The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties

The aim of this study was to develop simple sequence repeat (SSR) markers for Dendrobium varieties/species, many of which have medicinal and horticultural values. Two genomic DNA libraries of Dendrobium Sonia enriched with GA repeats and CA repeats were constructed. Fourteen polymorphic SSR markers were identified when screened against 42 popular commercial Dendrobium hybrids. The average allele number was 12.0 ± 1.9 and the observed heterozyosity was averaged at 0.70. All 42 hybrids tested, except for two tissue culture mutants, were uniquely identified with the markers used. Sibling hybrids were closely clustered. Hybrids were also closer to parents. These SSR markers can be used for molecular ecology research, genetic mapping and marker‐assisted breeding. They can also help protection for new Dendrobium varieties.     

Development of specific ITS markers for plant DNA identification within herbivorous insects

DNA-based techniques have proved to be very useful methods to study trophic relationships between pests and their natural enemies. However, most predators are best defined as omnivores, and the identification of plant-specific DNA should also allow the identification of the plant species the predators have been feeding on. In this study, a PCR approach based on the development of specific primers was developed as a self-marking technique to detect plant DNA within the gut of one heteropteran omnivorous predator (Macrolophus pygmaeus) and two lepidopteran pest species (Helicoverpa armigera and Tuta absoluta). Specific tomato primers were designed from the ITS 1-2 region, which allowed the amplification of a tomato DNA fragment of 332?bp within the three insect species tested in all cases (100% of detection at t=0) and did not detect DNA of other plants nor of the starved insects. Plant DNA half-lives at 25°C ranged from 5.8?h, to 27.7?h and 28.7?h within M. pygmaeus, H. armigera and T. absoluta, respectively. Tomato DNA detection within field-collected M. pygmaeus suggests dietary mixing in this omnivorous predator and showed a higher detection of tomato DNA in females and nymphs than males. This study provides a useful tool to detect and to identify plant food sources of arthropods and to evaluate crop colonization from surrounding vegetation in conservation biological control programs.     

Assignment of DNA markers to Nicotiana sylvestris chromosomes using monosomic alien addition lines

 A sesquidiploid hybrid (PPS, 2n=32) between Nicotiana plumbaginifolia (PP, 2n=20) and N. sylvestris (SS, 2n=24) was backcrossed to N. plumbaginifolia to produce monosomic alien addition lines. A total of 89 2n=21 plants, each containing two sets of N. plumbaginifolia chromosomes and a single N. sylvestris chromosome, were obtained in the BC₁ and BC₂ generations. These plants were classified into 12 groups based on morphological characteristics. The N. sylvestris chromosomes in these plants were identified by RFLP and karyotype analyses. Among the 84 probes tested, 20 could not detect N. sylvestris-specific DNA bands, and the remaining 64 were assigned to 9 normal and 6 aberrant synteny groups. The 9 normal synteny groups corresponded to chromosomes 2, 4, 5, 6, 7, 8, 9, 10 and 12, respectively. Four aberrant synteny groups were the result of chromosome translocations, and 2 were deletions. Received: 10 April 1996 / Accepted: 5 July 1996     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

Development of genome-wide SSR markers in horsegram and their use for genetic diversity and cross-transferability analysis

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] commonly known as kulthi or Madras gram is an important drought tolerant legume crop used as food and fodder in India and across the globe. Horsegram is tolerant to many biotic and abiotic stresses and considered a potential future food legume. Despite being a multiutility crop, insufficient genomic information is available in this species, which is otherwise required for genetic improvement. Hence, in the present work we used next-generation sequencing (NGS) technology for genome-wide development and characterization of novel simple sequence repeat (SSR) markers in horsegram. In all, 2458 SSR primer pairs were designed from NGS data and 117 SSRs were characterized in 48 diverse lines of horsegram. Cross-transferability of these markers was also checked in nine related legume species. The polymorphic SSRs revealed high diversity measures such as mean values of expected heterozygosity (He; 0.54), observed heterozygosity (Ho; 0.64), and polymorphism information content (PIC; 0.46). Analysis of molecular variance (AMOVA) revealed high degree of genetic variance within the populations. Dendrogram based on Jaccard’s similarity coefficient and principal component analysis (PCA) revealed two groups in the analyzed accessions. This observation was further confirmed by Bayesian genetic STRUCTURE analysis. The SSR markers developed herein can be used in diverse genetic analysis including association mapping in this crop and also in related legume crops with limited marker resources. Hence, this new SSR dataset can be useful for molecular breeding research in this underutilized pulse crop. In addition, genetic diversity estimates of analyzed germplasm can be important for devising future breeding programmes in horsegram.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

Development of microsatellite markers in potato and their use in phylogenetic and fingerprinting analyses.

Three genomic libraries were constructed using a mixture of DNA from Solanum phureja Juz. & Buk., and S. chacoense Bitt. Two of the libraries were enriched for ATT and GT repeats (a 27-fold enrichment was achieved). In total, 3500 clones of the conventional library, 1,000 of the library enriched for ATT, and 12,000 of the one enriched for GT were screened with five different repeat motifs, and a total of 18 primer pairs was obtained. Another group of 12 primer pairs was obtained from the SSR-containing sequences in the public databases (18 SSR-containing sequences were utilized). From among 30 newly developed primer pairs, 12 previously published ones, and 12 pairs developed for tomato, 7 were used to identify 12 different potato cultivars and introductions, and 12 were used to study phylogenetic distance among seven wild and cultivated potato species. Two SSR markers were sufficient to discriminate the 12 cultivars. The mean number of alleles per polymorphic locus was 5 for the 12 cultivars and 4.5 for the seven species. The results obtained in this study confirm those achieved in similar studies in other plant species regarding the abundance and use of SSR markers in identifying species and cultivars.     

