RAPD Molecular Markers Research Articles

Diversity within American cassava germ plasm based on RAPD markers

This work focuses on the genetic diversity of American cassava through RAPD molecular markers. The 126 genotypes studied were distributed on four geographical levels ranging from local to continental. Samples included ethnocultivars from the Santa Isabel community in the Brazilian Amazon, local cultivars collected in the State of São Paulo, native accessions from very diverse Brazilian regions, and representative accessions from the Centro Internacional de Agricultura Tropical (CIAT) core collection. Eighty-eight polymorphic bands were analyzed. Results revealed the weak genetic structure of the cassava analyzed. The pattern formed by the first two axes of the principal coordinates analysis (PCoA) revealed an overlapping of the São Paulo State genotype, the Brazilian group and the core collection accessions. The Santa Isabel ethnocultures formed a separate group. The weak genetic structure of cassava can be explained by the common practice of exchanging botanical material among small producers as well as by recombinations among genotypes. When the genotypes were analyzed using climatic data, the sample sites were found to be structured according to temperature and precipitation. RAPD markers proved very useful in the genetic diversity study, resulting in important implications for cassava germ plasm collections and genetic breeding.

Variation and Inheritance in Hemisexual Dogroses, Rosa Section Caninae

ABSTRACTDogroses, Rosa sect. Caninae (x=7, 2n=28, 35 or 42) grow wild in the Scandinavian countries. Hips from this section have a particular aroma and are used in Sweden for making a traditional dessert dish, rose hip soup. Commercial production has recently been initiated with field-grown selections from the Balsgård rose hip breeding program. The species are often used as rootstocks for ornamental roses and some of them could also be a potential source of resistance for e.g. blackspot. All species within the section have a unique meiosis with 7 bivalents regardless of ploidy level. Viable pollen cells contain 7 chromosomes and egg cells 21, 28 or 35 chromosomes. In a study of a pair of reciprocal crosses between R. dumalis X R. rubiginosa (both 2n=35), molecular RAPD markers showed that all 78 progeny plants inherited almost all of the seed-parent specific marker. In contrast, only half of the pollen-specific markers were transmitted to any of the progenies. Most of the pollen-specific markers were found in 75–90% of the progenies, but there was also one marker that was only found in 50% of the progenies and three markers that only appeared in 2–4 plants. The study also showed that the species were genetically very similar. Nine progeny plants have never received any of the pollen-specific markers and these plants were subjected to an extended study with additional molecular markers. Reproductive characters as pollen viability, amount and weight of seeds and fruit weight variables were studied on all the progeny plants. Pollen viability was approx 25% in the parent plants whereas the hybrid progenies had a pollen viablity of 0–10%. Of the nine progeny plants which lacked pollen-specific RAPD markers even in this extended study, seven had a pollen viability close to that found in their seed-parents. This suggests that facultative apomixis occurs in this section, but it is difficult to quantify its contribution to the already low genetic variability.

072 Backcross Breeding with RAPD Molecular Markers to Enhance Resistance to Common Bacterial Blight in Pinto Beans

Common bacterial blight (CBB) in common bean (Phaseolus vulgaris L.), caused by Xanthomonas campestris pv. phaseoli (Xcp), reduces bean yields and quality throughout the world. Pinto `Chase' is a high-yielding variety with moderate resistance to Xcp derived from great northern Nebraska #1 selection 27, whose resistance is derived from an unknown tepary (P. acutifolius) bean source. XAN-159 is a black mottled small seeded breeding line with different genes for high resistance to Xcp derived from a different tepary source (PI 319443). Our objective was to pyramid different genes for Xcp resistance from the donor parent XAN-159 into the rust-resistant recurrent parent Pinto `Chase' using the classical back-cross breeding method with confirmation of resistance using RAPD molecular markers. Resistance was confirmed in some BC2F2 generation plants. Seven RAPD markers and the V locus (flower color) previously identified were confirmed in the BC1 and BC2 populations. Smaller seed size, purple flower color, and black mottled seed coat color were coinherited with resistance to Xcp. However, a recombinant plant with enhanced CBB resistance and moderate-sized pinto seed was identified. Backcross breeding is being continued.

Morphological and genetic variation in Centrolepis paludicola and C. monogyna (Centrolepidaceae)

Centrolepis paludicola is a small cushion-forming herb which is listed as an endangered species. However, some taxonomists have questioned its species status and treat it as a subspecies of the common C. monogyna. Morphological measurements were taken on plants from 50 cushions collected from 31 sites throughout Tasmania, and included C. paludicola, C. monogyna and C. fascicularis as an outgroup to resolve this taxonomic issue. While C. fascicularis was distinct in all morphological analyses, the sympatric C. paludicola and C. monogyna could not be clearly distinguished on the basis of the reproductive and vegetative characters that are supposed to separate them. Centrolepis monogyna is henceforth interpreted as a morphologically variable species which includes C. paludicola. Once it was shown that these are best treated as one species, RAPD molecular markers were used to analyse genetic variation between populations. This analysis revealed a high level of polymorphism between cushions. As in the multivariate analysis of morphological data, C. monogyna and C. paludicola containing cushions were indistinguishable. However, there was a strong geographical structure to the genetic variation in the C. monogyna/C. paludicola group, consistent with a model in which gene flow between populations is limited and local differentiation has occurred.

