RAPD analysis of seed purity in a commercial hybrid cabbage (Brassica oleracea var. capitata) cultivar.

The use of random amplified polymorphic DNA (RAPD) markers for evaluating seed purity in a commercial F1-hybrid cabbage (Brassica oleracea var. capitata) cultivar is demonstrated. Genomic DNA isolated from single ungerminated seed was found to be suitable for RAPD analysis. DNA from F1-hybrid and its parental lines was subjected to RAPD screening with 36 random decamer arbitrary primers. A total of 241 scorable products were observed with 54 (22%) being polymorphic. The RAPD data showed that the parental lines of this commercial cabbage cultivar were not very closely related. Two primers were chosen for purity testing of the F1-hybrid seeds. The sib (inbred seed; seed from self-pollination of parental lines) contamination results obtained by RAPD analysis were comparable to the commonly used grow-out trial and isozyme analysis, hence showing that RAPD analysis can be used for seed purity testing of commercial hybrid cabbage seeds.     

Detection of DNA polymorphisms within the genusCattleya (Orchidaceae)

We have adapted methodology necessary for the detection of molecular polymorphisms in the orchid genusCattleya, namely, randomly amplified polymorphic DNA (RAPD). We report a high level of molecular variability among species; each of eight species examined exhibited a unique DNA fingerprint with nine out of ten arbitrary primers used in single-primer RAPD reactions. Among progeny of an intraspecificCattleya cross, 55 percent of major amplification products were found to segregate. Segregation of these markers facilitated the preliminary identification of several linkage intervals. The identification and mapping of DNA polymorphisms by the RAPD technique will facilitate the use of these taxa for the identification of species-specific and genus-specific traits, allow for the measurement of recombination and introgression in hybrid populations, and enable geneticists to address concordance (or lack thereof) in the processes of speciation, morphological evolution, and molecular change in a large and highly advanced plant family.     

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.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Identification of 27 <Emphasis Type="Italic">Porphyra</Emphasis> lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.     

Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

RAPD-based analysis of introgression of barley genetic material into the genome of alloplasmic wheat lines (Hordeum geniculatum All./ Triticum aestivum L.)

Genomes of three alloplasmic wheat lines obtained on the basis of barley--wheat hybrid Horderum geniculatum All. (2n = 28) x Triticum aestivum L. (2n = 42)(Pyrotrix 28) were examined using random amplified polymorphic DNA (RAPD) analysis. Line L-29 was obtained after first backcross of the initial hybrid with the wheat variety Pyrotrix 28 and ten subsequent self-pollinating generations. This line was represented by euploid plants with typical to the common wheat chromosome number (2n = 42), as well as by aneuploids, which contained an additional telocentric chromosome in the main karyotype (2n = 42 + t). Lines L-26 and L-27 were obtained by two backcrosses of one BC1 plant with the wheat variety Novosibirskaya 67 and one subsequent self-polination of one BC3 plant. Chromosome number in all these plants corresponded to 2n = 40 + 4t. RAPD analysis was carried out using seven primers, which were previously proved to be effective for identification of the barley genome fragments within hybrid genomes of alloplasmic lines. The presence of barley genome fragments in line L-29 was revealed by use of five primers, while in lines L-26 and L-27 these fragments were detected by use of one primer. The significant difference in the number of barley RAPD fragments in the genomes of alloplasmic lines obtained at different backcrossing stages suggests more intense displacement of barley genome during backcrossing compared to self-pollination in BC1 plants.     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

红莲型杂交稻(红莲2号)及其骨干亲本的RAPD分析与鉴定

利用RAPD技术,从248个随机寡核苷酸引物（10－mer）中筛出18个引物对红莲型杂交稻组合红莲2号及其亲本（T－07A、T－07B、YD6－05）,另6个红莲型胞质不育系的骨干恢复和汕优63及其亲本共14份水稻材料进行分析。共检测到173个多态性标记。聚类分析结果表明：不育系与保持系间因核背景相似,遗传差异很小;杂种（F1）的基因型更倾向于恢复系;恢复系与保持系间遗传距离的相对较大,但各恢复系之间的遗传距离较小。利用这些标记能有效地地区交组合中不育系,保持系、恢复系和杂种（F1）。     

Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.     

RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase,chelex 100, and gel purification of genomic DNA

We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.     

Identification and analysis of genetic variation among rose cultivars using random amplified polymorphic DNA

Identified germplasm is an important component for efficient and effective management of plant genetic resources. Traditionally, cultivars or species identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and analysis of genetic variation within 34 rose cultivars through random amplified polymorphic DNA (RAPD) markers. Analysis was made by using twenty five decamer primers. Out of twenty five, ten primers were selected and used for identification and analysis of genetic relationships among 34 rose cultivars. A total of 162 distinct DNA fragments ranging from 0.1 to 3.4 kb was amplified by using 10 selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 34 rose cultivars form 9 clusters. The first cluster consists of eight hybrid cultivars, three clusters having five cultivars each, one cluster having four cultivars, two clusters having three cultivars each and two clusters having one cultivar each. The genetic distance was very close within the cultivars. Thus, these RAPD markers have the potential for identification of clusters and characterization of genetic variation within the cultivars. This is also helpful in rose breeding programs and provides a major input into conservation biology.     

Comparison of essential oils and genetic relationship of Origanum × intercedens to its parental taxa in the island of Crete

Plants of the taxa Origanum onites and Origanum vulgare were allowed to cross-pollinate under natural open field conditions and to produce a mixed population of putative hybrid Origanum × intercedens and parent genotypes. Randomly collected plants were classified as putative hybrids or parent genotypes by inspection of their inflorescence. They were then subjected to analysis of their essential oil composition and were fingerprinted by randomly amplified polymorphic DNA (RAPD) fragments. DNA primers identifying the genotype as well as showing the distance of a particular putative hybrid plant from the parent genotypes were found. Alone or in combination with the essential oil composition they can be used as reliable tools for the genetic identification of the two parental taxa and the putative hybrid plants in natural populations.     

Biochemical and molecular markers for investigating the mode of reproduction in the facultative apomict Poa pratensis L.

Isozymes and random amplified polymorphic DNA (RAPD) markers were used for precocious identification of non-maternal plants in progenies of the facultative apomict Poa pratensis. Four progenies obtained from controlled crosses that showed different degrees of apomixis on isozyme analysis of phospho-gluco-isomerases, esterases and peroxidases were chosen for RAPD analysis to generate genomic fingerprints using species-specific primers. At an advanced vegetative stage, a morphological analysis was also performed and characteristics related to growth habit and leaf morphology were observed and recorded. On the basis of the isozyme and RAPD electrophoretic pattern and the morphological appearance, each plant was classified as maternal or aberrant. All three classes of genetic markers employed were able to identify plants that exhibited aberrant traits in the four progenies. Overall, the results of RAPD analysis supported those of isozyme and morphology studies. However, in each progeny, some plants which both isozyme and morphological analyses distinguished as of maternal origin were aberrant according to RAPD analysis. Therefore, the RAPD method proved the most precise screening technique. The greater cost of the molecular approach was offset by its higher accuracy. The use of either three isozyme systems or six primers for PCR amplification seems to be sufficient for reliable estimation of the degree of apomixis. Histological analyses were carried out and the aposporic development of the plant material studied.     

Cytological and molecular characterization of a novel monogenic dominant GMS in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5′-CTATGCCGAC-3′) gave a repeatable 1500-bp DNA polymorphic segment S243₁₅₀₀ between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243₁₅₀₀ is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.     

