Individual polymorphism by RAPD markers in spring generation of the Colorado beetle Leptinotarsa decemlineata (Say)]

Genetic diversity of Colorado potato beetle in a natural population from Fastovskii rayon, Kiev oblast was estimated using RAPD. The level of polymorphism for RAPD markers was far higher in the spring generation than in the sample obtained from the same locality in summer of the preceding year. The frequency of individual RAPD markers was found to differ in males and females of potato beetle.     

Genetic Structure of Populations of the Colorado Potato Beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The genetic structure of a Colorado potato beetle population from Kiev oblast was examined by cluster analysis of individual RAPD patterns. The obtained clustering indicates that the population is structured. This may be explained by adaptation to pyrethroid insecticides used for controlling the population size of this pest. Microevolutionary factors affecting the genetic structure of local populations of Colorado potato beetle are discussed.     

Genetic structure of populations of the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

The genetic structure of a Colorado potato beetle population from Kiev oblast was examined by cluster analysis of individual RAPD patterns. The obtained clustering indicates that the population is structured. This may be explained by adaptation to pyrethroid insecticides used for controlling the population size of this pest. Micro-evolutionary factors affecting the genetic structure of local populations of Colorado potato beetle are discussed.     

Assessment of genetic polymorphism in Colorado potato beetle Leptinotarsa decemlineata 8Say) by RAPD markers

Four decanucleotide primers were chosen to detect polymorphism for randomly amplified polymorphic DNA (RAPD) in the populations of Colorado potato beetle. The variability of RAPD patterns was studied in three oblasts (Kiev, Moscow and Kurgan) that are geographically distant and considerably differ in ecological and climatic conditions affecting the development of this beetle: Kiev, Moscow, and Kurgan oblasts. A high level of intrapopulation polymorphism exceeding interpopulation differences was revealed. RAPD-marker subsets differing in the frequency and distribution throughout the studied populations were revealed.     

RAPD-based genetic linkage maps of Tribolium castaneum.

A genetic map of the red flour beetle (Tribolium castaneum) integrating molecular with morphological markers was constructed using a backcross population of 147 siblings. The map defines 10 linkage groups (LGs), presumably corresponding to the 10 chromosomes, and consists of 122 randomly amplified polymorphic DNA (RAPD) markers, six molecular markers representing identified genes, and five morphological markers. The total map length is 570 cM, giving an average marker resolution of 4.3 cM. The average physical distance per genetic distance was estimated at 350 kb/cM. A cluster of loci showing distorted segregation was detected on LG9. The process of converting RAPD markers to sequence-tagged site markers was initiated: 18 RAPD markers were cloned and sequenced, and single-strand conformational polymorphisms were identified for 4 of the 18. The map positions of all 4 coincided with those of the parent RAPD markers.     

Molecular markers associated with leptinine production are located on chromosome 1 in Solanum chacoense

Leptines of Solanum chacoense are effective natural deterrents against the Colorado potato beetle. Leptines are the acetylated forms of the glycoalkaloids solanine and chaconine and are supposed to be synthesised via hydroxylated derivatives, called leptinines. Inheritance of leptinine production was studied in crosses of closely related S. chacoense genotypes. The segregation data supported a single-gene model for the inheritance of leptinine production. In the segregating F₁ population of a S. chacoense cross, AFLP, RFLP and RAPD markers segregating with the leptinine production have been identified. The locus involved in leptinine synthesis was localised to the short arm of chromosome 1 of the potato where a major QTL for solanidine production, and markers with tight linkage to leptine production, have been mapped before. Our data further support the previous finding that the short arm of chromosome 1 is involved in steroid alkaloid synthesis in potato, and suggest that the genes involved in leptinine and leptine production are tightly linked in S. chacoense. Received: 27 June 2000 / Accepted: 4 August 2000     

