match the CAPS results. This could be attributable to an uneven genomic distribution of ISSR markers as substantiated by a database search for ISSR binding sites in A. thaliana genomic DNA sequence files, or to the ISSR's different mode of evolution. We recommend CAPS markers for diversity analysis in A. thaliana because a careful selection of markers can ascertain an even representation of the entire genome.     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

 Inter-simple sequence repeat (ISSR) amplification was used to analyze microsatellite motif frequency in the rice genome and to evaluate genetic diversity among rice cultivars. A total of 32 primers, containing different simple sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties, representative of the diversity of cultivated rice (Oryza sativa L.). The ISSR analysis provided insights into the organization, frequency and levels of polymorphism of different simple sequence repeats in rice. The more common dinucleotide motifs were more amenable to ISSR analysis than the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif was more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISSR markers, suggesting which motifs would be better targets for microsatellite marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and isozyme markers. The ISSR fingerprint could be used to differentiate the genotypes belonging to either Japonica or Indica sub species of cultivated rice and to dissect finer levels of diversity within each subspecies. A higher percentage of polymorphic bands was produced with the ISSR technique than the AFLP method, based on a similar PCR reaction. Therefore, ISSR amplification proved to be a valuable method for determining genetic variability among rice varieties and for rapidly identifying cultivars. This efficient genetic fingerprinting technique would be useful for characterizing the large numbers of rice accessions held in national and international germplasm centers. Received: 25 May 1998 / Accepted: 17 September 1998     

石斛SSR标记的开发及可转移性分析

当前已开发的石斛（Dendrobium）SSR标记仅100余对,难以满足研究的需要。为开发更多石斛分子标记,本研究通过生物信息学方法从公共核酸数据库搜索石斛SSR。结果表明,GenBank收录的3599条石斛DNA序列经拼接获得1343条Uni—DNA序列。经搜索,共检测出283个SSR,分布于205条Uni—DNA序列,平均每2815bp含一个SSR。通过序列比对,剔除86条已开发引物的SSR—DNA序列,从剩余的119条序列设计76对引物,并在石斛属32个种间进行可转移性分析,结果显示47对引物得到有效扩增,种间可转移率为51．1％N95．7％,平均为75．9％。有效扩增引物中有46对在石斛种间具多态性,检测出等位基因数2～8个,平均4．0个。选取10对多态性引物扩增60份铁皮石斛资源,每对引物检测出等位基因数2～5个,平均3．4个。根据SSR扩增带型将60份铁皮石斛资源聚为5大类,同一类型内的表型较类群间接近。对DMl21扩增产物测序表明铁皮石斛种内的SSR等位变异由SSR重复次数差异造成,而石斛种间的SSR等位变异还包含SSR位点侧翼序列的插入缺失以及替换。     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998