Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species. Received: 6 October 2000 / Accepted: 13 March 2001     

Nuclear- and chloroplast-microsatellite variation in A-genome species of rice.

Simple sequence length polymorphism analysis was carried out to reveal microsatellite variation and to clarify the phylogenetic relationships among A-genome species of rice. Total DNA from 29 cultivars (23 Oryza sativa and 6 O. glaberrima) and 30 accessions of wild A-genome species (12 O. rufipogon, 5 O. glumaepatula, 2 O. longistaminata, 6 O. meridionalis, and 5 O. barthii) was used as a template for PCR to detect 24 nuclear and 10 chloroplast microsatellite loci. Microsatellite allelic diversity was examined based on amplified banding patterns. Microsatellites amplified clearly in all 59 accessions, with an average of 18.4 alleles per locus. The polymorphism information content (PIC) value ranged from 0.85 to 0.94, with an average of 0.89. At the species level, high average PIC values were observed in O. sativa (0.79) and O. rufipogon (0.80). For chloroplast microsatellites, the average number of alleles per locus and the average PIC value were 2.9 and 0.38, respectively. While the magnitude of diversity was much greater for nuclear microsatellites than for chloroplast microsatellites, they showed parallel patterns of differentiation for each taxonomic group. Using the ratio of common alleles (estimated as size of amplified fragments) as a similarity index, the average percentages of common microsatellite alleles were calculated between taxa. For both nuclear and chloroplast microsatellites, O. sativa showed the highest similarity values to O. rufipogon, and O. glaberrima was most similar to O. barthii. These data support previous evidence that these cultivars originated from the corresponding wild ancestral species.     

Isolation and characterization of microsatellite loci from Asparagus acutifolius (Liliaceae)

The isolation of molecular markers in Asparagus acutifolius, a wild edible plant species, is important to characterize local ecotypes that could be cultivated and preserved. We isolated and characterized polymorphic microsatellite loci from A. acutifolius by constructing and screening an enriched DNA library. Primer pairs were designed for 12 loci. Seven primer pairs worked well during amplification reactions and were tested on a wild population from Pontecagnano (SA), Italy. These loci showed a high level of genetic variability, with the numbers of alleles identified ranging from two to five and observed heterozygosity ranging from 0.20 to 0.73.     

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.     

Isolation and characterization of microsatellite loci in Lesquerella fendleri (Brassicaceae) and cross‐species amplification

Fifteen novel microsatellite primer pairs are presented for Lesquerella fendleri, which were developed from seven dinucleotide, five trinucleotide and three tetranucleotide microsatellite DNA loci. These loci were characterized for 40 individuals from 24 localities throughout the species range. The number of alleles observed per locus ranged from three to 16, the observed heterozygosity ranged from 0.175 to 0.750, and the polymorphic information content ranged from 0.218 to 0.889. Cross‐species transferability tested on nine species of Lesquerella and one species of the related genus Physaria indicates that these primer pairs may be useful for population genetic studies of other species in Lesquerella and possibly other closely related genera.     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.     

Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.)

Microsatellite markers containing simple sequence repeats (SSR) are a valuable tool for genetic analysis. Our objective is to augment the existing RFLP map of rice with simple sequence length polymorphisms (SSLP). In this study, we describe 20 new microsatellite markers that have been assigned to positions along the rice chromosomes, characterized for their allelic diversity in cultivated and wild rice, and tested for amplification in distantly related species. Our results indicate that the genomic distribution of microsatellites in rice appears to be random, with no obvious bias for, or clustering in particular regions, that mapping results are identical in intersubspecific and interspecific populations, and that amplification in wild relatives ofOryza sativa is reliable in species most closely related to cultivated rice but becomes less successful as the genetic distance increases. Sequence analysis of SSLP alleles in three relatedindica varieties demonstrated the clustering of complex arrays of SSR motifs in a single 300-bp region with independent variation in each. Two microsatellite markers amplified multiple loci that were mapped onto independent rice chromosomes, suggesting the presence of duplicated regions within the rice genome. The availability of increasing numbers of mapped SSLP markers can be expected to increase the power and resolution of genome analysis in rice.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Isolation and characterization of polymorphic microsatellites in the groundnut seed beetle,Caryedon serratus (Coleoptera,Bruchidae)

Caryedon serratus feeds on the seeds of four genera of wild Caesalpinioideae. It recently became a major pest of groundnut in Africa. To determine the extent of gene flow and isolation between wild and groundnut‐feeding populations, we isolated and characterized 10 microsatellite loci in this species. Polymorphism ranged from three to 15 alleles in the 80 individuals collected in four localities in Senegal on five host plants.     