Characterization and authentication of insect cell lines using RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to characterize 11 insect cell lines, including six from lepidoptera (five species), one from diptera and four from coleoptera (one species: Leptinotarsa decemlineata). Whatever the order and even when comparing two closely related species from the same genus (Spodoptera), the DNA fingerprints are very different from one species or from one primer to the other. On the other hand, two independently isolated cell lines from the lepidopteran Phthorimaea operculella produce nearly identical profiles with only minor differences. Finally, a statistical analysis based on Nei's similarity coefficient was performed on the fingerprints of four independent cell lines from the Colorado potato beetle, L. decemlineata. Each possesses a common recognizable pattern also found in field-collected insects, while also showing a series of polymorphic markers which allow one to distinguish each cell line from the three others. RAPD fingerprinting, together with the use of appropriate statistics, thus constitutes a highly specific method both for the authentication of the species from which a cell line was developed and for the individual characterization of each cell line from a given species.     

Mapping of resistance to the potato cyst nematode Globodera rostochiensis from the wild potato species Solanum vernei

A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F₁ population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.Abbreviations BSA bulked segregant analysis - RAPD random-amplified polymorphic DNA - RFLP restriction fragment length polymorphism - SCAR sequence-characterized amplified region     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Genetic variation in monoploids of diploid potatoes and detection of clone-specific random amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers have been used to study the genetic variation among androgenetic monoploids of diploid Solanum species. Cluster analysis of pairwise genetic distances was used to construct a genetic relationship among anther donor and anther-derived potato plants. The clustering based on Rogers' distances resembled classifications based on parental origins and hybrid combinations. Six of the 32 RAPD primers used resulted in the selective amplification of DNA fragments which were polymorphic between the two S. phureja parental clones, 1.22 and A95. It should be possible to construct a genetic linkage map, without making crosses, using monoploids derived from a single heterozygous diploid clone and RAPD markers.     

Analysis of the introgression of Solanum bulbocastanum DNA into potato breeding lines

Somatic hybrids have been obtained between potato and Solanum bulbocastanum PI 245310, a Mexican diploid (2n=2x=24) species. Through restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses it was found that the somatic hybrids contain each chromosome of the diploid parent and that the synteny of RFLP markers noted with tomato, potato and S. brevidens is largely maintained in S. bulbocastanum. RFLP analyses of BC1 progeny of two different hybrids indicated that a substantial number of markers were either lost or were heterozygous, in marked contrast with results previously noted with S. brevidens. A RAPD map for all 12 chromosomes of S. bulbocastanum was prepared and marker transmission was followed in three BC2 populations. Results with chromosomes 3, 8 and 10 from these populations are compared.     

几种基于PCR的分子标记在甘薯遗传多样性研究中的应用

遗传多样性是甘薯品种遗传改良的基础。由于分子标记具有数量极大、不受环境及基因表达与否的限制、多为共显性、不影响生物性状表现等优点,现已在甘薯遗传多样性研究中得到广泛应用。本文比较了RAPD、AFLP、SSR、ISSR和SRAP等几种基于PCR的分子标记方法,分别从遗传差异和亲缘聚类分析两方面,对它们在甘薯遗传多样性研究中的应用进行了综述。对比分析表明ISSR是一种共显性、成本较低、重复性好、多态性较高且非常有发展前途的分子标记,并已经被广泛应用到甘薯遗传多样性、物种亲缘关系、系统分类和辅助育种研究中。     

Segregation and Recombination of Solanum Brevidens Synteny Groups in Progeny of Somatic Hybrids with S. Tuberosum: Intragenomic Equals or Exceeds Intergenomic Recombination

The Solanum brevidens genome (2n = 2x = 24) was examined with randomly applied polymorphic DNA (RAPD) markers in a second backcross population derived from a S. brevidens + S. tuberosum somatic hybrid. RAPD markers cosegregated into 12 different S. brevidens synteny groups. Most synteny groups were nonrecombinant. However, nearly 40% of the S. brevidens synteny groups detected in this population were recombinant deletions that carried at least one, but not all, synteny group-specific RAPD markers. All S. brevidens synteny groups (except chromosome 5) were involved in recombination, and recombination occurred within most intervals between markers. About 20% of the recombinant S. brevidens synteny groups involved a single synteny group-specific marker. The inheritance of some single-marker representatives was followed in four BC(3) families. At least nine changes in S. brevidens synteny groups had occurred during backcrossing. Six of the nine changes involved translocation of S. brevidens markers between nonhomologous S. brevidens chromosomes, and three S. brevidens markers may have been introgressed into the potato genome.     

