Karyogenomics of species of the genus Linum L

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were identified and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Karyogenomics of species of the genus <Emphasis Type="Italic">Linum</Emphasis> L.

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were discovered and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.     

利用分子标记技术检测玉米杂种纯度研究

以12份玉米自交系及其组配的8个杂交种为材料,利用分子标记技术进行杂种纯度检测研究。有用RAPD标记方法,从300个随机引物中,筛选确定了8个杂交种种子纯度检测引物,制定了检测标准标记图谱。建立了海禾3纯度分析的SCAR标记,该SCAR标记与RAPD标记在种子纯度检测上结果完全一致。     

DNA fingerprinting of jute germplasm by RAPD

The genotype characteristic of cultivars was investigated, along with varieties of both of the jute species, Corchorus olitorius and Corchorus capsularis, in the germplasm collection at the Bangladesh Jute Research Institute (BJRI). DNA fingerprinting was generated for 9 different varieties and 12 accessions of jute cultivars by using random amplified polymorphic DNA (RAPD). A total of 29 arbitrary oligonucleotide primers were screened. Seven primers gave polymorphism within the varieties, and 6 primers detected polymorphism within the accessions that were tested. A dendrogram was engendered from these data, and this gave a distinct clustering of the cultivated species of jute. Therefore, we generated RAPD markers, which are species-specific. These primers can distinguish between C. olitorius and C. capsularis. From the dendrogram that we generated between the various members of these two species, we found the existing genetic classification that agrees with our molecular marking data. A different dendrogram showed that jute accessions could be clustered into three groups. These data will be invaluable in the conservation and utilization of the genetic pool in the germplasm collection.     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genome comparisons of three closely related flax species and their hybrids with chromosomal and molecular markers

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimum L., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienne Mill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1-3 microns), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. In L. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed that L. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifolium being closest to it.     

Locus-specific view of flax domestication history

Crop domestication has been inferred genetically from neutral markers and increasingly from specific domestication-associated loci. However, some crops are utilized for multiple purposes that may or may not be reflected in a single domestication-associated locus. One such example is cultivated flax (Linum usitatissimum L.), the earliest oil and fiber crop, for which domestication history remains poorly understood. Oil composition of cultivated flax and pale flax (L. bienne Mill.) indicates that the sad2 locus is a candidate domestication locus associated with increased unsaturated fatty acid production in cultivated flax. A phylogenetic analysis of the sad2 locus in 43 pale and 70 cultivated flax accessions established a complex domestication history for flax that has not been observed previously. The analysis supports an early, independent domestication of a primitive flax lineage, in which the loss of seed dispersal through capsular indehiscence was not established, but increased oil content was likely occurred. A subsequent flax domestication process occurred that probably involved multiple domestications and includes lineages that contain oil, fiber, and winter varieties. In agreement with previous studies, oil rather than fiber varieties occupy basal phylogenetic positions. The data support multiple paths of flax domestication for oil-associated traits before selection of the other domestication-associated traits of seed dispersal loss and fiber production. The sad2 locus is less revealing about the origin of winter tolerance. In this case, a single domestication-associated locus is informative about the history of domesticated forms with the associated trait while partially informative on forms less associated with the trait.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Identification,cloning and sequence analysis of a dwarf genome-specific RAPD marker in rubber tree [<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Muell.) Arg.]

High-yielding dwarf clones of Hevea brasiliensis are tolerant to wind damage and therefore useful for high-density planting. The identification of molecular markers for the dwarf character is very important for isolating true-to-type high-yielding dwarf hybrid lines in the early stage of plant breeding programs. We have identified a dwarf genome-specific random amplified polymorphic DNA (RAPD) marker in rubber tree. A total of 115 random oligonucleotide 10-mer primers were used to amplify genomic DNA by PCR, of which 19 primers produced clear and detectable bands. The primer OPB-12 generated a 1.4-kb DNA marker from both natural and controlled F₁ hybrid progenies (dwarf stature) derived from a cross between a dwarf parent and a normal cultivated clone as well as from the dwarf parent; it was absent in other parent (RRII 118). To validate this DNA marker, we analyzed 22 F₁ hybrids (13 with a dwarf stature and nine with a normal stature); the dwarf genome-specific 1.4-kb RAPD marker was present in all dwarf-stature hybrids and absent in all normal-stature hybrids. This DNA marker was cloned and characterized. DNA marker locus specificity was further confirmed by Southern blot hybridization. Our results indicate that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern based on the presence/absence of specific DNA markers than dye-stained gels or Southern blot analysis of RAPD blots using probes made from purified PCR products. Detection of RAPD markers in the hybrid progenies indicates that RAPD is a powerful tool for identifying inherited genome segments following different hybridization methods in perennial tree crops.     

