Cytoplasmic genetic diversity in bananas and plantains

Summary Concerns over yield declines in bananas and plantains due to the spread of Black Sigatoka disease in Musa have drawn attention to the collection of Musa germplasm and its use in conventional and biotechnological improvement programs. This report demonstrates the use of chloroplast DNA (cpDNA) restriction fragment length polymorphisms (RFLPs) for differentiating cytoplasms of various Musa clones. DNA was extracted from lyophilized leaf blade tissue and digested with either Eco R1, Hind III, Bam H1 or Pst I. Southern blots onto nylon membranes were probed with radioactively labeled heterologous orchid and lettuce cpDNA fragments. Among the 14 Musa clones examined, a single balbisiana and four acuminata-type cytoplasms were differentiated. The ability to distinguish between cytoplasms and to place plants within a cytoplasmic grouping demonstrates the usefulness of RFLP technology in evaluating diversity and determining the ancestry of Musa clones.     

The use of mitochondrial DNA polymorphisms to distinguish commercial cultivars of the broad bean, Vicia faba L.

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect mitochondrial DNA (mtDNA) variation among 9 commercial cultivars of Vicia faba L. The mitochondrial DNA was initially digested with 8 restriction endonucleases revealing complex banding patterns on ethidium bromide (EtBr) stained gels. However, no RFLPs were visualised from these complex profiles. Southern hybridisation using total digested mtDNA as a probe against mtDNA digested with the same restriction enzymes revealed a limited number of RFLPs which allowed the 9 cultivars to be consistently distinguished into two main cytoplasmic types. Southern hybridisation with 23 random mitochondrial clones covering 56 kb of the mitochondrial genome revealed considerable levels of polymorphisms. Of the 23 clones analysed, 12 detected at least 22 polymorphisms using 3 restriction enzymes among the cultivars analysed. These RFLPs allowed the 9 commercial cultivars analysed in this study to be distinguished into at least 6 separate groups. Most polymorphisms distinguished the cultivars into two main cytoplasmic groups.     

RFLP variation and genetic relationships in cultivated cucumber

Summary Two sets of cucumber (Cucumis sativus L.) germplasm were used to determine the potential use of restriction fragment length polymorphisms (RFLPs) for estimating genetic relationships. Sixteen accessions [15 domesticated variety sativus and one feral variety hardwickii (PI 183967)] of diverse origin were used to assess RFLP variation in cucumber, and to determine if genetic relationships based on RFLPs were similar to those obtained by isozyme analysis. Additionally, 35 commercial lines or cultivars were surveyed to determine genetic relationships among and within common cucumber types (narrow genetic base). The 16 accessions were surveyed with 440 low copy clones from two libraries (Pst I partial genomic and cDNA) using two restriction enzymes. Data from a subset of 104 random (mapped and unmapped) and a set of 30 mapped RFLPs were used to estimate genetic relationships among the 16 cultigens. Variability was low among RFLPs (33% of all probes) and putative alleles ( 2.2 polymorphic fragments/probe). RFLP variation between sativus lines and hardwickii (21±4%) was greater than among sativus lines (12±2%). RFLPs among the 16 accessions revealed genetic relationships which agree with those obtained using isozymes. Genetic relationships estimated using mapped and unmapped RFLPs were similar. The 35 elite lines were surveyed using a set of 40 RFLPs from 3 libraries (Pst I and EcoR I partial genomic and cDNA) to evaluate the discriminatory value of RFLPs among and between commercial cucumber types. The RFLP-derived genetic relationships among this germplasm were in agreement with predictions based on fruit type and pedigree information. Thus, RFLPs are a useful addition to the morphological characters and isozyme loci currently used for taxonomic classification and plant variety protection of cucumber.     

The potential of high-resolution BAC-FISH in banana breeding

The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively breeders must better understand the genetic differences of the potential crossing parents for introgression hybridization, but extensive genetic information is lacking. As an alternative to achieve information on genetic collinearity we make use of modern chromosome map technology known as high-resolution fluorescent in situ hybridization (FISH). This article presents the technical aspects and applications of such a technology in Musa species. The technique deals with BAC clone positioning on pachytene chromosomes of Calcutta 4 (Musa acuminata ssp. burmanicoides, A genome group, section Eumusa) and M. velutina (section Rodochlamys). Pollen mother cells digestion with pectolytic enzymes and maceration with acetic acid were optimized for making cell spread preparations appropriate for FISH. As an example of this approach we chose BAC clones that contain markers to known resistance genes and hybridize them for establishing their relative positions on the two species. Technical challenges for adapting existing protocols to the banana cells are presented. We also discuss how this technique can be instrumental for validating collinearity between potential crossing parents and how the method can be helpful in future mapping initiatives, and how this method allows identification of chromosomal rearrangements between related Musa species and cultivars.     