DNA divergence as a criterion of a sweet pepper (Capsicum annuum L.) selection for heterosis

The identification of perspective parental lines for the creation of high-yield hybrids is the most labor-consuming stage of selection, because it needs extensive trials of combining ability. Based on evaluation of the genetic divergence of the parental lines, the prediction accuracy of F₁ hybrids performance was investigated. The value of the divergence was calculated using biometric and molecular methods, such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD). Based on estimates of divergence, 10 lines were chosen for cyclic cross (scheme I) and testcross (scheme II). In most crosses, the F₁ hybrids were significantly superior to the parents in the main economically valuable traits. The level of heterosis was higher among hybrids of scheme I. Analysis of the relationship between parental divergence and F₁ performance showed that the hybrid productivity of scheme I was predetermined by ISSR divergence in 86%, and productivity was caused by RAPD divergence in 69%, whereas the F₁ yield of scheme II was not related to the value of genetic distances. Since the values of DNA divergence were closely associated both with midparent level and F₁ performance, we assumed that part of the polymorphic DNA fragments of the parental lines of scheme I is related to heterotic loci (HTL), which may be considered potential key markers for the heterotic selection of the sweet pepper.     

Interspecific and temporal variation in herbivore responses to hybrid willows

We studied herbivory of two species of willows (Salix sericea and S. eriocephala) and their interspecific hybrids to test alternative hypotheses concerning the effects of hybridization on plant resistance. Individually marked plants were identified using morphological traits in the field and random amplified polymorphic DNA (RAPD) band analysis was used to verify the genetic status of many parental and hybrid plants. The desities of 12 herbivore species on plants in the field were compared between two parents and their F₂-type hybrids. We found about equal support for the additive, dominance, and hybrid susceptibility hypotheses over 4 years. In one year, one species supported the hybrid resistance hypothesis. Guild membership was not a good predictor of similar responses of species to hybrid versus parental plants. There were marked differences in support for particular hypotheses among years for four herbivore species. This study demonstrates the diversity of responses of phytophages in response to interspecific hybridization, and indicates that year-to-year variation in relative resistance of hybrid plants can be important.     

Use of random amplified polymorphic DNA (RAPD) markers in the discrimination and verification of genotypes in Eucalyptus

We carried out four separate studies using random amplified polymorphic DNA (RAPD) markers to analyse samples of Eucalyptus supplied by several different organisations. The objective was to examine the reproducibility of the RAPD technique and its ability to discriminate between individual genotypes for verification of clonal identities. We found that RAPD profiles that are unique to a genotype can be generated reliably and simply and that even closely related genotypes can be distinguished. In addition, in each of the four studies, we detected cases where the plant material studied had been mis-sampled or mis-labelled (i.e. the RAPD profiles were not consistent with the identification numbers): (1) ramets of a Eucalyptus grandis clone were found to be derived from 2 different clones; (2) ramets labelled as 2 different Eucalyptus hybrid clones were found to be the same clone, owing to a mis-planted clonal hedge; (3) samples supplied as a single progeny of a controlled E. nitens cross were derived from two crosses involving different pairs of parents; (4) mis-labelling was detected for ramets of 4 of a set of 10 clones of E. grandis and E. camaldulensis. For three of the four studies, the detection of genotype mis-identifications was unexpected, suggesting that labelling or sampling errors during the handling of plant material are a frequent occurrence, with potentially serious economic consequences.     

Assessment of random amplified polymorphic DNA (RAPD) for determining the origin ofYoungia koidzumiana Kitamura (compositae)

We determined the parental species ofYoungia koidzumiana (a natural interspecific hybrid) using PCR and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. These markers, generated by three primers, were sufficient to distinguishYoungia sonchifolia, Youngia denticulata, Youngia chelidoniifolia, andY. koidzumiana. The electrophoresis profiles of the amplified products from each of the four species were then compared. Three primers produced a total of 42 scorable markers; nine were specific markers forY. denticulata andY. chelidoni-ifolia. The length of the amplified DNA fragments ranged from 370 to 2500 b p. The three primers revealed polymorphic bands, which were indicators of the parental species ofY. koidzumiana. These bands showed a combination of specific profiles forY. denticulata andY. chelidoniifolia. Our results also were comparable to the data obtained for flowering times, floret numbers, and chromosome numbers of the four species. Therefore, we suggest thatY. koidzumiana is a hybrid betweenY. denticulata andY. chelidoniifolia}, and that RAPD markers are well suited for assessing the origins of plant species.