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM.

A method to measure genetic distance between allogamous populations of alfalfa (Medicago sativa) using RAPD molecular markers

Alfalfa (Medicago sativa L.) is a forage legume of world-wide importance whose both allogamous and autotetraploid nature maximizes the genetic diversity within natural and cultivated populations. This genetic diversity makes difficult the discrimination between two related populations. We analyzed this genetic diversity by screening DNA from individual plants of eight cultivated and natural populations of M. sativa and M. falcata using the RAPD method. A high level of genetic variation was found within and between populations. Using five primers, 64 intense bands were scored as present or absent across all populations. Most of the loci were revealed to be highly polymorphic whereas very few population-specific polymorphisms were identified. From these observations, we adopted a method based on the Roger's genetic distance between populations using the observed frequency of bands to discriminate populations pairwise. Except for one case, the between-population distances were all significantly different from zero. We have also determined the minimal number of bands and individuals required to test for the significance of between-population distances.

Use of molecular marker techniques in seed testing by Brazilian seed companies

Seed market is becoming global and globalization is growing very fast. To compete favourably in this new global seed world, quality and cost are and will be certanly the key issues. High seed quality can only be obtained by a thorough control of the entire seed production process, step by step from planning to final delivery. That requires science, technology, expertise, experience, good management and certanly, the most important, an absolute and unconditional commitment with quality. Seed testing for quality assurance is one important step in the process of production of high quality seed. In the late years a considerable amount of research has been published, particularly on the use of some Polymerase Chain Reaction DNA based new technologies (RAPD, microsatelites, AFLP) for genetic purity determinations in seed testing. As far as we know, no Brazilian seed company is using, on regular basis, RAPD or other molecular marker techniques in the determination of genetic purity in seed testing. Most of these are using morphological or physiological traits expressed by seed, seedling or mature plant and/or electrophoresis of seed or seedling proteins/isoenzymes for that purpose. Main reasons for that are: DNA molecular marker techniques are relatively new; lack of specialized personnel to run DNA molecular marker assays on routine basis; higher cost/sample when compared to proteins/isoenzymes electrophoresis.

RAPD Marker Facilitated Analysis of Genetic Diversity among Capsicum Genetic Resources from the Asian Vegetable Research and Development Center

Significant effort has been made in the collection of Capsicum germplasm throughout the world for maintenance by genebanks. The largest Capsicum germplasm collection is held by the Asian Vegetable Research and Development Center (AVRDC), consisting of 6844 accessions and eight species. The paradox of any germplasm collection is that, as the number of accessions and the probability of preserving genetic variability increases, the ability of users to efficiently utilize this resource decreases. Genetic variation can be quantified using RAPD molecular marker allele frequency and allelic variation to understand the genetic structure and variation within and among populations. The comprehensive Capsicum collection held at the AVRDC provides an opportunity to sample a range of germplasm representative of the variability that exists in available Capsicum germplasm. Accessions were sampled from the AVRDC collection to represent the range of genetic variation available in Capsicum 1) based on cluster analysis using morphological traits among 1500 accessions and 2) based on pedigree information from the Capsicum breeding program. Our objectives include understanding the structure and magnitude of genetic diversity among these AVRDC accessions and comparing the genetic diversity within sub-populations of these accessions. RAPD fingerprints of these accessions were collected using markers dispersed over numerous linkage groups based on a genetic map we have constructed. RAPD band frequencies and RAPD band diversity were used to test differences among and within sub-populations. The understanding of the distribution of genetic variation among and within these sub-populations will be useful for prioritizing collection, conservation, and sampling of these genetic resources.