Relationships among ancestral species of sugarcane revealed with RFLP using single copy maize nuclear probes

Summary DNA restriction fragment length polymorphism (RFLP) analysis was performed on 50 wild and old cultivated sugarcane accessions. Ninety-four maize low copy nuclear DNA sequences of known chromosomal position were screened for hybridization to digested sugarcane genomic DNA blots. Seventy-five (80%) gave very strong hybridization signals and usually yielded many bands and detected profuse polymorphism. Twenty-nine probes and 36 probe/enzyme combinations were selected on the basis of the scorability of the banding profiles. A total of 1110 fragments were separately identified among the 50 genotypes. Multivariate analyses of the data allowed the separation of the three basic species, Saccharum spontaneum, S. robustum and S. officinarum, showed that S. spontaneum had structure which could be related to the geographic origin of the clones and supported current hypotheses on the origin of secondary species S. barberi and S. sinense. The use of more probes did not improve the resolution between the various species examined but identified a few key polymorphisms which were not accounted for by current phylogenetic hypotheses and can guide future analyses. RFLPs in sugarcane will be useful essentially for depicting the genomic constitution of modern varieties of interspecific origin.     

Characterisation of genetic variation between Vitis vinifera cultivars from central Chile using RAPD and Inter Simple Sequence Repeat markers

The origins and authenticity of many grape cultivars (Vitis vinifera) used for wine production around the world is unclear and the subject of some controversy. In this study, DNA fingerprints generated by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR) analyses were used to compare the four most widely planted V. vinifera cultivars in Chile (viz. Cabernet Sauvignon, Cabernet Franc, Merlot and Carmenere). Material obtained from France was used as an external reference. Both techniques were able to distinguish between the cultivars studied although the resolving power of ISSR profiles was higher than that of RAPDs, suggesting that the latter would be more suited for use on a wider range of cultivars. Surprisingly, however, variability was observed between clones of Merlot, the original Chilean clone and the representative clone from France. Furthermore, the high degree of divergence between the two sources (64% similarity) suggests that the French Merlot is not even a close relative of the stock in Chile. Interestingly, the latter was derived directly from the original French founder clones of the Merlot cultivar. No variation was found within the Chilean Merlot clone using either ISSR or RAPD analyses. These results indicate that French and Chilean vines grown for Merlot production represent different genotypes. The history of Merlot cultivation and the implications of these findings are explored. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of the degree of genetic variation in beet based on RFLP analysis and the taxonomy of Beta

Summary Clones derived from Beta vulgaris and Beta maritima were assayed for their ability to detect restriction fragment length polymorphisms (RFLPs) in different beet accessions. The clones able to detect polymorphism were used as genetic markers to assess the degree of genetic variation existing between and within different species of the genus Beta. The data support the current taxonomy of the Beta vulgaris section, while the great genetic similarity found between Beta webbiana and Beta procumbens indicates that they could belong to the same species.Enough variation was found between Beta vulgaris cultivars, allowing the isolation of a sufficient number of genetic markers for the construction of detailed genetic maps.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Genetic diversity among Paspalum spp. as determined by RFLPs

The genus Paspalum is characterized by over 400 species that are indigenous to a wide range of stressful habitats and marginal environments. Fifty-one accessions representing 29 Paspalum species were analyzed for DNA restriction fragment length polymorphisms (RFLPs). Fifteen random genomic probes were used in combination with restriction enzyme EcoRI to detect RFLPs, and data were analyzed phenetically. Hybridization with the 15 selected clones resulted in the detection of 261 RFLPs. Among the 261 restriction fragments scored, 204 (78.2%) were phenetically informative. Extensive RFLP variation was found between the species studied. Species affinities based on RFLP data were found to be in close agreement with previously determined relationships based on both morphological and cytological characteristics. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of image analysis for variety testing of mushroom

Summary A restriction enzyme map of the ribosomal RNA genes (rDNA) in Solanum tuberosum cultivars Golden Wonder and Desiree has been constructed. An heterologous probe pTa 71 containing the rDNA derived from wheat was used to detect and map the corresponding region in potato genomic DNA fragments. rDNA repeats of cultivars Desiree and Golden Wonder are similar with respect to their target sites for restriction enzymes, however, these cultivars have diverged with respect to the length of their intergenic spacer (IGS) sequences. IGS length variants may reflect the different breeding systems for Golden Wonder and Desiree. Furthermore, some of the 25S genes of both cultivars appear to have a 100–150bp insertion/deletion near their 3 end. The presence of RFLPs (restriction fragment length polymorphisms) in the IGS region could be used as a DNA fingerprint for potato cultivar identification; the ability to recognise IGS length variants may be of significance to potato phylogenetics and breeding.     

