Identification, genetic localization, and allelic diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp.).

Selectively amplified microsatellite polymorphic locus (SAMPL) analysis is a method of amplifying microsatellite loci using generic PCR primers. SAMPL analysis uses one AFLP primer in combination with a primer complementary to microsatellite sequences. SAMPL primers based on compound microsatellite sequences provided the clearest amplification patterns. We explored the potential of SAMPL analysis in lettuce to detect PCR-based codominant microsatellite markers. Fifty-eight SAMPLs were identified and placed on the genetic map. Seventeen were codominant. SAMPLs were dispersed with RFLP markers on 11 of the 12 main linkage groups in lettuce, indicating that they have a similar genomic distribution. Some but not all fragments amplified by SAMPL analysis were confirmed to contain microsatellite sequences by Southern hybridization. Forty-five cultivars of lettuce and five wild species of Lactuca were analyzed to determine the allelic diversity for codominant SAMPLs. From 3 to 11 putative alleles were found for each SAMPL; 2-6 alleles were found within Lactuca sativa and 1-3 alleles were found among the crisphead genotypes, the most genetically homogeneous plant type of L. sativa. This allelic diversity is greater than that found for RFLP markers. Numerous new alleles were observed in the wild species; however, there were frequent null alleles. Therefore, SAMPL analysis is more applicable to intraspecific than to interspecific comparisons. A phenetic analysis based on SAMPLs resulted in a dendrogram similar to those based on RFLP and AFLP markers.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.     

Sixteen polymorphic microsatellite markers from Zizania latifolia Turcz. (Poaceae)

Sixteen polymorphic microsatellite markers were isolated and identified in Zizania latifolia Turcz. (Poaceae), a perennial aquatic plant widespread in Eastern Asia. The microsatellite-enriched library was constructed using the fast isolation by AFLP of sequences containing repeats method. These markers revealed two to 14 alleles, with an average of 5.6 alleles per locus. The observed and expected heterozygosities varied from 0.071 to 0.690 and from 0.174 to 0.812, respectively. These markers will be useful for studying of gene flow and evaluating the genetic diversity of the Zizania latifolia population.     

Genetic variation of Avicennia marina (Forsk.) Vierh. (Avicenniaceae) in Vietnam revealed by microsatellite and AFLP markers

Genetic variation of Avicennia marina in the costal area of Vietnam was examined using microsatellite and AFLP markers. By using five microsatellite loci a total of 21 alleles were detected. The average number of alleles per locus per population ranged from 1.667 to 3.000. The observed heterozygosity varied from 0.180 to 0.263, with an average of 0.210 indicating relatively low level of genetic variation comparing to the previous studies on A. marina in the worldwide range. The expected heterozygosity was larger than the observed heterozygosity leading to positive inbreeding coefficients in all the six populations. Highly significant departures from Hardy-Weinberg Equilibrium were detected in four populations. AFLP analysis revealed a total of 386 loci, of which 232 (60.1%) were polymorphic. In congruent with microsatellite markers relatively low levels of genetic variation were detected at both gene and nucleotide levels (H = 0.086; pi = 0.0054). Reduced level of genetic variation was found in the central population, and in the southern populations. Both microsatellite and AFLP markers revealed large genetic differentiation (F(ST) = 0.262 and 0.338, respectively) indicating strong genetic structure among regional populations. Pairwise genetic distance by AFLP showed two populations in the north and the other two in the south are closely related each other.     

Isolation and characterization of microsatellite markers for the endangered Monimopetalum chinense (Celastraceae)

We describe the isolation and characterization of 13 polymorphic microsatellite loci in the endangered Monimopetalum chinense (Celastraceae) using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol in this paper. The 13 loci were tested in 20 individuals from two populations. The number of alleles per locus varied from 2 to 8, with an average of 3.8. The expected and observed heterozygosities ranged from 0.05 to 0.87 and from 0.05 to 0.55, respectively. Two of the 13 loci were biased from Hardy–Weinberg equilibrium. No significant linkage disequilibrium was found among all loci. These polymorphic SSR markers will be useful in studying population genetics of M. chinense.     

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

 Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942 cM with the average distance between adjacent markers of approximately 10 cM. The maximum distance allowed between markers is 27.5 cM. About 11% of the intervals (20 out of 173) are over 20 cM (but less than 27.5 cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed. Received: 12 March 1997 / Accepted: 20 May 1997     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Genetic Analysis of Selected Strains of Eastern Oyster (Crassostrea virginica Gmelin) Using AFLP and Microsatellite Markers

Amplified fragment length polymorphisms (AFLPs) and microsatellite markers were used to examine genetic variation and divergence in 4 selected strains (DBH, NEH, FMF, and CTS) and 1 wild population (DBW) of the eastern oyster Crassostrea virginica Gmelin. Eighty-six AFLP markers (from 3 primer pairs) and 5 microsatellite loci were used for the analysis of 30 oysters from each of the 5 populations. Microsatellite loci were considerably more variable than AFLPs. The observed heterozygosity ranged from 0.560 to 0.640 across populations for microsatellites, and from 0.186 to 0.207 for AFLPs. Both F_st and _PT of microsatellite data and _PT statistics of AFLP data revealed significant divergence between all pairs of populations. There was no significant reduction in heterozygosity in all 4 selected strains; however, the number of alleles per locus was considerably lower in the selected strains than in the wild population. Two strains subjected to long-term selection for disease resistance shared frequency shifts at a few loci, which deserve further analysis to determine if they are linked to disease-resistance genes.     

Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship

Microsatellite amplification was performed on cassava (Manihot esculenta) and six other different species (all wild) of the Manihot genus. We used ten pairs of microsatellite primers previously developed from cassava, detecting 124 alleles in a sample of 121 accessions of the seven species. The number of alleles per locus ranged from four to 21 alleles, and allelic diversity was greater in the wild species than in cassava. Seventy-nine alleles, including unique ones, were detected in the wild species but were not found in the crop. The lower level of heterozygosity in some wild species probably resulted from a combination of fine-scale differentiation within the species and the presence of null alleles. Overall, microsatellite primers worked across the genus, but, with increasing genetic distance, success in amplifying loci tended to decrease. No accession of M. aesculifolia, M. carthaginensis, and M. brachyloba presented a banding pattern at locus Ga-140; neither did one appear for M. aesculifolia at locus Ga-13. Previous work with amplified fragment length polymorphism (AFLP) markers and this microsatellite analysis show that these three wild taxa are the most distant relatives of the crop, whereas the wild forms M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana appear to be the closest.     

Development and characterization of polymorphic microsatellite markers in Momordica charantia (Cucurbitaceae)

? Premise of the study: Polymorphic microsatellite markers were developed for Momordica charantia L. to investigate the genetic diversity and population structure within and between M. charantia and its four related species (Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L.). ? Methods and Results: Using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) method, 16 polymorphic microsatellite loci were identified in 36 individuals of M. charantia. Across all the M. charantia samples, the number of alleles per locus ranged from three to eight. Seven primers successfully amplified in the four related species. ? Conclusions: These markers will be useful to study population ecology and population differentiation among M. charantia species and its related species.     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

Microsatellite markers for woolly monkeys (Lagothrix lagotricha) and their amplification in other New World primates (Primates: Platyrrhini)

Seven polymorphic microsatellite loci were identified for woolly monkeys (Lagothrix lagotricha) from an ‘enriched’ genomic library. For a wild population of 66 animals, these markers averaged over 10 alleles per locus and provided a combined probability for excluding a random individual from parentage of over 98%. These loci were screened in up to 13 other genera of New World monkeys, and many were variable in multiple taxa. Few other platyrrhine‐specific microsatellite markers have been identified; thus, these loci should prove valuable for studying the population genetic structure and mating system not just of Lagothrix but also of other neotropical primates.     

Isolation and characterization of polymorphic microsatellite loci from Yellowcheek (Elopichthys bambusa)

Nine yellowcheek (Elopichthys bambusa) microsatellite loci were isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Three to eight alleles per locus were detected in 29 samples collected from five populations of E. bambusa. The mean number of alleles was 5.6 ± 1.9 and the level of observed heterozygosities ranged from 0.415 to 0.843. These are the first microsatellite loci characterized from E. bambusa that can be used for estimating genetic diversity, population structure and parentage analysis.     

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.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Development and characterization of polymorphic microsatellite markers in Rhododendron simsii (Ericaceae)

A set of 14 polymorphic microsatellite markers has been developed and characterized using FIASCO (fast isolation by AFLP of sequences containing repeats) methods for the garden plant, Rhododendron simsii Planch. Forty‐one R. simsii individuals showing large morphological differences were used to identify these markers. The total number of alleles for each locus ranged from 2 to 6, with an average value of 3.643. The expected heterozygosities and observed heterozygosities ranged from 0.137 to 0.778 and 0.000 to 0.769, respectively. Eight loci exhibited significant deviations from Hardy‐Weinberg equilibrium. Among these microsatellites, seven could successfully be transferred to four related species (R. pulchrum, R. hybrida, R. alutaceum and R. molle (Blume) G. Don). These novel microsatellite markers could be further used for assessment of genetic diversity and population structure, phylogeographic analysis and marker‐assisted selection for R. simsii and other Rhododendron species.     

Development of microsatellite loci for the invasive weed Wedelia trilobata (Asteraceae)

? Premise of the study: Microsatellite markers were developed to help elucidate the population genetics of the invasive species Wedelia trilobata. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) repeats protocol, 23 sets of primers for amplifying microsatellite loci were identified in W. trilobata, 10 of which showed polymorphism (two to five alleles per locus) in samples of two populations of W. trilobata, one from China and one from Peru. Six of these loci were successfully amplified from samples of the native congener W. chinensis, with expected sizes. ? Conclusions: These markers may be useful for further investigation of population genetics of Wedelia trilobata and other congener species.     

Isolation and characterization of microsatellite markers from reed parrotbill, Paradoxornis heudei (Aves: Timaliidae)

Eleven microsatellite markers were obtained from reed parrotbill, Paradoxornis heudei, using the fast isolation by AFLP of sequences containing repeats (FIASCO) method as part of an effort to compare levels of genetic diversity in different populations of China. Polymorphism levels ranged form 5 to 11 alleles (mean = 9.27) using 32 reed parrotbills, with the observed heterozygosity ranging from 0.037 to 0.80. Six loci were significant deviated from HWE and nine loci showed linkage equilibrium. The utility of these loci on two other Passeriformes species, vinous-throated parrotbill (P. webbianus) and oriental great reed warbler (Acrocephalus orientalis), were also tested.