Confirmation of cross-fertilization using molecular markers in ornamental passion flower hybrids

Several interspecific Passiflora hybrids are produced in the northern hemisphere for the ornamental plant market. In Brazil, production of passion flower hybrids is limited to the introgression of genes into the main cultivated species, yellow passion fruit, to be used as rootstocks. Confirmation of hybridization in the initial developmental stage is important for breeding perennial and sub-perennial plants, such as passion flowers, reducing time and costs in plant stock maintenance. In order to obtain F? hybrids with ornamental potential, four species of Passiflora (P. alata, P. gardneri, P. gibertii, and P. watsoniana) from the Active Germplasm Bank at UESC were hybridized. Flower buds, in pre-anthesis, of the genitors were previously protected, and the female buds were emasculated. To confirm hybridization, the genomic DNA of the genitor species and the supposed hybrids was extracted and RAPD primers were used to obtain molecular markers and select passion flower interspecific hybrids. Eight primers were used to confirm hybrids derived from P. gardneri with P. alata, P. watsoniana with P. alata, P. watsoniana with P. gardneri, and P. gardneri with P. gibertii; 75, 50, 45, and 46% of the informative bands, respectively, confirmed the hybrid nature of these plants. The RAPD technique was effective in the early identification of hybrids; this will be useful for development of hybrid Passiflora progeny.     

Genetic analysis of Indian mulberry varieties through molecular markers

India is one of the countries where sericulture is being practiced traditionally. Due to the higher economic return and the greater employment potential, attempts are being made to increase the productivity by developing high yielding mulberry varieties. At the present, Mysore local, Bomaypiasbari, Kanva-2, Bilidevalaya, Kajli, S1, BC(2)59, C776, RFS-175, S-36 and Victory-1 are being cultivated extensively in different parts of India for rearing the silkworm Bombyx mori L. Using 17 random amplified polymorphic DNA (RAPD) and 11 inter-simple sequence repeat (ISSR) primers the genetic relationships among these varieties were analyzed. The RAPD and ISSR primers revealed more than 75% polymorphism among the varieties. The genetic similarity estimated from RAPD markers varied from 0.645, between Kajli and Victory-1 to 0.887, between Kanva-2 and Bilidevalaya. Similarly, the genetic similarity estimated from the ISSR markers ranged from 0.600, between Kajli and Victory-1, to 0.873 between Kanva-2 and BC(2)59. The dendrogram constructed from these markers grouped the varieties into three major groups comprising the low yielding, medium yielding and high yielding. The low genetic similarity between the group of varieties originating from the eastern regions with that of the southern region encourages formation of extensive breeding programs between these groups as to transfer the high yield potential of the southern varieties to the low yielding but highly adaptive eastern varieties.     

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70?C100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

陕油8号种子纯度的RAPD鉴定研究

从杂交油菜“陕油8号”及其亲本中提取基因组DNA,用100个RAPD随机引物进行扩增,从中筛选出3个可将亲本和子代区分的引物BA208、BA1090、BA497。BA208产生亲本互补的特征带BA208-1050bp、BA2081250bp;BA1090产生母本特征带BA1090-700bp,BA497产生父本特征带BA497-870bp,上述谱带均在子代中出现。以BA208产生的特征谱带作为分子标记对杂交油菜种子纯度鉴定得到了一致的结果,并与大田纯度检测结果一致。BA497可将“陕油8号”与当地4个主栽品种有效区分。此外,还对双引物共同鉴定杂交种子纯度问题进行了初步探讨。     

Analysis of genetic diversity among garden- and field-pea genotypes of higher Indian Himalayas

Genetic diversity among 31 genotypes of field and garden pea including primitive cultivated forms and widely cultivated varieties of India was studied using 40 random decamer and 9 ISSR primers. A total of 274 amplicons were detected using both types of markers, which amplified 192 RAPD and 82 ISSR amplicons. Average number of bands amplified per primer was higher in case of ISSR (9.1) as compared to RAPDs (4.8). ISSR primers also exhibited higher average polymorphism (89.0%) and resolving power (4.50) than RAPDs (72.4%, 1.87, respectively). Genetic similarity estimates based on the pooled data of both types of markers using Jaccard??s coefficient ranged from 0.58 to 0.85 delineating considerable diversity among the pea genotypes studied. The 31 genotypes clustered in two major groups based on pooled data. Popular cultivars of garden and field pea of the region exhibited high similarities among themselves. However, primitive cultivated forms collected from the higher Indian Himalayas were diverse from the current varieties and hold potential in pea breeding programmes.     

Application of RAPD Analysis in the Study on the Origin of Chinese Cultivated Chrysanthemum

The RAPD ( random amplified polymorphic DNA) markers generated by 22 20-ruer primers, discriminated 7 wild Dendraathema species, 14 cultivars and 5 interspecific hybrids by banding profiles. Of 224 bands observed, 34 bands (15%) were polymorphic. Cluster analysis based on UPGMA method indicated that: of 7 wild species, D. indicum, D. vestitum and D. nankingense were closest to the cultivated groups while D. zawadskii was most distinct. Genetic distances between the former three species and cuhivars were less than 0.40 but those between D. zawadskii and cuhivars were more than 0.50. According to the RAPD analysis and previous research, the authors discussed the origin of Chinese cultivated chrysanthemum and proposed that the cuhivars of this genus may derive from D. indicum, D. vestitum and D. nankingense. The results have provided basic research data for study on the origin of Chinese cultivated chrysanthemum.     

The effect of DNA reamplification on RAPD-PCR results

The results of reamplification of flax DNA with random primers are represented. It was shown that eight of 17 decameric primers used in the experiments had an reamplification effect: the intensity of the bands increased; new bands and/or new polymorphic bands appeared in the gel sample. The results confirm the effectiveness and necessity of reamplification in case of uncertain or ambiguous interpretation of RAPD analysis making possible to select the highly-informative primers.