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PubMed2002

Development of microsatellite markers from an ectomycorrhizal fungus, Tricholoma matsutake, by an ISSR-suppression-PCR method.

An inter-simple sequence repeat (ISSR)-suppression-PCR technique established to develop microsatellite markers of plant species was applied to an ectomycorrhizal fungus, Tricholoma matsutake. Six polymorphic SSR markers were developed. All six polymorphic SSR markers were single-locused and co-dominant. Alleles produced by these six single-locused markers ranged from two to nine per locus and the expected heterozygosities were calculated as values from 0.098 to 0.803. The results indicated that the ISSR-suppression-PCR technique was effective and applicable to the development of microsatellite markers from ectomycorrhizal fungi. Furthermore, the six microsatellite loci did not amplify DNA from any other ectomycorrhizal species investigated, except for Tricholoma nauseosum (Swedish matsutake) and Tricholoma fulvocastaneum, suggesting that population genetics and reproduction of T. matsutake could be investigated by the SSR markers developed in the present study.

Basidiomycota↗

PubMed2004

Population genetic structure of an ectomycorrhizal fungus Amanita manginiana in a subtropical forest over two years.

The population genetic structure of the late-stage fungus Amanita manginiana in a natural forest in Dujiangyan, southwest China was examined over two years using inter-simple sequence repeat (ISSR) markers. Seven ISSR primers were used and 170 bands were obtained in this population: 134/160 and 135/153 bands were polymorphic for sporocarps of 2001 and 2002, respectively. Each sporocarp represented a single genet in 2001 and 2002, and no identical genets were found between the two years. The results of genetic similarity comparison, using unweighted pair group method with arithmetic means, and analysis of molecular variance, indicated that although genetic variances were mainly within individuals of the same year the genetic variance between years was statistically significant (P<0.001). Relationships between genetic similarity and spatial distance of pairwise sporocarps were also found to be different in the two years. The differences in genetic structure and genetic similarity between individuals of the two years implied that the sporocarps were not likely to be derived from continuous generations, i.e., the sporocarps collected in 2002 were not developed from sexual spores dispersed by sporocarps of 2001. We suggest that the life-cycle traits of ectomycorrhizal (ECM) fungi should be considered in genetic studies on ECM fungal populations.

Amanita↗

PubMed2005

Spatial distribution of ectomycorrhizal Basidiomycete Russula subsect. Foetentinae populations in a primary dipterocarp rainforest.

The spatial distribution of basidiocarps of the ectomycorrhizal Basidiomycete Russula subsect. Foetentinae was assessed in a primary forest in the Western Ghâts (India) dominated by the ectomycorrhizal tree species Vateria indica and Dipterocarpus indicus. Over a 7,700-m(2) sampling area, both trees and basidiocarps of Russula subsect. Foetentinae were mapped during the first month of the 2002 rainy season. First-order spatial analysis revealed that the distribution of the 45 collected carpophores was highly aggregated, with 60% of all basidiocarps located at a distance lower than 1 m from the nearest one. The genetic structure of the Russula subsect. Foetentinae population was studied by inter-simple sequence repeat polymorphism analysis using three primers. Eighteen of the 45 genotypes were represented by single basidiocarps. Twenty-seven basidiocarps were identified as belonging to 11 genets or separated ramets. Five genets were small, with diameters ranging from 0.5 to 5 m. The six others were large, with a diameter ranging from 31 m to a maximum measured distance of 70 m. In spite of the lack of data concerning the reproductive biology of this species, the presence of large genets suggests that mature stands may shelter well-spread underground mycelium, crucial for durable interaction with plant partner.

Basidiomycota↗

PubMedDate not supplied

High frequency and large number of polymorphic microsatellites in cultured shrimp, Penaeus (Litopenaeus) vannamei [Crustacea:Decapoda].