A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing

• Premise of the study: Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation. Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia. • Methods and Results: Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flanking primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further characterized by genotyping Dyckia samples at the level of populations and species. Three, five, and six cpSSRs were polymorphic among four individuals of D. limae, 12 individuals of D. dissitiflora, and 12 of D. pernambucana, respectively, with two to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per locus. Ten primer pairs cross-amplified with bromeliad genera from five subfamilies. • Conclusions: The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.     

Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers

To study the transferability of rice (Oryza sativa L.) genome data, we used amplified consensus genetic markers to analyze the phylogenetic relationships among several species and genera in Gramineae. Ten accessions representing five grass genera (Oryza, Zea, Setaria, Triticum, and Phyllostachys) were used. According to the genetic distances, a cluster tree was constructed. The relationships among the five genera could be simply described as ((Oryza + (Zea + Setaria)) + Triticum) + Phyllostachys. The results suggest that the genetic distance between rice and maize (Z. mays L.) or rice and millet (Setaria italica L.) is closer than that between rice and wheat (Triticum aestivum L) or rice and bamboo.     

Microsatellites and RFLP probes from maize are efficient sources of molecular markers for the biomass energy crop Miscanthus

A survey of Gramineae markers was carried out with the aim of developing cost-effective methods for the molecular analysis of Miscanthus species. Ten out of twenty Gramineae RFLP probes from ”anchor” sets hybridized well to Miscanthus DNA while all 15 maize probes tested cross-hybridized successfully, showing similar patterns in both species. Cross-taxa amplification of maize microsatellite primers was then tested. This showed that 57 out of 76 (75%) give highly reproducible amplification with Miscanthus DNA. Amplification products differed in size from those in maize but there was no bias toward higher or lower molecular weights. Microsatellite polymorphism produced by 17 primer pairs was studied in detail in a panel of 11 Miscanthus clones belonging to the species Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus ×giganteus and Miscanthus condensatus. Intra- and inter-specific length polymorphisms were frequent between the tested Miscanthus clones with length polymorphisms being found for all primer pairs, detecting 3–22 alleles. Polymorphism information content (PIC) values for microsatellites ranged from 0.48 to 0.94 with an average of 0.83. Species-specific amplicons were produced by two microsatellites. Genetic similarity coefficients of the Miscanthus clones ranged from 0.35 to 0.92, with an average of 0.57. Five polymorphisms were studied in a segregating population, where they showed Mendelian inheritance. In addition, two microsatellite markers mapping 1.3-cM apart on maize chromosome 7 were linked in Miscanthus at an estimated distance of 8 cM, suggesting collinearity. The high transferability of microsatellite markers from maize will enhance the power and resolution of genome analysis in Miscanthus. Received: 14 April 2000 / Accepted: 9 June 2000     

Malaysian weedy rice shows its true stripes: wild Oryza and elite rice cultivars shape agricultural weed evolution in Southeast Asia

