Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

Assessment of genetic diversity and phylogenetic relationship among grouper species Epinephelus spp. from the Saudi waters of the Arabian Gulf

Understanding of fish genetic characterization plays a vital role in the conservation and utilization of fish genetic resources of grouper species. The present study was carried out to assess the genetic diversity and phylogenetic relationships in five grouper species, Epinephelus spp. from eastern Saudi Arabian coast using two molecular marker systems, inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. In total, 219 individuals grouper specimens (Epinephelus tauvina, E. coioides, E. bleekeri, E. malabaricus, and E. areolatus) were genotyped with 10 ISSR and 11 SSR selected primers. The ISSR produced 94 DNA fragments, of which 44 were polymorphic with an average of 2.13 fragment per primer. While SSR primers generated 107 alleles, all of them were polymorphic with an average 9.72 per primer. ISSR and SSR techniques demonstrated a high level of gene diversity and genetic distances illustrated by UPGMA dendrograms among the grouper species. The results proved that the SSR markers were highly informative and efficient in detecting genetic variability and relationships of the Epinephelus spp.     

Comparative analysis of microsatellite loci in chicken and turkey.

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.     

芜菁SSR引物的开发及多态性分析

目的:用于芜菁的简单序列重复(SSR)标记数量有限,初步探索多态性较好SSR的结构特性,开发更多的SSR引物,有助于芜菁的研究。方法:开发680对新的SSR引物,以2个芜菁品种为模板进行多态性扩增,筛选出多态性较好的引物。结果:565(83.1%)对引物在2种芜菁之间能够有效扩增,有多态性的引物有141对(20.7%);SSR的长度与其多态性水平之间没有直接的线性关系,但SSR基序碱基数与重复次数和多态性水平之间存在一定的联系,即3碱基为基序、7次重复的SSR多态性较好。结论:SSR的类型与引物的多态性之间有一定的联系。     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

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。     

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.     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

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.     

Rapid development of gene-tagged microsatellite markers from bacterial artificial chromosome clones using anchored TAA repeat primers

We developed a technique to improve the efficiency of producing TAA repeat microsatellite markers linked to interspecific conserved genes. Template DNA was prepared from cultures derived from single bacterial artificial chromosome (BAC) colonies using a simple alkaline lysis miniprep. The presence of conserved genes in each BAC clone was verified by sequencing with gene-specific primers. The BAC templates were directly sequenced using short tandem repeat-anchored primers (STRAPs), consisting of TAA repeats with one or two unique 3' terminal bases. At least one STRAP provided sufficient 3' flanking sequence from each clone for the design of a BAC-specific primer. The BAC-specific primer was used to sequence back through the tandem repeat and obtain 5' flanking sequence, and a second BAC-specific primer was designed for microsatellite genotype analysis. This technique quickly provided microsatellite markers with an average of 15 tandem repeats for the BAC clones tested. The identification of polymorphic microsatellite loci in these clones permits the identification of alleles linked to candidate genes, placement of conserved genes on genetic linkage maps, and integration of linkage and physical maps.     

Development and transferability of apricot and grape EST microsatellite markers across taxa

EST microsatellite markers were developed in apricot (Prunus armeniaca L.) and grape (Vitis vinifera L.). cDNA libraries from either apricot leaves or grape roots were used in an enrichment procedure for GA and CA repeats. The transferability of EST simple sequence repeat (SSR) markers from apricot and grapevine to other related and unrelated species was examined. Overall, grape primers amplified products in most of the Vitaceae accessions while the apricot primers amplified polymorphic alleles only in closely related species of the Rosaceae. In this taxonomic family, ten EST SSR loci were tested, and one single primer pair, PacB22, was amplified across species and sections in the Prunoideae and Maloideae. Sequencing of EST SSR loci in other species and genera confirmed a higher level of conservation in the microsatellite motif and flanking regions in the Vitaceae compared to the Rosaceae. Two distinct fragments of the PacB22 locus amplified across the Malus and Pyrus genera; however, while the coding region was highly conserved, the microsatellite repeat motif was no longer present. The banding pattern was explained by base substitution and insertion/deletion events in the intronic region of PacB22. This study includes the determination of the degree of polymorphism detected among species and genera in two unrelated taxonomic families and the evaluation of the information provided by the microsatellite repeats and the flanking regions.     

