Nuclear RFLP between pepper cultivars (Capsicum annuum L.)

Summary Forty one nuclear probes, distributed across the different linkage groups of the previously published map, were used to examine restrition fragment length polymorphism between cultivated peppers. Total DNA from thirteen accessions of Capsicum annuum var. annuum and one accession of C. baccatum var. pendulum was separately cut with ten restriction enzymes. The analyses were restricted to only one enzyme per probe to reduce the polymorphism redundancy. Nei & Li's genetic distances between accessions were calculated from the 141 resultant nuclear DNA restriction fragments. The genetic variation was larger between C. annuum and C. baccatum than between C. annuum cultivars. Large fruited related accessions closely clustered together. Distances between the small fruited cultivars were larger than within the bell pepper group. A correspondence analysis performed on differences between the global RFLP patterns of each accessions of C. annuum revealed the particular genomic structure of four small fruited cultivars: Criollo de Morelos 334, H3, Perennial and Doux Long des Landes. The percentage of probes revealing at least one RFLP with at least one enzyme ranged from zero to fifty percent for all the pairwise comparisons of C. annuum accessions. 82 presumptive loci were detected with a mean number of 1.46 alleles per locus within C. annuum and 1.83 within all the accessions. This result indicates that molecular markers will be more usable in intraspecific study of C. annuum than isozyme markers.     

Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Bermudagrass cultivar identification by use of isoenzyme electrophoretic patterns

Summary Isoenzyme analysis has been demonstrated as an effective tool for definitive identification of plant cultivars, but it has not been applied to pasture bermudagrass (Cynodon spp.) cultivars in the USA. Polyacrylamide gel electrophoresis (PAGE) was used to study five isoenzyme systems in mitochondrial, microsomal, and soluble cell fractions of actively growing leaves, stems, and roots of seven vegetatively-propagated pasture bermudagrass (Cynodon spp.) cultivars used in the southern half of the USA. Peroxidase, esterase, and, with one exception, acid phosphatase successfully differentiated between the cultivars in all leaf and stem cell fractions. Fewer cultivar differences were found for amino- and endo-peptidases. Only peroxidase and acid phosphatase were resolved from root cell fractions; and only the microsomal fraction differentiated between all cultivars. Within plant parts, cultivars were distinguishable on the basis of peptidase banding in some cellular fractions, but not in others. Plant part and subcellular fraction-specific isoenzyme variations suggest the existence of multiple molecular forms of various enzymes within the same plant.Journal Article 5746 of the Okla. Agric. Exp. Stn.     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. 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.     

Natural variation in fruit abscission-related traits in apple (Malus)

Abscission or retention of ripening fruit is a major component of seed dispersal strategies and also has important implications for horticultural production. Abscission-related traits have generally not been targeted in breeding efforts, and their genetic bases remain mostly unknown. We evaluated 144 Malus accessions representing wild species, domestic cultivars, and hybrids for abscission-related traits. We found that seasonal timing of fruit abscission in wild species and hybrids showed a broad distribution similar to that seen for domestic cultivars, and that internal ethylene concentration at the time of abscission varied by over three orders of magnitude. Wild species, domestic cultivars, and hybrids all included representatives that showed abscission of fruit prior to substantial production of ethylene, as well as accessions that retained fruit for a significant period of time following ethylene production. For all accessions that retained fruit, fruit removal resulted in abscission of the pedicel, and exogenous ethylene promoted abscission, suggesting that the abscission zone was functional. Our results suggest important roles for mechanisms independent of fruit ethylene production in abscission.     

Selection of wild Cicer accessions for the generation of mapping populations segregating for resistance to ascochyta blight

In order to determine genetically diverse parents for the generation of mapping populations segregating for resistance to ascochyta blight in wild Cicer species, the genetic diversity between a selection of resistant and susceptible accessions was assessed using molecular markers. Twenty Cicer accessions — comprising eight C. reticulatum accessions, six C. echinospermum accessions, five C. bijugum accessions, and one C. arietinum accession — were compared using a combination of seven RAPD primers and seven ISSR primers. A total of 231 polymorphic bands were scored and used to determine the genetic distances between accessions using Jaccard similarity coefficients. The most genetically diverse parents for the generation of intraspecific and interspecific populations segregating for resistance to ascochyta blight are reported. This revised version was published online in July 2006 with corrections to the Cover Date.     