A total of 1479 recombinant clones were obtained from a Sau3A-digested genomic library of Penaeus (Litopenaeus) vannamei and used for probe hybridization. Of the 251 clones that tested positive to one or more of the probes and were sequenced, 173 (69%) contained 573 simple sequence repeats, or microsatellites, with 3 or more repeats. The frequency of microsatellites with 3, 5, and 10 or more repeats was 1 in 0.94 kb, 1 in 2.78 kb, and 1 in 5.94 kb, respectively. To increase the number of polymorphic markers for mapping, 136 primer sets that flanked microsatellites containing single or multiple motifs with 3 or more repeats were designed and tested. Of the 136 primers, 93 (68.0%) were polymorphic in cultured shrimp, with polymorphism information content (PIC) values ranging from 0.195 to 0.873, and observed heterozygosities ranging from 10% to 100%. These markers are being used along with other markers to construct a linkage map for P. vannamei.

Animals↗

PubMed2004

Bioinformatic mining of type I microsatellites from expressed sequence tags of channel catfish (Ictalurus punctatus).

Gene-derived markers are pivotal to the analysis of genome structure, organization, and evolution and necessary for comparative genomics. However, gene-derived markers are relatively difficult to develop. This project utilized the genomic resources of channel catfish expressed sequence tags (ESTs) to identify simple sequence repeats (SSRs), or microsatellites. It took the advantage of ESTs for the establishment of gene identities, and of microsatellites for the acquisition of high polymorphism. When microsatellites are tagged to genes, the microsatellites can then be used as gene markers. A bioinformatic analysis of 43,033 ESTs identified 4855 ESTs containing microsatellites. Cluster analysis indicated that 1312 of these ESTs fell into 569 contigs, and the remaining 3534 ESTs were singletons. A total of 4103 unique microsatellite-containing genes were identified. The dinucleotide CA/TG and GA/TC pairs were the most abundant microsatellites. AT-rich microsatellite types were predominant among trinucleotide and tetranucleotide microsatellites, consistent with our earlier estimation that the catfish genome is highly AT-rich. Our preliminary results indicated that the majority of the identified microsatellites were polymorphic and, therefore, useful for genetic linkage mapping of catfish. Mapping of these gene-derived markers is under way, which will set the foundation for comparative genome analysis in catfish.

Animals↗

PubMed2005

Development of EST-SSR markers by data mining in three species of shrimp: Litopenaeus vannamei, Litopenaeus stylirostris, and Trachypenaeus birdy.

We report on the data mining of publicly available Litopenaeus vannamei expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers and on their transferability between related Penaeid shrimp species. Repeat motifs were found in 3.8% of the evaluated ESTs at a frequency of one repeat every 7.8 kb of sequence data. A total of 206 primer pairs were designed, and 112 loci were amplified with the highest success in L. vannamei. A high percentage (69%) of EST-SSRs were transferable within the genus Litopenaeus. More than half of the amplified products were polymorphic in a small testing panel of L. vannamei. Evaluation of those primers in a larger testing panel showed that 72% of the markers fit Hardy-Weinberg equilibrium, which shows their utility for population genetic analysis. Additionally, a set of 26 of the EST-SSRs were evaluated for Mendelian segregation. A high percentage of monomorphic markers (46%) proved to be polymorphic by singles-stranded conformational polymorphism analysis. Because of the high number of ESTs available in public databases, a data mining approach similar to the one outlined here might yield high numbers of SSR markers in many animal taxa.

Animals↗

PubMed2005

Characterization of EST-derived microsatellites in the wheat genome and development of eSSR markers.

