Identification of sugarcane hybrids is difficult when selections are based solely on morphological traits. Our objective was to combine morphological traits and molecular marker analysis to select F1 hybrids from two separate crosses between Djatiroto, a clone of Saccharum spontaneum, and elite sugarcane clones, LCP 85-384 (Cross 97-3144) and CP 62-258 (Cross 97-3146). The maternal inflorescences of Djatiroto were emasculated by submersion in a circulating 45°C hot-water tank for 10 min to minimize self-fertilization. Cross 97-3144 produced 4.7 g of seeds with 338 viable seeds per gram and Cross 97-3146 produced 2.4 g of seeds with 166 viable seeds per gram. After greenhouse germination, 96 progeny from each cross were evaluated in a field plot. Evaluations were conducted on the ratoon crops for stalk diameter (mm), juice Brix (percentage soluble solids), and a randomly amplified polymorphic DNA (RAPD) marker OPA-11-366 that was reproducibly amplified through PCR from the elite clones, but not the maternal S. spontaneum clone. Fifty progeny (52.1%) from Cross 97-3144 and 36 progeny (37.5%) from Cross 97-3146 inherited the RAPD marker. Five putative F1 progeny were selected from each cross, namely US 99-43, US 99-44, US 99-45, US 99-46 and US 99-47 from Cross 97-3144, and US 99-48, US 99-49, US 99-50, US 99-51 and US 99-52 from Cross 97-3146, based on their relatively larger stalk diameter, higher Brix and inheritance of the RAPD marker. The hybrid nature of these selected progeny was verified with sugarcane microsatellite markers. This is the first report of the development of Saccharum hybrids with the cytoplasm of S. spontaneum for breeding purpose through a combination of conventional and molecular breeding approaches. Availability of these F1 hybrids could enhance the genetic diversity of Saccharum germplasm and has enabled sugarcane geneticists and breeders to explore the possible contribution of S. spontaneum cytoplasm in the development of new sugarcane cultivars.

Proso millet occurs both as a crop and a weed in North America. In 1970, an olive-black seeded biotype called ‘wild proso millet’ was found as an aggressive weed in row crops in Minnesota and Wisconsin and has since spread over a large area. We used Random Amplified Polymorphic DNA (RAPD) to assess genetic relationships among biotypes, measure genetic variation within wild proso millet across its range, and detect hybridization between wild proso millet and crop biotypes of proso millet. We found 97 RAPD genotypes among 398 individuals: 69 wild proso millet genotypes, 26 crop and crop-like weed genotypes, and two hybrid genotypes. Five RAPD markers consistently differentiated wild proso millet from crop cultivars and crop-like weeds. About 10% of the genotypes had at least one marker of the other type, suggesting possible hybridization between wild proso millet and crop biotypes. Most genotypes occurred in only one or two of the over 100 populations tested. The most widespread wild proso millet genotype occurred in 12 populations distributed in North Dakota, Minnesota, Illinois, and Wisconsin. More genetic variation exists among populations of wild proso millet than expected for a plant that presumably experienced a severe genetic bottleneck only 20 generations ago. Hypermutation rates and crossing between wild proso millet and crop cultivars could not account for the degree of genetic variation found in wild proso millet. The pattern of genetic variation among wild proso millet populations suggests multiple introductions of wild proso millet to North America.

Genetic variation was assessed in a range of populations of wild mustard (Sinapis arvensis L.) using random amplified polymorphic DNA (RAPD) analysis. Sixty markers were used to assess the extent of genetic variation in wild mustard populations sampled throughout 12 different locations in the United Kingdom, including herbicide treated and untreated sites. In addition, selected sites were sampled over two consecutive seasons. Individual plant analysis was required, since a high degree of intra-population genetic variation was observed. The apparent extent of genetic variation in a population at one site increased when the results from two consecutive seasons were assessed. The range of genetic variation was as high in the herbicide-treated populations as in those which were untreated. Genetic diversity was maintained in populations of wild mustard where rotational cropping and herbicide use was practiced. These results confirm the utility of RAPD for the determination of genetic variation in outcrossing species where no prior genotypic knowledge is available.