Inter simple sequence repeat analysis of genetic relationships in the genus Vigna

The utility of inter simple sequence repeat (ISSR) DNA polymorphisms to distinguish taxa within the genus Vignawas investigated. Nineteen primers, most containing either aGA or CA repeat, generated amplification products that differed among the taxa examined. The ISSR polymorphisms produced by 15 of these primers were very effective for distinguishing taxa at the species level or below. The Vigna unguiculataaccessions analyzed formed a cohesive group and appeared to be most closely related to V. triphylla and V. reticulata.In contrast, ISSR analysis was not able to clearly differentiate subgeneric divisions within Vigna. We attribute this loss of resolution at the subgeneric level to the high rate of evolution of the sequences we examined. Several probable instances of misclassification or hybrid origin of an accession were identified.     

Levels of host plant resistance to banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae), in African Musa germplasm

Forty-five Musa clones, including endemic and introduced cultivars plus hybrids, were evaluated for resistance against the banana weevil, Cosmopolites sordidus, in a field trial in Uganda. The predominant groups of staple crops, East African highland bananas (Musa spp. AAA) and plantains (Musaspp. AAB), as well as plantain-derived hybrids (AAB × AA), showed the highest levels of susceptibility to this pest. These were followed by dessert bananas (Musa spp. AAA), exotic bananas (Musa spp. ABB) and finally diploids of M. acuminata (AA). Hybrids of banana origin were highly resistant. Some East African highland cultivars, especially brewing types (e.g., Kabula, Bagandeseza, Ediirira), showed intermediate levels of resistance. Among the non-highland bananas, high levels of resistance were observed in Yangambi-Km5 (AAA), Cavendish (AAA), Gros Michel (AAA), Kayinja (ABB, Pisang Awak subgroup), Ndiizi (AB, Ney Poovan subgroup)and Kisubi (Ney Poovan subgroup). The highest resistance was observed in banana hybrids TMB2×7197-2, TMB2×8075-7 and the wild banana Calcutta-4 (AA). These were considered the best sources of resistance for a weevil resistance-breeding programme with the two hybrids commonly used as improved male parents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of DNA probes that reveal polymorphisms among closely related Phaseolus vulgaris lines

Summary Analyses of genetic diversity within populations could be of great benefit to plant genetic resources conservation. In order to identify genetic markers that are variable within populations, the genome of Phaseolus vulgaris was screened with several DNA sequences in order to identify hypervariable sequences. Polymorphisms were observed between Middle American and Andean cultivars using the protein III tandem repeat of the M13 phase and the 33.15 human minisatellite. Extensive differences were observed when the DNA of two divergent lines—BAT93 and Jalo EEP558, of Middle American and Andean origin, respectively—were digested with HinfI, TaqI, HaeIII and hybridized with the 33.15 human minisatellite. Similarly, numerous polymorphisms were observed when the M13 protein III tandem repeat region was hybridized with TaqI digests of these cultivars. Polymorphism was also detected among sister lines of two F₆ backcross materials involving Middle American and Andean lines when genomic DNA was digested with TaqI and hybridized with M13 tandem repeat region. In addition, polymorphism was observed among Porrillo cultivars that resulted from selection within a single landrace population. Whereas only one isozyme difference had been observed previously among the Porrillo cultivars, eleven restriction fragments detected by the M13 protein III tandem repeat sequence differentiated these cultivars. Ribosomal DNA also hybridized to several polymorphic bands on TaqI and EcoRI genomic Southern blots of the F₆ backcross material. Only one polymorphism was observed with EcoRI-digested genomic DNA of BAT93 and Jalo EEP558 was hybridized with microsatellite (GACA)₄. This probe might be useful in ascertaining relationships at the species and subspecies level, and as a marker in mapping studies. Our results show that both the human 33.15 minisatellite and M13 should be useful probes to detect within-population variability in common bean.     