EST-derived microsatellites or simple sequence repeats (eSSR) occur in expressed sequence tags (EST). Here we report characteristics of eSSRs in the wheat genome, construction of consensus chromosome bin maps of SSR-containing ESTs ((SSR)ESTs), and development of eSSR markers for the 21 wheat chromosomes. A Perl script known as MISA was used to identify eSSRs in wheat ESTs available in the database http://wheat.pw.usda.gov/cgi-bin/ace/search/wEST ). Among 492,832 ESTs from the database, 36,520 (7.41%) contained 43,598 eSSRs. This is equivalent to 1 eSSR per 5.46 kb EST sequence. About 60% of the eSSRs were trinucleotides, 19.7% were mononucleotide, 16.7% were dinucleotides, and the remaining approximately 3% consisted of tetra-, penta-, and hexanucleotides. Among the identified eSSRs, (CCG/CGG)n is the most frequent (20.5%) followed by (A/T)n at 13.6%, (AAC/GTT)n at 11.7%, and (AG/CT)n at 8.7%. Among ESTs previously mapped to wheat chromosome bins, a total of 1,010 eSSR loci were derived from 341 (SSR)ESTs. Consensus chromosome bin maps showing the chromosome locations of (SSR)ESTs, SSR sequence motif, and cDNA library were constructed. A chi(2) test indicated that the distribution pattern of eSSR loci was generally similar to that of the original mapped ESTs in the wheat genome. Forty-eight (SSR)ESTs were converted into PCR-based eSSR markers, and 266 eSSR loci were mapped to specific chromosome arms using wheat cytogenetic stocks. The average polymorphism information content (0.45+/-0.16) of eSSR markers was lower than that reported for genomic SSRs (0.54+/-0.19), but higher than RFLPs (0.30+/-0.27). The eSSR markers were transferable among related Triticeae species, Triticum aestivum, T. durum, T. dicoccoides, Hordeum spontaneum, H. vulgare, and Secale cereale. The results confirm the presence of SSRs in expressed genes of wheat and demonstrate another application of ESTs in genomics research. eSSRs will be useful for gene tagging, gene cloning, and comparative genomics studies of cereal crops.

Chromosome Mapping↗

PubMed2005

Complex microcolinearity among wheat, rice, and barley revealed by fine mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat.

A major quantitative trait locus (QTL), Qfhs.ndsu-3BS, for resistance to Fusarium head blight (FHB) in wheat has been identified and verified by several research groups. The objectives of this study were to construct a fine genetic map of this QTL region and to examine microcolinearity in the QTL region among wheat, rice, and barley. Two simple sequence repeat (SSR) markers (Xgwm533 and Xgwm493) flanking this QTL were used to screen for recombinants in a population of 3,156 plants derived from a single F(7) plant heterozygous for the Qfhs.ndsu-3BS region. A total of 382 recombinants were identified, and they were genotyped with two more SSR markers and eight sequence-tagged site (STS) markers. A fine genetic map of the Qfhs.ndsu-3BS region was constructed and spanned 6.3 cM. Based on replicated evaluations of homozygous recombinant lines for Type II FHB resistance, Qfhs.ndsu-3BS, redesignated as Fhb1, was placed into a 1.2-cM marker interval flanked by STS3B-189 and STS3B-206. Primers of STS markers were designed from wheat expressed sequence tags homologous to each of six barley genes expected to be located near this QTL region. A comparison of the wheat fine genetic map and physical maps of rice and barley revealed inversions and insertions/deletions. This suggests a complex microcolinearity among wheat, rice, and barley in this QTL region.

Fusarium↗

PubMed2005

Comparative analysis of expressed sequence tags from different organs of Vitis vinifera L.

Expressed sequence tags (ESTs) are providing a valuable approach to sampling organism-expressed genomes, especially when studying large genomes such as those of many plants. We report on the comparison of 8,647 ESTs generated from six different grape (Vitis vinifera L.) organs: berry, root, leaf, bud, shoot and inflorescence. Clustering and assembly of these ESTs resulted in 4,203 unique sequences and revealed that at this level of EST sampling, each organ shares a low percentage of transcripts with the others. To define organ relationships based on EST counts, we calculated a distance matrix of pairwise correlation coefficients between the libraries which indicated bud, inflorescence and shoot as a group distinct from the other organs considered in this study. A putative function was identified for about 85% of the unique sequences. By assigning them to specific functional classes, we were able to highlight strong differences between organs in the metabolism, protein biosynthesis and photosynthesis categories. This grape EST collection has also proven to be a valuable source for the development of 'functional' simple sequence repeats (SSRs) markers: a total of 405 SSRs have been identified. EST sequences and annotation results have been organised in the IASMA-grape database, freely available at the address http://genomics.iasma.it.