The genetic diversity of 39 clones of common reed originating from different geographical areas of Iran were evaluated using morphological and RAPD analyses. High level of morphological variation was observed among clones. The 16 primers used in this study amplified 149 scorable RAPD loci among which 123 were polymorphic (83.1%). A dendrogram was prepared on the basis of a similarity matrix of RAPD data using the unweighted pair-group method with arithmetic averages (UPGMA) algorithm and separated the 39 clones into four groups, which mainly were in accordance with geographical origins. The results of the morphological comparison mostly corresponded with the results of RAPD analysis. It is possible that these variations among clones will affect successful management of common reed using chemical or the other methods of control.

Recent advances in molecular methods and statistical analyses provide weed scientists with powerful tools for examining the genetic structure of weedy plant populations. The value of these studies depends on effective sampling protocols; however, there is little consensus on how to sample plant populations for genetic diversity analyses. In this review, we draw on published literature that incorporates sampling theory and spatial statistics in population genetic analyses to identify key factors to consider when designing a sampling strategy. We discuss how sampling design is affected by research objectives, biology of the study species, population structure, marker choice, and the genetic parameters to be investigated, and we offer suggestions on defining sampling units and developing sampling protocols.

A set of universal and degenerate primers has been designed to clone (by polymerase chain reaction [PCR]) the conserved domains of the acetolactate synthase (ALS) gene where mutations confer resistance to ALS herbicides in plants. These primers were successful in cloning conserved domains of ALS in all monocotyledonous and dicotyledonous plants tested to date, as well as that of bacteria. Total genomic DNA was used as the source of target DNA because no introns were found in the sequences to be amplified. The design of the universal primers was performed after subtle modifications of the consensus degenerate hybrid oligonucleotide primers approach, which implies the synthesis of hybrid oligonucleotide primers containing fixed clamp 5′ and degenerate core 3′ sequences. Optimizations of PCR reactions were done according to a Taguchy approach described for the first time with degenerate oligonucleotides. This method optimizes a PCR reaction using four variables (deoxynucleoside triphosphate, DNA, primers, and Mg2+) under three different concentrations per variable using just nine reactions. The ALS herbicide-binding domains from many susceptible and resistant plants can be cloned and sequenced in a few hours by using only 100 mg of starting plant material, like one leaf or several small seedlings or seeds.

Technological advances in molecular biology have contributed substantially to our understanding of plant genetic diversity. Early studies of allozyme variation employing protein electrophoresis revealed that plant populations have high levels of genetic diversity, most of the variation at polymorphic loci is found within populations, and geographic range and breeding system explain the largest proportion of variation in genetic diversity. With the discovery of restriction endonucleases, the first DNA-based markers allowed the detection of variation in DNA sequences in plant population studies. More recently, techniques that utilize the polymerase chain reaction have allowed a more representative assessment of genetic variation in plants by screening multiple loci distributed throughout the genome. The analyses reveal sufficient polymorphism for the examination of fine-scale genetic differences among individuals. Information on plant genetic diversity is also emerging from studies of plant genome structure. Comparative genetic mapping studies of members of the Brassicaceae, Poaceae, and Solanaceae show that gene content is highly conserved between closely related species, although gene order on a chromosomal segment may differ between species. Comparative sequencing studies reveal higher degrees of diversity at the microstructural (less than 1 million base pairs) level than predicted at the genetic map level and suggest that genes are densely packed in gene-rich regions, rather than randomly distributed along chromosomes in species with large genomes. Sequencing of the entire genomes of rice and Arabidopsis thaliana will help identify genes controlling agronomically important traits, improve our understanding of genetic variation for fitness-related traits in wild plant populations including weed species, resolve evolutionary relationships among plant taxa, and potentially revolutionize current ideas on plant diversity and evolution.

Resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides in populations of eastern black nightshade from Ontario has been linked to an Ala205Val substitution in the AHAS enzyme. The aim of this study was to determine the mechanism of inheritance of AHAS inhibitor resistance and the genetic relationships among resistant (R) and susceptible (S) eastern black nightshade populations from Ontario. Homozygous R and S parental populations were crossed and the inheritance was analyzed in F1 (S × R), reciprocal F1 (R × S), F2, and backcross (S × F1) progenies after application of imazethapyr at 150 g ai ha−1. Compared to parental lines, the progenies were rated as R, intermediate (I), and S phenotypes. All the F1 progenies were of the I phenotype. The backcross progenies segregated in a 1:1 (S:I) ratio, and the F2 families segregated in a 1:2:1 (R:I:S) ratio. These results indicate that a single nuclear gene, with incomplete dominance, controls resistance to AHAS-inhibiting herbicides in R population of eastern black nightshade. Random amplified polymorphic DNA (RAPD) markers were screened among 25 R and S populations. The genetic relationship of R and S populations based on RAPD profiles generated from six RAPD primers indicated four groups of populations in which resistance seems to have arisen independently. However, based on similarity coefficients, resistance within three of the groups could have arisen by gene flow. Both similar local selection pressure and gene flow could explain the spread of the Ala205Val substitution in R populations of eastern black nightshade in Ontario.

In India 14 seagrass species can be found with monospecific genera (Enhalus, Thalassia and Syringodium), Cymodocea with two species and Halophila and Halodule represented by more than two taxonomically complex species. Considering this, the present study was made to understand the level and pattern of genetic variability among these species collected from Tamilnadu coast, India. Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the level of polymorphism existing between the species. Out of the 12 primers tested, 10 primers amplified 415 DNA fragments with an average of 41.5 fragments per primer. Of the total 415 amplified fragments only 123 (29.7%) were monomorphic and the remaining 292 (70.3%) were polymorphic for Indian seagrass species. Among the 10 primers used four are identified as the key primers capable of distinguishing all the Indian seagrasses with a high degree of polymorphism and bringing representative polymorphic alleles in all the tested seagrasses. From the present investigation, this study shows that the RAPD marker technique can be used not only as a tool to analyse genetic diversity but also to resolve the taxonomic uncertainties existing in the Indian seagrasses. The efficiency of these primers in bringing out the genetic polymorphism or homogeneity among different populations of the Halophila and Halodule complex still has to be tested before recommending these primers as an identification tool for Indian seagrasses.

Memecylon wayanadense Ratheesh, Sivu & Pradeep, a new species of Melastomataceae from the Wayanad forests of Kerala, India, is described and illustrated. The new species is allied to Memecylon angustifolium, M. rivulare and M. sivadasanii but differs in habit, leaf shape, sclereid type, inflorescence type and position, and the shape and size of the sepals and petals. An UPGMA analysis of 20 RAPD primers resulted in two major clusters with Memecylon sivadasanii in one cluster and M. rivulare, M. angustifolium and M. wayanadense in the second cluster. Memecylon wayanadense forms a subgroup within the second cluster.

Thrips are small insects (0.5–3.0 mm) with distinct habits and life histories characterized by haplodiploid sex determination. In general, low levels of genetic diversity have been reported in haplodiploid insects, although most reports focus on the order Hymenoptera. Therefore, we used RAPD markers to evaluate the structure and both inter- and intra-population genetic variability of Gynaikothrips uzeli (Thysanoptera: Phlaeothripidae). Six populations, three from Paraná state, southern Brazil, and three from Bahia, northeastern Brazil, were studied. Similarly to other haplodiploid insects, the genetic diversity of G. uzeli was reduced. This result is putatively related to the haplodiploid sex determination system, which yields little genetic variation, and to ecological traits of the studied species, such as the low dispersal abilities and life mode in leaf galls. All individuals were homogeneously clustered in their respective collection sites, forming two main groups in which populations from similar environments were more closely related. The analyzed populations were highly structured, and the genetic variation was higher among than within populations.

Knowledge of the genetic diversity of germplasm of breeding material is invaluable in crop improvement programmes. Frequently, qualitative and quantitative data are used separately to assess genetic diversity of crop genotypes. While assessing diversity based on qualitative and quantitative traits separately, there may occur a problem when the degree of correspondence between the clusters formed does not agree with each other. This study compares five different procedures of clustering based on the criterion of weighted average of observed proportion of misclassification in black gram genotypes using qualitative, quantitative traits and mixture data. The INDOMIX- and PRINQUAL-based clustering procedures, i.e. INDOMIX and PRINQUAL methods in conjunction with the k-means clustering procedure, show better performance compared with other clustering procedures, followed by clustering based on either quantitative or qualitative data alone. The use of the INDOMIX- and PRINQUAL-based procedures can help breeders in capturing the variation present in both qualitative and quantitative trait data simultaneously and solving the problem of ambiguity over the degree of correspondence between clustering based on either qualitative or quantitative traits alone.

