Towards a genus-wide reference linkage map for Eucalyptus based exclusively on highly informative microsatellite markers

A novel set of 50 highly polymorphic microsatellite markers were developed and mapped on existing RAPD framework maps of Eucalyptus grandis and E. urophylla. Together with the twenty previously developed microsatellite markers, these were used to align the existing maps for the two most commercially important Eucalyptus species in the tropics. Sixty-three microsatellite markers were placed on the E. grandis map in 11 linkage groups, and 53 on the E. urophylla map distributed in 10 linkage groups. Approximately 66% of the microsatellite markers segregated in a fully informative fashion, allowing the establishment of colinear syntenic linkage groups between the two maps. The 50 new microsatellite markers were highly informative, with an average of 14 alleles per locus, and average expected heterozygosity between 0.82 and 0.87. Furthermore, within the subgenus Symphyomyrtus, to which the vast majority of commercially important Eucalyptus species belong, these markers display on average 90% transportability. This set of 70 mapped microsatellite markers represents a significant step toward the development of a genus-wide reference linkage map for Eucalyptus. These highly multiallelic and transportable markers constitute a powerful tool for QTL discovery and validation, and can be used in directed searches for QTL allele variation across Eucalyptus pedigrees.     

Development of 50 gene-associated microsatellite markers using BAC clones and the construction of a linkage map of swine chromosome 4

The development of informative polymorphic markers is essential for QTL mapping. We developed 50 microsatellite markers from BAC clones containing genes that were predicted to map swine chromosome 4 (SSC4) according to comparative analysis between human and swine chromosomes, and constructed a linkage map that consisted of 37 markers including 24 markers closely linked to genes in BAC clones. Microsatellite markers were developed by direct-sequencing of BAC clones and our results demonstrated that this method was effective for developing microsatellite markers in specific regions on chromosomes. Effective development of microsatellite markers closely linked to genes can further accelerate the comparative studies of chromosomes between different species.     

Isolation and characterization of microsatellite loci of Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta)

Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta) is a gardening plant with a native, fragmentary distribution in Taiwan. In this study, we described the development of eleven microsatellite loci in L. fordii for genetic studies. These new markers were tested in 16 individuals of three populations. The number of alleles ranged from 1 to 5 and the expected heterozygosity from 0.41746 to 0.72222. Four of the nine polymorphic loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. The microsatellite markers have also been proved as informative genetic markers for other 15 Lycopodium species.     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Development, characterization, and transferability to other Solanaceae of microsatellite markers in pepper (Capsicum annuum L.).

A novel set of informative microsatellite markers for pepper (Capsicum annuum L.) is provided. Screening of approximately 168 000 genomic clones and 23 174 public database entries resulted in a total of 411 microsatellite-containing sequences that could be used for primer design and functional testing. A set of 154 microsatellite markers originated from short-insert genomic libraries and 257 markers originated from database sequences. Of those markers, 147 (61 from genomic libraries and 86 from database sequences) showed specific and scoreable amplification products and detected polymorphisms between at least 2 of the 33 lines of a test panel consisting of cultivated and wild Capsicum genotypes. These informative markers were subsequently surveyed for allelic variation and information content. The usefulness of the new markers for diversity and taxonomic studies was demonstrated by the construction of consistent phylogenetic trees based on the microsatellite polymorphisms. Conservation of a subset of microsatellite loci in pepper, tomato, and potato was proven by cross-species amplification and sequence comparisons. For several informative pepper microsatellite markers, homologous expressed sequence tag (EST) counterparts could be identified in these related species that also carry microsatellite motifs. Such orthologs can potentially be used as reference markers and common anchoring points on the genetic maps of different solanaceous species.     

Identification and mapping of microsatellite markers linked to a root-knot nematode resistance gene (rkn1) in Acala NemX cotton (Gossypium hirsutum L.)

Host-plant resistance is the most economic and effective strategy for root-knot nematode (RKN) Meloidogyne incognita control in cotton (Gossypium hirsutum L.). Molecular markers linked to resistance are important for incorporating resistance genes into elite cultivars. To screen for microsatellite markers (SSR) closely linked to RKN resistance in G. hirsutum cv. Acala NemX, F₁, F₂, BC₁F₁, and F_2:7 recombinant inbred lines (RILs) from intraspecific crosses and an F₂ from an interspecific cross with G. barbadense cv. Pima S-7 were used. Screening of 284 SSR markers, which cover all the known identified chromosomes and most linkage groups of cotton, was performed by bulked segregant analysis, revealing informative SSRs. The informative SSRs were then mapped on the above populations. One co-dominant SSR marker CIR316 was identified tightly linked to a major resistance gene (designated as rkn1), producing amplified DNA fragments of approximately 221 bp (CIR316a) and 210 bp (CIR316c) in Acala NemX and susceptible Acala SJ-2, respectively. The linkage between CIR316a marker and resistance gene rkn1 in Acala NemX had an estimated distance of 2.1–3.3 cM depending on the population used. Additional markers, including BNL1231 with loose linkage to rkn1 (map distance 25.1–27.4 cM), BNL1066, and CIR003 allowed the rkn1 gene to be mapped to cotton linkage group A03. This is the first report in cotton with a closely linked major gene locus determining nematode resistance, and informative SSRs may be used for marker-assisted selection.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