Development of sex-linked PCR markers for gender identification in Actinidia

 Two sex-linked random amplified polymorphic DNA (RAPD) markers identified from Actinidia chinensis were converted into sequence-characterised amplified regions (SCARs) for the large-scale screening of Actinidia breeding populations. Initial SCAR primers converted one RAPD (SmX) into a dominant marker, but the other (SmY), which was potentially more useful because of its linkage to the male determining ‘Y’ locus, failed to retain polymorphism. This difficulty was overcome by cloning and sequencing the alternate ‘allele’ from female plants, and then designing ‘allele’-specific primers that utilised nucleotide differences between the sexes. Using a quick squash-blot method of DNA extraction, the SCAR primers were tested in 120 A. chinensis plants to determine their gender. The system is now in use for large-scale screening of seedling populations in the Actinidia breeding programme. The sex-linked SCAR primers also functioned with plants from some other geographically separate accessions of A. chinensis and with plants in the closely related polyploid species A. deliciosa, but did not amplify a sex-linked band in more distantly related species of Actinidia. Received: 27 December 1997 / Accepted: 5 March 1998     

DNA markers for identification of Pseudomonas syringae pv. actinidiae

The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv. actinidiae, as well as similar strains that were isolated from kiwifruits. The primer C24 detected a fragment that is specific in P. syringae pv. actinidiae. This fragment was cloned. The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae using the cloned fragment as a probe. The sequence size of the cloned fragment was determined as 675 bp. A DNA Database search suggested that the fragment was a novel one. Approximately 9 kb of a single fragment was detected only in the P. syringae pv. actinidiae by a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae. Similar strains were also detected with the use of the cloned fragment as a probe. Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P. syringae pv. actinidiae as a single copy. A pair of primers that produced a 492 bp single fragment (only in the strains of P. syringae pv. actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P. syringae pv. actinidiae. The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree.     

Development of microsatellite markers in Fragaria, their use in genetic diversity analysis, and their potential for genetic linkage mapping.

We have developed 21 new microsatellites in the model diploid perennial species Fragaria vesca from an enriched genomic library developed using F. vesca 'Ruegen'. The transferability of the primer pairs to other Fragaria species was high; all 31 primer pairs produced amplicons in 3 accessions of the octoploid strawberry Fragaria x ananassa, whereas 24 (77%) amplified a product in 7 other diploid Fragaria species. We analysed the allelic variation among 15 F. vesca accessions using the 21 microsatellites reported here and 10 F. vesca microsatellites described previously. The level of polymorphism detected at these microsatellite loci was high; five loci were monomorphic. Only two microsatellites were required to unambiguously discriminate among the 15 F. vesca accessions. A preliminary survey of segregation in an F2 progeny indicates that 20 of the 26 polymorphic loci (77%) could be mapped.     

Genome-wide identification of intron fragment insertion mutations and their potential use as SCAR molecular markers in the soybean

Introns often have a high probability of mutation as a result of DNA insertions and deletions (indels). In this study, 503 introns with exon-derived insertions were identified using a comprehensive search of the soybean genome. Of the 375 pairs of PCR primer sets designed for the loci in question, 161 primer sets amplified length polymorphism among nine soybean varieties and were identified as soybean gene-intron-driven functional sequence characterized amplified region (SCAR) markers. These SCAR markers are distributed among all 20 of the soybean chromosomes, and they developed from numerous genes involved in various physiological and biochemical processes that influence important agronomic traits of the soybean. The development of these novel gene-driven functional SCAR markers was fast and cost effective, and their use will facilitate molecular-assisted breeding of the soybean.     

Eukaryotic DNA methylases and their use for in vitro methylation

DNA methylases from mouse and pea have been purified and characterized. Both are high molecular mass enzymes that show greater activity with hemimethylated than unmethylated substrate DNA. Both methylate cytosines in CpG preferentially, but not exclusively and show similar kinetics of methylation, which makes it difficult to saturate all possible sites on the DNA, but procedures are described that circumvent this problem.     

Limbal stem cells, a review of their identification and culture for clinical use

The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.     

Development of swine-specific DNA markers for biosensor-based halal authentication

The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.     

Plasmid DNA of Agrobacterium tumefaciens detected in a presumed habituated tobacco cell line

Summary Tabac anergié tissue has been used by many investigators as a habituated tobacco cell line. In this study, we have found sequences which are homologous to approximately 9 megadaltons of an Agrobacterium octopine-type plasmid in DNA isolated from Tabac anergié tissue. These T-DNA sequences were shown to be the same core DNA sequences found to be present in several other unorganized tumor lines which have been studied in our laboratory. The T-DNA was also shown to be integrated into plant DNA. Thus, the Tabac anergié tissue is a crown gall tumor induced by a strain of Agrobacterium tumefaciens containing an octopinetype plasmid. These results indicated that the plants regenerated from 40 clones of the Tabac anergié line by Sacristán and Melchers (1977) were actually reverted plants obtained from an unorganized crown gall tumor, kept in culture for more than 25 years. It is necessary to re-evaluate observations and conclusions which have been based upon Tabac anergié being a habituated tissue.     

Cultivar identification of Korean rice based on DNA markers for blast resistance

Cultivar identification of seven Korean domestic rice using DNA markers related to blast resistance was conducted. By PCR analyses using six markers, which we developed in a previous study, and one newly-developed marker for pib, it became possible to differentiate the seven cultivars from each other. This result should contribute not only to cultivar identification but also, to molecular breeding of blast resistance for Korean rice.