Simple sequence repeats (SSR) and random amplification of polymorphic DNA (RAPD) molecular markers were used to assess the genetic diversity of 80 isolates of Phytophthora infestans in potato (Solanum tuberosum) from Fujian, Heilongjiang, Hebei and Inner Mongolia Provinces in China. Polymorphism was identified by 13 SSR primers and 14 RAPD primers in the isolates of P. infestans in potato. A total of 76 bands were amplified by SSRs, with the percentage of polymorphic bands (PPB) being 78.9% and the similarity coefficient ranging between 0.00 and 0.42. A total of 189 bands were amplified by RAPDs, with the percentage of polymorphic bands being 95.2% and the similarity coefficient ranging between 0.04 and 0.66. Analysis of genetic diversity showed that there exists higher genetic variation in the Fujian population in comparison to the populations of Heilongjiang, Hebei and Inner Mongolia. Nei's genetic identity analysis indicates that the genetic similarity between populations of Heilongjiang and Inner Mongolia is the highest and that between Fujian and Hebei is the lowest. A cluster analysis revealed that isolates from Fujian, in the south of China, are distantly related to those from Heilongjiang, Hebei and Inner Mongolia in the north, and the Fujian population is distributed among more groups than the other three, exhibiting a higher genetic diversity.

This research aimed to describe the genetic and phenotypic diversity of 74 Spanish Brachyspira hyodysenteriae field isolates, to establish epidemiological relationships between the isolates and to confirm the presence of tiamulin-resistant isolates in Spain. For these purposes, we performed biochemical tests in combination with diagnostic PCR analysis for the identification of Brachyspira spp. and for detection of the smpA/smpB gene. We also used antimicrobial susceptibility tests, random amplified polymorphic DNA (RAPD) and a new pulsed-field gel electrophoresis (PFGE) protocol. The combination of RAPD and PFGE allowed the study of epidemiological relationships. Both indole-negative and tiamulin-resistant isolates of B. hyodysenteriae are reported in Spain for the first time. The genetic analyses indicated a relationship between these Spanish isolates and indole-negative isolates previously obtained from Germany and Belgium.

Random amplified polymorphic DNA markers (RAPD) were used to visualize the genetic diversity within and between infrapopulations of Schistosoma mansoni recovered from the natural vertebrate host, Rattus rattus, trapped at an insular Guadeloupean focus. Phenotypes were characterized by the sex of the parasites and by 8 polymorphic markers generated by 3 primers. Among the 212 parasite individuals recovered from 10 infected rats, 78 genotypes were characterized. All the hosts naturally infected harboured multiple parasite genotypes with a maximum diversity of 28 genotypes/host. Phenotypic and genotypic diversity calculated by Shannon-Wiener's indices and Lynch and Milligan's estimators respectively is, on average, greater within than between hosts. Considering the very low snail infection rates observed in this focus and the rapid turnover of the vertebrate hosts, our results suggest that the high mobility of the vertebrate host and/or plurimiracidial snail infections could be factors responsible for parasite genetic diversity within hosts.

Eight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

The random amplified polymorphic DNA (RAPD) technique was successfully used to produce genetic fingerprints distinguishing between Trichinella spiralis and Trichinella britovi. The same patterns were obtained from purified and crude DNA preparations of pooled and single muscle larvae. RAPD fingerprinting was applied to muscle larvae preserved under different conditions and recovered from different hosts. Larvae recovered from fresh and frozen meat and stored at – 20 °C for a long time or under 70% ethyl alcohol at room temperature for 30 d gave good and reproducible results. Single larvae recovered from a naturally infected wild boar and from a human biopsy gave fingerprints congruent to those obtained from T. britovi reference strains. The results prove that RAPD analysis is a quick method to distinguish between the autochthonous Trichinella species of Central-Southern Europe in less than 1 d after the detection of the infection. If necessary, the biological material can be frozen or stored under 70% ethyl alcohol at room temperature and sent to laboratories able to perform the RAPD analysis. The RAPD technique requires no prior knowledge of the molecular biology of the organism to be investigated and therefore appears to be a promising tool in parasitology for the identification of sibling species.