New microsatellite markers for bananas (Musa spp)

Thirty-four microsatellite markers (SSRs) were identified in EST and BAC clones from Musa acuminata burmannicoides var. Calcutta 4 and validated in 22 Musa genotypes from the Banana Germplasm Bank of Embrapa-CNPMF, which includes wild and improved diploids. The number of alleles per locus ranged from 2 to 14. The markers were considered highly informative based on their polymorphism information content values; more than 50% were above 0.5. These SSRs will be useful for banana breeding programs, for studies of genetic diversity, germplasm characterization and selection, development of saturated genetic linkage maps, and marker assisted selection.     

Isolation and Characterization of the Bovine Microsatellite Loci

Microsatellite loci were isolated using five repetitive probes for Korean native cattle. Eleven microsatellite loci were developed based on a biotin hybrid capture method, and enrichment of the genomic libraries (AAAT, TG, AG, T, and TGC repeats) was performed using Sau3AI adapters. The isolated markers were tested in two half-sib Korean cattle families and four imported breeds (Angus, Limousine, Holstein, and Shorthorn). Nine informative microsatellite loci were observed, and two microsatellite loci were revealed as monomorphic in Korean cattle. In the imported breeds, however, all of the markers were informative. In total, 213 alleles were obtained at the 11 loci across five breeds, and the average number of alleles found per locus, considering all populations, was 4.26. Heterozygosity was 0.71 (expected) and 0.57 (observed). The range of the polymorphic information content for the markers in all cattle populations was 0.43-0.69. Eleven percent of genetic variation was attributed to differentiation between populations as determined by the mean F (ST) values. The remaining 89% corresponded to differences among individuals. The isolated markers may be used to identify and classify the local breeds on a molecular basis.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Polymorphic microsatellite markers in the outbred CFW and ICR stocks for the generation of speed congenic mice on C57BL/6 background

Reliable definition of the phenotype of particular alleles is carried out in the genetic background of inbred strains. Appearance of mutations in outbred mice therefore requires the generation of congenic mice. The aim of this study was the establishment of a list of polymorphic microsatellite markers which can be used in a polymerase chain reaction (PCR)-based marker-assisted selection protocol (MASP) to allow the use of the two common outbred stocks, CFW and ICR, as donor animals for the fast generation of congenic C57BL/6 mice. The selection of informative microsatellite markers was carried out to provide a simple evaluation of the PCR products by conventional agarose gel electrophoresis. Outbred mice from three suppliers were examined. In total, 153 microsatellite loci were analysed. Here we present 76 and 70 microsatellite markers polymorphic for the outbred ICR and CFW stocks compared to C57BL/6. At least three microsatellite loci per chromosome were chosen as informative markers for the autosomal genome, giving rise to a maximum marker distance of 58 cM. Thus, additional individual markers have to be selected for the respective outbred mouse which is chosen as a donor animal.     

Comparative analysis and development of microsatellite markers on swine (Sus scrofa) chromosome 1qter

Several quantitative trait loci (QTL) have been detected on SSC1qter (Sus scrofa chromosome 1qter), including QTL for the number of vertebrae, as reported in our previous study. To provide the tools for analysis of QTLs on SSC1qter, we constructed a comparative map of swine and human. In addition, we identified 26 swine STSs and mapped 16 of them on SSC1qter using the INRA - University of Minnesota porcine radiation hybrid (IMpRH) panel. We screened a BAC library using these swine STSs and developed 35 new polymorphic microsatellite markers from the BAC clones, of which 26 were informative in our reference family. We also mapped nine microsatellite markers we had isolated previously. Consequently a total of 44 new polymorphic microsatellite markers were located within a 60-cM region of SSC1qter, spanning from SW1092 to the telomere.     

Seventy microsatellite markers from Persea americana Miller (avocado) expressed sequence tags

Expressed sequence tags for Persea americana Mill. were investigated to expand upon the number of informative microsatellite markers available for avocado. Seventy informative loci were discovered using 24 P. americana var. americana Mill. accessions. The number of alleles detected ranged from two to 17 and averaged 7.1 alleles per locus. These primers successfully amplified products in different varieties of P. americana, hybrids and a related species, Persea schiedeana. These primers will be useful for characterizing germplasm, determining genetic relationships of cultivated accessions, and for marker‐assisted development of root rot‐tolerant P. americana var. americana rootstock material.     

