Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley

BACKGROUND: Earlier comparative maps between the genomes of rice (Oryza sativa L.), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were linkage maps based on cDNA-RFLP markers. The low number of polymorphic RFLP markers has limited the development of dense genetic maps in wheat and the number of available anchor points in comparative maps. Higher density comparative maps using PCR-based anchor markers are necessary to better estimate the conservation of colinearity among cereal genomes. The purposes of this study were to characterize the proportion of transcribed DNA sequences containing simple sequence repeats (SSR or microsatellites) by length and motif for wheat, barley and rice and to determine in-silico rice genome locations for primer sets developed for wheat and barley Expressed Sequence Tags. RESULTS: The proportions of SSR types (di-, tri-, tetra-, and penta-nucleotide repeats) and motifs varied with the length of the SSRs within and among the three species, with trinucleotide SSRs being the most frequent. Distributions of genomic microsatellites (gSSRs), EST-derived microsatellites (EST-SSRs), and transcribed regions in the contiguous sequence of rice chromosome 1 were highly correlated. More than 13,000 primer pairs were developed for use by the cereal research community as potential markers in wheat, barley and rice. CONCLUSION: Trinucleotide SSRs were the most common type in each of the species; however, the relative proportions of SSR types and motifs differed among rice, wheat, and barley. Genomic microsatellites were found to be primarily located in gene-rich regions of the rice genome. Microsatellite markers derived from the use of non-redundant EST-SSRs are an economic and efficient alternative to RFLP for comparative mapping in cereals

DNA-based identification of Lentinula edodes strains with species-specific primers

Lentinula edodes is among the five globally cultivated edible mushrooms, which are wood decaying spore bearing Basidiomycetes possessing separate hyphae. Specific identification of this fungus from others in the division Basidiomycota using specific primers enables a fast and accurate detection through polymerase chain reaction (PCR). As a prelude to additional nutritional and sequence characterization research, we have developed a species specific PCR assay for this fungus after screening four primer-pairs and two universal primer pairs. The primer-pair LE1F/R was specific in amplifications of ATCC-defined L. edodes strains and did not amplify DNA from six medicinally and nutritionally important fungal reference strains, Oyster (Pleurotus ostreatus), Maitake (Grifola frondosa), Enoki (Flammulina velutipes), Baby bella (Agaricus bisporus), Porcini (Boletus edulis), and Chanterelle (Cantharellus cibarius). However, amplifications using the universal primers were positive for all six strains. This assay will therefore serve to validate morphology-based identifications of L. edodes strains.Keywords: Lentinula edodes, LE1F/R, species-specific primer

BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in upland cotton.

Acknowledgments “Dedicated to Dr. Ramesh Kantety, a mentor, colleague and friend”. We would like to acknowledge the support offered by Padmini Sripathi during data analysis and submissions. Author Contributions Conceived and designed the experiments: RVK JZY. Performed the experiments: RB ZX SM GBW. Analyzed the data: RB. Contributed reagents/materials/analysis tools: RVK RB JZY RJK BAR. Wrote the manuscript: RB. Revised the manuscript: RB RVK JZY RGP BAR GCS. Advised the research: RVK JZY RGP BAR GCS.Author Contributions Conceived and designed the experiments: RVK JZY. Performed the experiments: RB ZX SM GBW. Analyzed the data: RB. Contributed reagents/materials/analysis tools: RVK RB JZY RJK BAR. Wrote the manuscript: RB. Revised the manuscript: RB RVK JZY RGP BAR GCS. Advised the research: RVK JZY RGP BAR GCS.Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.Yeshttp://www.plosone.org/static/editorial#pee

Inter-simple sequence repeats as genetic markers and mapping genes for grain milling quality in maize