DNA, Complementary↗

PubMed2005

Genetic and in silico comparative mapping of the polyphenol oxidase gene in bread wheat (Triticum aestivum L.).

Polyphenol oxidases (PPOs) are involved in the time-dependent darkening and discolouration of Asian noodles and other wheat end products. In this study, a doubled haploid (DH) population derived from Chara (moderately high PPO activity)/WW2449 (low PPO activity) was screened for PPO activity based on L-DOPA and L-tyrosine assays using whole seeds. Both these assays were significantly genetically correlated (r = 0.91) in measuring the PPO activity in this DH population. Quantitative trait loci (QTLs) analysis utilising a skeleton map enabled us to identify a major QTL controlling PPO activity based on L-DOPA and L-tyrosine on the long arm of chromosome 2A. The simple sequence repeat (SSR) marker GWM294b explained over 82% of the line mean phenotypic variation from samples collected in both 2000 and 2003. Four SSR markers were validated for PPO linkage in genetically diverse backgrounds and proven to correctly predict the PPO activity in more than 92% of wheat lines. Physical mapping using deletion lines of Chinese Spring has confirmed the location of the GWM294b, GWM312 and WMC170 on chromosome 2AL, between deletion breakpoints 2AL-C to 0.85. In order to identify functional gene markers, data searches for alignments between rice BAC/PAC clones assembled on chromosome 1 and 4, chromosome 7, and (1) the wheat expressed sequence tags mapped in deletion bin (2AL-C to 0.85) and (2) the coding sequence of a previously cloned wheat PPO gene were made and found significant sequence similarities with the PPO gene or common central domain of tyrosinase. Available PPO gene sequences in the National Centre for Biotechnology Information (NCBI) database have revealed that there is a significant molecular diversity at the nucleotide and amino acid level in the wheat PPO genes.

Alleles↗

PubMed2005

Physical molecular maps of wheat chromosomes.

In bread wheat, a set of 527 simple sequence repeats (SSRs) were tried on 164 deletion lines, leading to a successful mapping of 270 SSRs on 313 loci covering all 21 chromosomes. A maximum of 119 loci (38%) were located on B subgenome, and a minimum of 90 loci (29%) mapped on D subgenome. Similarly, homoeologous group 7 carried a maximum of 61 loci (19%), and group 4 carried a minimum of 22 loci (7%). Of the cited 270 SSRs, 39 had multiple loci, but only eight of these detected homoeologous loci. Linear order of loci in physical maps largely corresponded with those in the genetic maps. Apparently, distances between each of only 26 pairs of loci significantly differed from the corresponding distances on genetic maps. Some loci, which were genetically mapped close to the centromere, were physically located distally, while other loci that were mapped distally in the genetic maps were located in the proximal bins in the physical maps. This suggested that although the linear order of the loci was largely conserved, variation does exist between genetic and physical distances.

Chromosomes, Plant↗

PubMed2005

Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting.

Amentotaxus, a genus of the Taxaceae, represents an ancient lineage that has long existed in Eurasia. All Amentotaxus species experienced frequent population expansion and contraction over periodical glaciations in Tertiary and Quaternary. Among them, Amentotaxus argotaenia complex consists of three morphologically alike species, A. argotaenia, Amentotaxus yunnanensis, and Amentotaxus formosana. This complex is distributed in the subtropical region of mainland China and Taiwan where many Pleistocene refugia have been documented. In this study, genetic diversity and population structuring within and between species were investigated based on the inter-simple sequence repeats (ISSR) fingerprinting. Mean genetic diversity within populations was estimated in three ways: (1) the percentage of polymorphic loci out of all loci (P) (2) Nei's unbiased expected heterozygosity (He), and (3) Shannon's index of phenotypic diversity. For a total of 310 individuals of 15 populations sampled from the three species, low levels of ISSR genetic variation within populations were detected, with P=4.66-16.58%, He=0.0176-0.0645 and Hpop=0.0263-0.0939, agreeing with their seriously threatened status. AMOVA analyses revealed that the differences between species only accounted for 27.38% of the total variation, whereas differences among populations and within populations were 57.70 and 14.92%, respectively, indicating substantial isolation between the patch-like populations. A neighbor-joining tree identified a close affinity between A. yunnanensis and A. formosana. Genetic drift due to small population size, plus limited current gene flow, resulted in significant genetic structuring. Low levels of intra-populational genetic variation and considerable inter-populational divergence were also attributable to demographic bottlenecks during and/or after the Pleistocene glaciations.