Weedy rice is a close relative of domesticated rice (Oryza sativa) that competes aggressively with the crop and limits rice productivity worldwide. Most genetic studies of weedy rice have focused on populations in regions where no reproductively compatible wild Oryza species occur (North America, Europe and northern Asia). Here, we examined the population genetics of weedy rice in Malaysia, where wild rice (O. rufipogon) can be found growing in close proximity to cultivated and weedy rice. Using 375 accessions and a combined analysis of 24 neutral SSR loci and two rice domestication genes (sh4, controlling seed shattering, and Bh4, controlling hull colour), we addressed the following questions: (i) What is the relationship of Malaysian weedy rice to domesticated and wild rice, and to weedy rice strains in the USA? (ii) To what extent does the presence of O. rufipogon influence the genetic and phenotypic diversity of Malaysian weeds? (iii) What do the distributions of sh4 and Bh4 alleles and associated phenotypes reveal about the origin and contemporary evolution of Malaysian weedy rice? Our results reveal the following: independent evolutionary origins for Malaysian weeds and US strains, despite their very close phenotypic resemblance; wild‐to‐weed gene flow in Malaysian weed populations, including apparent adaptive introgression of seed‐shattering alleles; and a prominent role for modern Malaysian cultivars in the origin and recent proliferation of Malaysian weeds. These findings suggest that the genetic complexity and adaptability of weedy crop relatives can be profoundly influenced by proximity to reproductively compatible wild and domesticated populations.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

DNA barcoding of Oryza: conventional,specific, and super barcodes

Key message

We applied the phylogenomics to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

Abstract

Rice (genus Oryza) is one of the most important crops in the world, supporting half of the world’s population. Breeding of high-yielding and quality cultivars relies on genetic resources from both cultivated and wild species, which are collected and maintained in seed banks. Unfortunately, numerous seeds are mislabeled due to taxonomic issues or misidentifications. Here, we applied the phylogenomics of 58 complete chloroplast genomes and two hypervariable nuclear genes to determine species identity in rice seeds. Twenty-one Oryza species were identified. Conspecific relationships were determined between O. glaberrima and O. barthii, O. glumipatula and O. longistaminata, O. grandiglumis and O. alta, O. meyeriana and O. granulata, O. minuta and O. malampuzhaensis, O. nivara and O. sativa subsp. indica, and O. sativa subsp. japonica and O. rufipogon. D and L genome types were not found and the H genome type was extinct. Importantly, we evaluated the performance of four conventional plant DNA barcodes (matK, rbcL, psbA-trnH, and ITS), six rice-specific chloroplast DNA barcodes (psaJ-rpl33, trnC-rpoB, rps16-trnQ, rpl22-rps19, trnK-matK, and ndhC-trnV), two rice-specific nuclear DNA barcodes (NP78 and R22), and a chloroplast genome super DNA barcode. The latter was the most reliable marker. The six rice-specific chloroplast barcodes revealed that 17% of the 53 seed accessions from rice seed banks or field collections were mislabeled. These results are expected to clarify the concept of rice species, aid in the identification and use of rice germplasms, and support rice biodiversity.

     

Potential of (GATA)n microsatellites from rice for inter- and intra-specific variability studies

Background

The microsatellite, (GATA)_n has been frequently used for DNA fingerprinting. However, very few attempts have been made to analyze (GATA)_n-containing loci in rice.

Results

Three polymorphic (GATA)_n-harboring loci viz. OS1A6, OS1H10 and OS2E7, containing 7–13 repeat motifs were identified from a genomic library of a cultivated rice, Oryza sativa var. Basmati-370 using oligonucleotide probe (GATA)₄. When (GATA)_n flanking primers were used to screen 26 wilds (representing different genomes of rice), 16 cultivars, 47 Indian elite rice varieties and 37 lines resistant/susceptible to bacterial blight, up to 22 alleles were obtained at an individual locus. Also, interestingly the bacterial blight resistant lines clustered into a separate group from the remaining rice genotypes, when a dendrogram was constructed based on the polymorphism obtained at the three loci. This may be due to the partial homology of the clones OS1H10 and OS2E7 to regions encoding O. longistaminata receptor kinase-like protein and pathogenesis-related protein. The ability of these O. sativa flanking primers to amplify DNA of maize, wheat, barley and oat indicates that these (GATA)_n-containing loci are conserved across different cereal genera.

Conclusions

The large allele number obtained reveals the potential of (GATA)_n-containing loci as powerful tools to detect simple sequence length polymorphism (SSLP). The (GATA)_n-flanking primers were not only useful in distinguishing between closely related genotypes, but could also be used for cross-species amplification and are also conserved across different cereal genera. These loci could also cluster the bacterial blight resistant/susceptible lines into different groups based on the resistance genes present in them.