High-throughput DNA markers, such as inter-simple sequence repeats (ISSRs), are more efficient than RFLPs. ISSRs result from PCR amplification of inter-microsatellite sequences and target multiple loci in the genome. We investigated the usefulness of ISSRs in applied maize (Zea mays L.) breeding. Our specific objectives were to (i) assess the genetic diversity among popcorn and dent corn inbreds with ISSRs, (ii) map ISSR markers relative to RFLP markers using the University of Missouri-Columbia immortalized F\sb2 population, (iii) use ISSRs in marker-assisted backcrossing of earliness from dent corn inbred A662 into popcorn inbreds HP86-19 and HP72-11, and (iv) identify RFLP, SSR and ISSR markers associated with grain milling quality in dent corn. Among 19 popcorn and 8 dent corn inbreds, ISSRs yielded an average of 54 bands/primer/inbred, and revealed 73% polymorphism among popcorn and 87% polymorphism among dent corn inbreds. Principal component and cluster analyses classified the dent and popcorn inbreds into their respective heterotic groups. Among 54 immortalized F\sb2 lines, ISSR amplification generated 239 markers which were mapped to all arms of the 10 chromosomes of maize with an average marker interval of 2.2 cM. In a ((HP86-19 X A662) X HP86-19) BC\sb1 population, we identified a BC\sb1 line with $\u3e$92% recurrent parent (RP) background that was used to generate advanced backcross generations. ISSR marker analysis of BC\sb2 and BC\sb3 generations resulted in a line, with 99.6% RP background, that flowers $\ge$10 days earlier than the RP. In a (B73 X HS99-423) F\sb2 population, 70 RFLP, 20 SSR, and 124 ISSR markers were used to map QTL for grain milling quality. The F\sb{2:3} progenies were grown at West Lafayette, IN, and Slater, IA, in 1994. Seventy five QTL were identified, 65 of which were specific to one environment. Different QTL were involved in wet milling and in kernel composition, indicating that the grain composition alone does not predict the milling performance. QTL associated with wet milling qualities were different from those involved in kernel density and seed weight. Our results indicate that maize hybrids with enhanced milling quality can be obtained with the use of DNA markers

Principal Component Analysis and Molecular Characterization of Reniform Nematode Populations in Alabama

U.S. cotton production is suffering from the yield loss caused by the reniform nematode (RN), Rotylenchulus reniformis. Management of this devastating pest is of utmost importance because, no upland cotton cultivar exhibits adequate resistance to RN. Nine populations of RN from distinct regions in Alabama and one population from Mississippi were studied and thirteen morphometric features were measured on 20 male and 20 female nematodes from each population. Highly correlated variables (positive) in female and male RN morphometric parameters were observed for body length (L) and distance of vulva from the lip region (V) (r = 0.7) and tail length (TL) and c′ (r = 0.8), respectively. The first and second principal components for the female and male populations showed distinct clustering into three groups. These results show pattern of sub-groups within the RN populations in Alabama. A one-way ANOVA on female and male RN populations showed significant differences (p ≤ 0.05) among the variables. Multiple sequence alignment (MSA) of 18S rRNA sequences (421) showed lengths of 653 bp. Sites within the aligned sequences were conserved (53%), parsimony-informative (17%), singletons (28%), and indels (2%), respectively. Neighbor-Joining analysis showed intra and inter-nematodal variations within the populations as clone sequences from different nematodes irrespective of the sex of nematode isolate clustered together. Morphologically, the three groups (I, II and III) could not be distinctly associated with the molecular data from the 18S rRNA sequences. The three groups may be identified as being non-geographically contiguous

Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, <i>Rotylenchulus reniformis</i>

<div><p>The <i>18S</i> rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of <i>18S</i> rRNA gene from a single reniform nematode (RN) <i>Rotylenchulus reniformis</i> labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the <i>18S</i> rRNA gene that can clearly distinguish the two <i>18S</i> rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing <i>R. reniformis</i> sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the <i>18S</i> rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.</p></div

Nematode species belonging to Tylenchoidea, GenBank accession numbers, and length of <i>18S</i> rRNA sequence.

<p>Nematode species belonging to Tylenchoidea, GenBank accession numbers, and length of <i>18S</i> rRNA sequence.</p

Oligonucleotide primers for sequencing of the RN <i>18S</i> rRNA gene.

<p>Oligonucleotide primers for sequencing of the RN <i>18S</i> rRNA gene.</p