Minisatellite Repeats↗

PubMed2006

Gene polymorphisms for elucidating the genetic structure of the heavy-metal hyperaccumulating trait in Thlaspi caerulescens and their cross-genera amplification in Brassicaceae.

Genetic polymorphism was investigated in Thlaspi caerulescens J. & C. Presl at 15 gene regions, of which seven have been identified to putatively play a role in heavy-metal tolerance or hyperaccumulation. Single nucleotide and length polymorphisms were assessed at four cleaved amplified polymorphic sequences (CAPS) and 11 simple sequence repeat (microsatellite) loci, respectively. The utility of these loci for genetic studies in T. caerulescens was measured among seven natural populations (135 individuals). Fourteen loci rendered polymorphism, and the number of alleles per locus varied from 2 to 5 and 1 to 27 for CAPS and microsatellites, respectively. Up to 12 alleles per locus were detected in a population. The global observed heterozygosity per population varied between 0.01 and 0.31. Additionally, cross-species/genera amplification of loci was investigated on eight other Brassicaceae (five individuals per population). Overall, 70% of the cross-species/genera amplifications were successful, and among them, more than 40% provided intraspecific polymorphisms within a single population. This indicates that such markers may, as well, allow comparative population genetic or mapping studies between and within several Brassicaceae, particularly for genes involved in traits such as heavy-metal tolerance and/or hyperaccumulation.

Alleles↗

PubMed2006

Use of molecular markers and flow cytometry to preserve ancient Annurca apple germplasm.

The old Annurca apple cultivar (Malus domestica), particularly appreciated for its peculiar flavor and crispy flesh, was studied in order to preserve its ancient germplasm. Twelve clones of Annurca were analyzed using random amplified polymorphic DNA (RAPD) and simple-sequence repeat (SSR) markers. Two out of 30 RAPD primers and nine out of ten SSR primers were able to discriminate all the clones analyzed. Data were confirmed by measuring DNA content using flow cytometry. The results provide a good procedure to improve germplasm field management, in order to removing redundant material in the Annurca collection. This represents an efficient way to create a data bank in order to preserve the genetic variability of the Annurca cultivar.

DNA Primers↗

PubMed2005

Secale cereale inter-microsatellites (SCIMs): chromosomal location and genetic inheritance.

The polymerase chain reaction (PCR) was used to locate Sectale cereale (inter-simple sequence repeat ISSR) or Secale cereale inter-microsatellite (SCIM) markers using wheat-rye addition lines in order to develop a set of molecular markers distributed on the seven rye chromosomes. The number of SCIM markers located on 1R, 2R, 3R, 4R, 5R, 6R and 7R chromosomes were 4, 3, 12, 3, 2, 9 and 8, respectively. Therefore, a total of 41 new SCIMs were located on the seven rye chromosomes. The segregation of the 63 different SCIM markers in three different F2 was studied. The observed ISSR segregations were the 3:1 (50.7%), the 15:1 (12.7%) and the 1:1 (14.2%). The linkage analysis carried out indicated that seven of the segregating SCIMs were linked to chromosome 7R and two were linked to chromosome 4R. The use of the SCIM markers as a source of molecular markers that could be linked to interesting genes or other important agronomic traits is discussed.

Chromosome Mapping↗

PubMedDate not supplied

Genetic spatial clustering: significant implications for conservation of wild soybean (Glycine soja: Fabaceae).