QTL mapping ofFusarium moniliforme ear rot resistance in maize. 1. Map construction with microsatellite and AFLP markers

To map the QTLsof Fusarium moniliforme ear rot resistance inZea mays L., a total of 230 F₂ individuals, derived from a single cross between inbred maize lines R15 (resistant) and Ye478 (susceptible), were genotyped for genetic map construction using simple sequence repeat (SSR) markers and amplified fragment length polymorphism (AFLP) markers. We used 778 pairs of SSR primers and 63 combinations of AFLP primers to detect the polymorphisms between parents, R15 and Ye478. From the polymorphic 30 AFLP primer combinations and 159 SSR primers, we scored 260 loci in the F₂ population, among which 8 SSR and 13 AFLP loci could not be assigned to any of the linkage groups. An integrated molecular genetic linkage map was constructed by the remaining 151 SSR and 88 AFLP markers, which distributed throughout the 10 linkage groups of maize and spanned the genome of about 3463.5 cM with an average of 14.5 cM between two markers. On 4 chromosomes, we detected 5 putative segregation distortion regions (SDR_s), including 2 new ones (SDR₂ and SDR₇). The other 3 SDR_s were located near the regions where gametophyte genes were mapped, indicating that segregation distortion could be partially caused by gametophytic factors.     

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.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers for the guava rust fungus, Puccinia psidii

We developed and characterized 15 polymorphic microsatellite markers present in the genome of the guava rust fungus, Puccinia psidii. The primers for these microsatellite markers were designed by sequencing clones from a genomic DNA library enriched for a simple sequence repeat (SSR) motif of (AG). All these 15 primer pairs successfully amplified DNA fragments from a sample of 22 P. psidii isolates, revealing a total of 71 alleles. The observed heterozygosity at the 15 loci ranged from 0.05 to 1.00. The SSR markers developed would be useful for population genetics study of the rust fungus.     

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     

Sequence Polymorphism Discovery in Wheat Microsatellite Flanking Regions using Pyrophosphate Sequencing

Sequence polymorphisms such as insertion/deletions (indels) and single nucleotide polymorphisms (SNP) are suitable for automated analysis of molecular markers and useful for cultivar identification, genetic mapping and trait association. While they are abundant, their initial discovery, comprising detection, validation and characterisation of sequence polymorphisms, is time consuming and expensive. This is especially true for multi-allellic hexaploid wheat. We investigated simple sequence repeat (SSR) flanking regions as a source for sequence polymorphisms in wheat. SSRs have a potentially high polymorphic frequency, there are a large number of highly characterised markers available, tested primers are published and most are single locus. Of 126 markers investigated, polymorphisms were found in 33 (26%) when tested in 10 wheat varieties. No new primers needed to be designed, the published primer sequences were used as PCR primers and then as sequencing primers. Polymorphism was detected by resequencing using a modification of pyrophosphate sequencing (Pyrosequencing^®) which yielded quality sequencing from the first base after the primer with up to 80 bases of information. Our method of pyrophosphate sequencing of SSRs, although not suitable for full-length sequencing, is an attractive method to directly find sequence polymorphism in varieties of interest using the abundant, well characterised and published SSR markers.     

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.     

Distribution of Simple Sequence Repeat and AFLP Markers in Molecular Linkage Map of Rice

SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) are PCR-based molecular markers developed in recent years. In this study, the authors analyzed the polymorphisms, inheritance and distribution of SSR and AFLP markers using an F2 population from a cross between cultivar "Aijiao Nante" ( Oryza sativa L. ssp. indica) and an accession of the common wild rice ( O. rufipogon Griff). A total of 200 new markers were obtained including 28 SSR and 172 AFLP markers. Six of the 28 SSR markers were developed by National Key Laboratory of Crop Genetic Improvement (NKLCGI) using DNA sequences from GenBank and the other 22 were from data published previously. The 172 AFLP markers were from a total 228 polymorphic bands amplified using 25 selected primer combinations. Mapping of the 200 new markers using NKLCGI' S previously developed RFLP map based on the same F2 population resolved these markers to all 12 rice chromosomes. Integration of the SSR and AFLP markers into the RFLP map resulted in a high density molecular linkage map containing 612 polymorphic loci.     

Inheritance pattern of RAPD markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae).

Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.