Development and initial evaluation of diversity array technology for soybean and mungbean

Diversity Array Technology (DArT), a technique for quickly generating large numbers of molecular markers, was established for two legume crops, soybean (Glycine max) and mungbean (Vigna radiata). For each crop, two genomic complexity reduction methods, utilizing PstI/TaqI and PstI/BstNI restriction digests, were selected for DNA clonal library development and for the isolation in each case of 7,680 DArT clones from genomic representations of pooled DNA samples. While the PstI/BstNI method produced more polymorphic clones than PstI/TaqI for the soybean library, there was no significant difference between the two methods for the mungbean library. Polymorphism frequencies in mungbean were around twice those in soybean, reflecting greater diversity in the mungbean samples. Even so, there were still nearly 1,500 unique polymorphic clones identified for soybean. The DArT marker transferability from soybean to mungbean (13.6%) was nearly five times higher than that from mungbean to soybean (3.1%). The percentage of DArT marker transferability between mungbean and several other Vigna species ranged from 3.4 to 20.2%. The genetic similarities among 11 diverse Vigna spp. samples, evaluated using the DArT mungbean library, were consistent with published information on these taxa. The results indicated that for soybean and mungbean, the DArT technique is an effective tool for marker generation in terms of speed and the numbers of markers identified. The transferability of markers between soybean and mungbean indicated that DArT may be useful for comparative genomic studies, while the ability of the mungbean library to discriminate between related Vigna taxa suggested that DArT may also be useful for studies of genetic diversity.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and characterization of sources for photo‐ and thermo‐insensitivity in Vigna species

Cultivation of the same varieties of mungbean and blackgram across different seasons and locations is constrained by their photo‐ and thermo‐sensitive behaviour. Developing insensitive genotypes, which can fit well across all seasons, requires robust donors which would provide genes imparting this trait. This study was undertaken to identify such donors in the Vigna species. Forty‐eight accessions belonging to 13 Vigna species and eight released cultivars were evaluated under natural field conditions. Among these, two accessions, viz. V. umbellata (IC251442) and V. glabrescens (IC251372) were found photo‐ and thermo‐insensitive as these were able to flower and set pods at temperatures as high as 43.9°C and as low as 2.7°C. Pollen viability studies indicated viable pollen (>75% at 2.7°C and >85% at 41.9°C) and normal pollen tube growth at both the extremes of temperature. The identified V. glabrescens accession has long, constricted pods and dark green, mottled seeds while V. umbellata has smooth, curved pods and shining, oval, large seeds. Both these accessions can be utilized in developing photo–thermo insensitive genotypes in cultivated Vigna species.     

Molecular stability of the ribosomal RNA genes in Solanum tuberosum plants recovered from slow growth and cryopreservation

Summary Ribosomal gene (rDNA) probes have been used to assess genomic changes in plants of (1) S. tuberosum cv Desiree subjected to slow growth and (2) S. tuberosum cv Golden Wonder recovered from cryopreservation. Restriction fragment length polymorphisms (RFLPs) were detected in two out of 16 DNA samples extracted from plants derived from slow growth, control plants did not show RFLP differences. Plants recovered from cryopreserved shoot-tips of Golden Wonder were unchanged in their ribosomal gene RFLP profile compared to the untreated controls. The use of rDNA probes as tools to assess stability, and the possible detrimental effects of slow growth, somaclonal variation, cryoprotectants, and freezing injury are discussed.     

Evaluation of random amplified polymorphic DNA (RAPD) markers in Hevea brasiliensis

The applicability of random amplified polymorphic DNA (RAPD) markers in the cultivated rubber tree, Hevea, was evaluated using 43 decamer oligonucleotide primers in a set of 24 clones selected in different South-East Asian countries. A total of 220 0.35–3.5 kb DNA fragments were amplified, of which 111 were polymorphic. Of these, 80 fragments (RAPD markers) which were repeatable and clearly scorable across all genotypes were used to estimate genetic distances among the clones tested. The estimated genetic distances ranged from 0.05 (RRII 308 and PB 5/51) to 0.75 (RRIC 100 and SCATC 88–13). A mean genetic distance of 0.5 indicates a rather high genetic variability among the tested clones. As expected, because of the breeding history of Hevea, UPGMA cluster analysis and Principal Coordinate Analysis (PCoA) indicated the absence of a distinct geographical grouping. The possible application of RAPD markers for clone identification and also for analysis of genetic relationships among Hevea clones is discussed.