Knowledge of spatial patterns of genetic variation within populations of wild relative species has significant implications with respect to sampling strategies for ex situ and in situ conservation. To study spatial genetic structure of wild soybean (Glycine soja Sieb. et Zucc.) at the fine scale, three natural populations in northern China were analyzed using inter-simple sequence repeat (ISSR) fingerprints for estimating kinship coefficients. A regression analysis of kinship coefficients against spatial distances revealed that individuals occurring close together tended to be more genetically related. The Sp statistic further indicated a comparable spatial pattern among the three wild soybean populations with similar Sp values (mean = 0.0734, varied from 0.0645 to 0.0943) detected across the three populations. Genetic patches were on average ca. 20 m in size, and the effective neighborhood sizes varied between 10 and 15 m. The spatial genetic structure evident in the wild soybean populations may be attributed to the restricted seed dispersal and predominant inbreeding mating system of this species. The detection of family structure in the populations of wild soybean has a significant implication for the effective conservation of the important genetic resources.

Breeding↗

PubMed2005

Genetic variability and genetic structure of wild and semi-domestic populations of tasar silkworm (Antheraea mylitta ) ecorace Daba as revealed through ISSR markers.

The genetic diversity in the wild and semi-domestic populations of Daba ecorace of Antheraea mylitta was studied to ascertain the distribution of variability within and among populations of semi-domestic bivoltine (DB), trivoltine (DT) and nature grown wild populations (DN) with inter-simple sequence repeat (ISSR) markers. A total of 138 markers were produced among 56 individuals of the three populations, of which 98% were polymorphic. For the individual populations, the percentage polymorphism was 58.69, 52.9 and 77.54 for DB, DT and DN, respectively. Average number of observed (1.791+/- 0.408) and effective alleles (1.389+/-0.348) was also high in the wild populations in comparison to the bivoltine and trivoltine semi-domestic populations. Genetic diversity (H(t)) in DB, DT and DN was 0.180+/- 0.033, 0.153+/- 0.032 and 0.235+/- 0.033, respectively and within-population genetic diversity (H(s)) ranged from 0.166 to 0.259 with a mean of 0.189. Mean gene differentiation (G(ST)) was found to be 0.25. Shanon's diversity index was 0.278, 0.237 and 0.361 for DB, DT and DN and overall it was 0.391. Gene flow (N(m)) among the populations was 1.509. The dendrogram produced by UPGMA with Dice's genetic distance matrices resulted in the formation of three major clusters separating the three populations. Considerable intra- and inter-population variability is found in all three populations. The population structure analysis further suggests that the semi-domestic populations of Daba ecorace are at the threshold of differentiating themselves. The high genetic variability present within wild Daba population of A. mylitta is of much importance for conservation as well as utilization in systematic breeding program.

Alleles↗

PubMed2004

SNPs, SSRs and inferences on cassava's origin.

Despite its importance as a staple food throughout the tropics, the root crop cassava (it Manihot esculenta ssp. esculenta) has traditionally not been a major focus of research. One basic question about cassava that remained unresolved until recently concerns the crop's origin. This paper describes analyses of SNPs (single nucleotide polymorphisms) and SSR (simple sequence repeat) variation as a means of tracing cassava's evolutionary and geographical origins. Genetic diversity was examined in a sample of 20 cassava varieties that are representative of germplasm diversity within the crop, and in 212 individuals collected from wild populations of two closely related Manihot species. SNP and indel variation was examined in portions of two low copy nuclear genes, BglA and Hnl. Inferences from these genes were compared to those from previously examined loci, including the low copy nuclear gene G3pdh and 5 SSR loci. For all genes examined, SNPs and SSR alleles are shared between domesticated cassava and a specific geographical subset of wild Manihot populations, which suggests the following: (1) Cassava was likely domesticated from a single wild Manihot species, M. esculenta ssp. flabellifolia, rather than from multiple hybridizing species, as traditionally believed; and (2) the crop most likely originated in the southern Amazon basin.

Aldehyde-Lyases↗