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.     

Genetic Diversity of Landraces in Gossypium arboreum L. Race sinense Assessed with Simple Sequence Repeat Markers

Asiatic cotton (Gossypium arboreum L.) is an "Old World" cultivated cotton species, the sinense race of which is planted extensively in China. This species is still used in the current tetraploid cotton breeding program as an elite germplasm line, and is also used as a model for genomic research in Gossypium. In the present study, 60 cotton microsatellite markers, averaging 4.6 markers for each A-genome chromosome,were chosen to assess the genetic diversity of 109 accessions. These included 106 G. arboreum landraces,collected from 18 provinces throughout four Asiatic cotton-growing regions in China. A total of 128 alleles were detected, with an average of 2.13 alleles per locus. The largest number of alleles, as well as the maximum number of polymorphic loci, was detected in the A03 linkage group. No polymorphic alleles were detected on chromosome 10. The polymorphism information content for the 22 polymorphic microsatellite loci varied from 0.52 to 0.98, with an average of 0.89. Genetic diversity analysis revealed that the landraces in the Southern region had more genetic variability than those from the other two regions, and no significant difference was detected between landraces in the Yangtze and the Yellow River Valley regions. These findings are consistent with the history of sinense introduction, with the Southern region being the presumed center of origin for Chinese Asiatic cotton, and with subsequent northeastward extension to the Yangtze and Yellow River Valleys. Cluster analysis, based on simple sequence repeat data for 60 microsatellite loci, clearly differentiated Vietnamese and G. herbaceum landraces from the sinense landrace. No relationship between inter-variety similarity and geographical ecological region was observed. The present findings indicate that the Southern region landraces may have been directly introduced into the provinces in the middle and lower Yangtze River Valley, where Asiatic cotton was most extensively grown, and further race sinense crops were subsequently produced.     

Development of Rainbow Trout Microsatellite Markers from Repeat Enriched Libraries

The efficiency of developing polymorphic microsatellite markers from 2 repeat enriched libraries was evaluated. Thirty-six polymorphic microsatellite markers were developed for rainbow trout, 27 of which were informative in a mapping family. The ability of each marker to amplify genomic DNA from other salmonids was also observed. Received March 1, 2001; accepted May 1, 2001.     

Evaluation of Microsatellite Markers for Genome Mapping in Indian Mustard (Brassica juncea L)

Microsatellites are robust markers for genome mapping, gene tagging and marker assisted selection. The genus Brassica, having a large and complex genome, requires such type of markers for various applications in genetics and breeding. A set of 202 microsatellite markers were used to screen two parental genotypes of Indian mustard (Brassica juncea) namely, ‘Varuna’, an indigenous cultivar and BEC144, an exotic collection from Poland, of which 36 (17.8%) were informative and usable for segregation analysis. The polymorphic markers detected heterozygosity in advanced generation recombinant inbred lines (RILs) developed earlier from the cross Varuna × BEC144 with a varying frequency that ranged from 0% to 23.5%. Normal Mendelian segregation for majority of microsatellite markers was observed. Eleven markers showed significant deviation from the expected 1:1 segregation ratio. Twelve markers were assigned to six different linkage groups of Indian mustard genome map. The level of polymorphism between the parents and the percentage of useful informative markers as observed in this study, suggested that many more markers are needed to achieve a reasonable coverage of mustard genome. This is the first report on the evaluation of microsatellite markers for genome mapping in B. juncea.     

A new method for identifying informative genetic markers in selectively bred rats

Microsatellite length polymorphisms are useful for the mapping of heritable traits in rats. Over 4000 such microsatellites have been characterized for 48 inbred rat strains and used successfully to map phenotypes that differ between strains. At present, however, it is difficult to use this microsatellite database for mapping phenotypes in selectively bred rats of unknown genotype derived from outbred populations because it is not immediately obvious which markers might differ between strains and be informative. We predicted that markers represented by many alleles among the known inbred rat strains would also be most likely to differ between selectively bred strains derived from outbred populations. Here we describe the development and successful application of a new genotyping tool (HUMMER) that assigns “heterozygosity” (Het) and “uncertainty” (Unc) scores to each microsatellite marker that corresponds to its degree of heterozygosity among the 48 genotyped inbred strains. We tested the efficiency of HUMMER on two rat strains that were selectively bred from an outbred Sprague-Dawley stock for either high or low activity in the forced swim test (SwHi rats and SwLo rats, respectively). We found that the markers with high Het and Unc scores allowed the efficient selection of markers that differed between SwHi and SwLo rats, while markers with low Het and Unc scores typically identified markers that did not differ between strains. Thus, picking markers based on Het and Unc scores is a valuable method for identifying informative microsatellite markers in selectively bred rodent strains derived from outbred populations.