Effect of crop rotation distance on populations of Colorado potato beetle (Coleoptera: Chrysomelidae): development of areawide Colorado potato beetle pest management strategies

The Colorado potato beetle, Leptinotarsa decemlineata (Say), overwinters adjacent to field edges and infests nearby fields in the spring, primarily by walking. Crop rotation is known to be an effective cultural control against Colorado potato beetle populations limiting spring infestations. Spatial separation is an important consideration in optimizing the rotational effect because long-distance rotations have been shown to reduce Colorado potato beetle infestations. To determine the effect of long-distance rotations on Colorado potato beetle populations in commercial Wisconsin potato production, a geographic area of >18,200 ha (>45,000) acres in southern Portage County, Wisconsin, was selected as an experimental area for areawide pest management of the Colorado potato beetle. From 1997 to 1999, beetle populations at edges within each potato field in this region were determined by spring and fall field sampling. The rotational distance between current and previous potato fields was measured and analysis was run between the distance and Colorado potato beetle populations. Long-distance rotations of >400 m were an effective cultural control management strategy to limit adult beetle infestations in the spring. This strategy can be optimized when collaborating growers are able to maximize their rotational distances by coordinating their rotational schemes within large areawide, geographic locations. Deploying long-distance rotations within a geographic area over many years would limit Colorado potato beetle populations and could result in a significantly reduced Colorado potato beetle populations entering fields in the spring.     

Analysis of the applicability of molecular markers linked to the PVY extreme resistance gene Rysto, and the identification of new markers

In this study molecular markers linked to the Rysto gene, which originates from the wild potato species Solanum stoloniferum and confers extreme resistance against PVY, were identified and the applicability of recently published Rysto, markers was analyzed. Three RAPD markers covering a total distance of 8.60 cM were detected in this experiment. The closest of these markers was located 0.53 cM from the gene. From among the published markers only one had diagnostic value in the experimental plant material, and mapped 2.95 cM from the gene, on the side opposite the RAPD markers developed in the present study. All the markers analyzed were present in Solanum stoloniferum accessions, irrespective of their resistance, indicating that these sequences are linked to the locus and not exclusively to the dominant allele of the Rysto gene in the wild species. The inapplicability of several published markers indicates that the genetic background is decisive in this tetraploid and highly heterozygous species. This means that it may be necessary to develop markers from the breeding material itself, until the resistance gene is not cloned and cannot be used as a selection marker in marker-assisted selection.     

Resistance potential of colorado potato beetle (Coleoptera: Chrysomelidae) to novaluron

Novaluron (Rimon 10 EC), a novel insect growth regulator, could play an important role in future management programs for Colorado potato beetle, Leptinotarsa decemlineata (Say). Studies were conducted to determine the potential of Colorado potato beetle to develop resistance to novaluron before its widespread use in Colorado potato beetle management. Second instars of an imidacloprid-resistant Colorado potato beetle strain exhibited reduced susceptibility (2.5-fold) to novaluron. The toxicity of novaluron to this strain was synergized by S,S,S-tributyl phosphorotrithioate (DEF) but not by piperonyl butoxide (PBO), suggesting that esterase-based detoxification mechanisms were responsible for novaluron resistance. Bioassays with treated potato foliage found that a single low- or medium-rate novaluron application was highly persistent under field conditions, resulting in up to 85% mortality of second instars 5 wk after treatment. Thus, intense selection pressure for novaluron-resistant Colorado potato beetle may continue long after population densities have been reduced below an economic threshold level. In a national survey, the susceptibility of second instars to a novaluron diagnostic dose was determined for 27 different field populations collected from six Canadian provinces in summer 2003. Despite no previous exposure to novaluron, mortalities at the diagnostic dose ranged from 55 to 100%. Although novaluron has several characteristics that should delay resistance development in insect pests, these results highlight the need for judicious use of the compound in management of Colorado potato beetle.     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.