Gloriosa superba L. is a highly valued indigenous medicinal plant. It is available throughout India with noticeable variations in morphology and colchicine content. In the present study, morphological, phytochemical, cytological and molecular data are employed to screen the polymorphism, if present, in five different populations of G. superba L. Based on gross morphological traits, the plants can be grouped into two clusters: Amtala (AM) and Baruipur (BR) plants belonging to one cluster, and Siliguri (SG), Darjeeling (DJ) and Sikkim (SK) plants belonging to the other. Colchicine content of the tubers ranged from 0.06% in AM plants to 0.37% in BR plants. Cytological analysis revealed that the SK plants showed 44 chromosomes, i.e. tetraploid status, whereas the other four were diploid. Assessment of the karyomorphology of the four diploid populations indicated a close similarity between AM and BR plants, whereas the SG plants exhibited slightly different karyomorphology and lacked sm-type chromosomes. Random Amplified Polymorphic DNA (RAPD) analysis revealed 76% polymorphism among the populations. Cluster analysis using RAPD resulted in three main cluster groups; BR and SG plants clustered together indicating that they are most closely related when compared with other populations.

A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance as well as to encourage investment and innovation in plant breeding and to ensure that the improvements in productivity and quality imparted by breeders are delivered to the farmer and, ultimately, to the consumer. Traditionally, morphological comparisons have formed the basis for genetic purity evaluations. However, replicated field observations are time-consuming, expensive and unreliable. Morphology cannot provide information on the purity of specific genetic attributes that relate to grain quality or to pest or herbicide resistance bred into varieties. Biochemical assays, including isozymes, can distinguish varieties within several species. Isozymes have been routinely used in checking seed-lot purity in maize (Zea mays L.) for the past 20 years. Newer DNA-based technologies such as restriction fragment length polymorphisms and more recently developed methods that use the polymerase chain reaction can allow even more discriminative and faster identification of varieties. However, none of the DNA methods have replaced biochemical methods for seed purity assays, other than in a relatively select group of crops with very high seed value, due to their high datapoint cost. It will require further miniaturization, automation and enhanced capabilities to process numerous samples simultaneously before newly developed methods supplant biochemical methods for routine usage in purity testing. New varieties that have major genes for herbicide or insect resistance incorporated within them require purity assays during product development and following seed production of the commercial variety. Immunological or DNA sequence assays can be developed and automated systems are required to process hundreds of thousands of individuals. Ultra-high, micro-array technologies and single-molecule detection systems are now under development. These technologies offer the promise that adequate distinction and high sample throughput will be combined. New methods may eclipse the capabilities of biochemical methodologies, thereby potentially raising genetic purity standards and enabling farmers and consumers better to utilize and benefit from increasingly productive varieties that are bred from a more diverse base of genetic resources.

Experiments were performed to determine if RAPD profiles developed using total DNA isolated from soybean seed could be affected by the physiological state or the quality of the seed. RAPD profiles were developed using template DNA isolated from a single seed lot of soybean (Glycine max L. cv. Kirby). High quality seeds were used to produce four populations varying in either quality or physiological state: untreated control seed ambient temperature and humidity storage for 12 months, accelerated aging at 41°C and 100% relative humidity for 48 h, and controlled hydration (seed priming). One hundred and eighty-eight primers were used to create separate RAPD profiles from total DNA isolated from each set of seed and from soybean leaf tissue. Sixteen polymorphisms from 14 primers were identified as a result of seed treatments. Six primers showed nine polymorphisms in RAPD profiles of ambient-stored seed DNA, while four and two primers produced polymorphisms in reactions using accelerated aged or primed-seed template DNA, respectively. Two primers showed a polymorphic fragment in vegetative DNA not observed in any of the seed DNA samples. Ten of the observed polymorphisms were due to the appearance of a DNA fragment in response to a specific seed treatment while six were the result of the treatment-induced loss of a DNA fragment. The six polymorphisms resulting from the loss of a major fragment were all due to ambient-temperature seed storage. Results were reproducibly obtained from multiple DNA isolations using three separate DNA isolation procedures involving either multiple seed or a single seed as the template source. Therefore, genetically identical seed can consistently display RAPD polymorphisms as a response to the environmental exposure.