RAPD Molecular Markers for the Gene Controlling Seedling Lethality and Plant Crippling in Common Beans

The genes involved in F1 seedling abnormal development and lethality in inter-gene pool crosses have been designated as Dl1 (MesoAmerican=MA) and Dl2 (Andean=A) (Shii et al., 1980, J. Hered. 71:218–222). The different degrees of leaf crippling (C) in segregating populations of crosses was due to the interaction between the Dl1 or Dl2 loci, growing environment, and the lcr allele (Singh and Molina, 1996, J. Hered., In press). The objective was to identify RAPD markers linked to the genes for crippling (lcr) and seedling lethality (Dl) using the bulked segregation analysis procedure for F2 of MA × A crosses. Crosses were made between C lines, FB 10413-24-2, WA 7807-305, and TY 5578-220 and normal (N) parents and tester stocks for Dl1 and Dl2 genes. The F2 FB 10413-24-2 × Carioca segregated 13 N:3C. F3 families segregated 3N:1C. RAPD marker OPB-10 was linked to Lcr at 31.2 cM. F3 families segregated 1N:3C. RAPD marker OPO16 was linked to Dl1 at 27 cM. The F2 WA-7807-305 × Rio Tibagi segregated 3N:1C. RAPD marker OPS-03 was linked to Lcr at 32.6 cM.

RAPD molecular markers and genetic diversity among 40 cultivars of <i>Brassica napus</i> in China

RAPD molecular markers and genetic diversity among 40 cultivars of <i>Brassica napus</i> in China

Predicting quantitative variation within rice germplasm using molecular markers

Diverse Asian rice (Oryza sativa) germplasm has been used to identify associations between various quantitative traits and RAPD molecular markers using multiple regression analysis. This has allowed us to predict for other samples of germplasm their performance for traits such as culm length and number, days to flowering, grain width, and panicle and leaf length using only RAPD marker data. Such predictive capability is possible because of the availability of extensive diversity held in genebanks, and can be used in the future to facilitate the exploitation of that biodiversity. More specifically the methodology could facilitate crop improvement by rapid ideotype prediction. For the mapping and isolation of QTLs (genes controlling quantitative traits) the method would provide information to guide the selection of parental material for hybridization and markers expected to show linkage to QTLs. It may also be possible that these associations could lead the way towards marker-assisted selection during breeding programs. In the future, this demonstration of association between markers and easily measured traits could also be extended to the study of important adaptive traits, such as stress tolerance, found either within germplasm collections or in natural populations.

The taxonomic characterisation of annual Beta germplasm in a genetic resources collection using RAPD markers

Annual beets in the genus Beta section Beta represent an important genetic resource. Representative accessions of annual beets from a beet germplasm collection were analysed using RAPD to assess the patterns of variation and relationships among them. Using arbitrary primers, markers showing variation across accessions were identified. A dendrogram of similarity was produced using these molecular markers. All the accessions analysed were classified into three major groups corresponding to species or subspecies macrocarpa, adanensis and maritima. Macrocarpa was shown to be the most divergent group in this section. Using RAPD molecular markers, it was possible to ascribe an accession to one of three taxonomic groups and overcome much of the confusion encountered when morphological traits are used for identification. The group of maritima was found to be more polymorphic than either the group of macrocarpa or adanensis at both accession and subspecies levels.

RAPD MARKERS ASSOCIATED WITH SINGLE GENE TRAITS IN IPOMOEA TRIFIDA

Ipomoea trifida (2X = 30) is purported to be the wild Ipomoea species most closely related to the commercially grown Ipomoea batatas (sweetpotato, 6X = 90). The two species can be crossed with much difficulty, but seed occur rarely. Ipomoea trifida has been shown to possess some agronomically desirable traits that are missing in sweetpotato (e.g., sweetpotato-weevil resistance). Attempts to locate morphological markers in the diploid trifida that would serve as indicators of successful crosses with sweetpotato resulted in the identification of two traits controlled by single genes: nectary color and male sterility. Both traits require flowering to identify, and flowering is often difficult to induce in Ipomoea species. An analysis of I. trifida accessions using RAPD molecular markers was undertaken. Using a segregant population resulting from crossing a green nectary, fertile plant with a yellow nectary, male, sterile plant, RAPD analysis resulted in clear markers for both the nectary color trait and the male sterility trait. These traits now can be identified in the absence of flowering plants.

INTER- AND INTRA-SPECIFIC VARIATION AMONG FOUR IPOMOEA SPECIES AS DETECTED BY RAPD ANALYSIS

To understand the prospects of applying the RAPD technique to assay genetic diversity in Ipomoea, four species (I. batatas, I. trifida, I. triloba, and I. ×leucantha) were analyzed for RAPD molecular markers. Six accessions of each species were used. Significant RAPD polymorphisms were detected within each species. Of 20 primers used, nine produced clear scorable polymorphic bands. The number of polymorphic bands produced per primer ranged from two to nine. Pair-wide genetic distance was calculated based on “band sharing”. The SAS-CLUSTER procedure was used to build a hierarchical species dendrogram. The four species were clearly separated by the clustering, which agrees with their existing taxonomic relationship. This study shows that RAPD analysis can be a powerful tool for identifying duplicates of germplasm acessions and for assessing genetic diversity. The procedures are relatively inexpensive and easy to perform and could be valuable in preliminary assessment of field genebank collections to separate species and indicate duplications in collected material.