Geographical differentiation of Asian taro, Colacasia esculenta (L.) Schott, detected by RAPD and isozyme analyses

Geographical differentiation and phylogenetic relationships of Asian taros, Colocasia esculenta (L.) Schott, and related species were analyzed by random amplified polymorphic DNA (RAPD) and isozymes of 13 enzyme systems with special interest in the accessions from the Yunnan area of China, which supposedly has served the secondary center of taro diversification and dispersal into the temperate Far East Asia. The RAPD analysis was found to be better suited for detecting genetic differences within taros and among its related species. However, both RAPD and isozyme analyses estimated quite similar genetic relationships within taros and between related species. Genetic differentiation was evident in the taro accessions of Nepal, Yunnan and other Asian areas; but, phylogenetic relationships between the differentiated taro groups were not clearly determined. When taro cultivation was introduced to a new area, only a small fraction of genetic variability in heterogeneous taro populations was transferred possibly causing random differentiation among locally adapted taro populations. Some of the Yunnan and Japanese accessions were found to be direct descendants of the common triploid population. Further analysis on taros from the eastern China and Korea is necessary to clarify the Yunnan's role in taro diversification and dispersal. The significant local differentiation in Asian taros was clearly demonstrated by RAPD and isozyme analyses in this study, and the results of this study will serve as a base to establish evolutionary and genetic relationships among Asian taros. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of DNA amplification fingerprinting techniques to study genetic relationships of white lupin germplasm

DNA amplification fingerprinting (DAF) techniques were used to evaluate genetic relationships among 24 randomly selected white lupin (Lupinus albus L.) accessions originating from four endemic regions. Parameters affecting DAF were optimized to obtain high resolution and reproducible banding patterns. DNA amplification was tolerant to a wide range of both template and primer concentrations but sensitive to Mg²⁺ concentration, and annealing temperature and time. Pure template DNA was found to be essential for obtaining reproducible banding patterns. Primer annealing occurred at multiple sites, permitting detection of polymorphism using only a single primer. Of 56 octamer primers tested, 22 yielded highly polymorphic DAF bands that could be used to distinguish closely related accessions. Genetic similarity coefficients were calculated and a dendrogram, using the unweighted pair group method with arithmetic average (UPGMA), was constructed. A high level of genome diversity was found among the accessions, but several from the Middle East and West Africa tended to cluster together. The results support the future use of DAF markers for the characterization and identification of white lupin germplasm.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

DNA fingerprinting in soybean (Glycine max (L.) Merrill) with oligonucleotide probes for simple repetitive sequences

Summary Soybean DNA fingerprints were analyzed by digoxigenin-labeled oligonucleotide probes complementary to simple repetitive sequences. The clearest and most polymorphic patterns were obtained with (AAT)₆ as a probe, with which all 47 soybean cultivars tested could be distinguished. However, DNA fingerprints of individuals within cultivars showed the same pattern. Using (CT)₈, (GAA)₅ or (AAGG)₄ as probes, clear polymorphic patterns among cultivars and accessions in the subgenus Soja (Glycine max and Glycine soja) were not observed, while quite different patterns were found in accessions in the subgenus Glycine. The results suggest that G. max and G. soja are closer in their genome structure. DNA fingerprints of reciprocal crosses between cultivars and accessions in the subgenus Soja were similar, and contained bands of both parents. In an F₂ population from these crosses, such bands segregated in a Mendelian fashion.     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity of wild coffee (Coffea arabica L.) using molecular markers

Genetic diversity was studied using RAPD markers among119 coffee (Coffea arabica L.) individuals representing 88 accessions derived from spontaneous and subspontaneous trees in Ethiopia, the primary centre of species diversity, six cultivars grown locally in Ethiopia, and two accessions derived from the genetic populations Typica and Bourbon, spread in the 18^th century, which gave rise to the most currently grown cultivars. Twenty-nine polymorphic fragments were used to calculate a similarity index and construct dendrograms. The Ethiopian material was separated from the Typica- and Bourbon-derived accessions and classified in four groups: one with most of the collected material from southwestern Ethiopia and three from southern and southeastern Ethiopia. Almost all detected diversity was found in the southwestern group while the southern and southeastern groups presented only 59% of identified markers. The genetic distances were low between the southwestern group and the southern and southeastern groups, and between the southwestern group and the Typica- and Bourbon-derived accessions. The cultivated coffee derived from the genetic populations Typica and Bourbon appeared little differentiated from wild coffee growing in the southwest. The results supported the hypothesis that southwestern Ethiopian coffee trees could have been introduced recently in the south and southeast. A separate analysis of the 80accessions classified in the southwestern group allowed identifying particular spontaneous- and subspontaneous-derived accessions and redundancies in the collected material from southwestern Ethiopia. RAPD markers did not detect any within-collection polymorphism except for two trees that were identified as off-types in the CATIE field genebank. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of genetic variation in Lachenalia bulbifera (Hyacinthaceae) using RAPDs

RAPD (randomly amplified polymorphic DNA) analyses were carried out on 21 accessions of Lachenalia bulbifera (Cyrillo) Engl. Five pre-selected primers produced an average of 88% polymorphisms. Fifteen of the 21 accessions could be identified using the five primers. In a pairwise comparison genetic distance values ranging from 0.11 to 1.08 were obtained. These values reveal a high amount of variation within the species. The genetic distance values within the tetraploid and hexaploid groups on the south coast were low, but values were high between the groups on the south coast and those on the west coast. A dendogram was constructed from the RAPD banding profiles, using UPGM cluster analysis. The dendogram clusters certain accessions together. These clusters are supported by their geographical locality and chromosome data. The hexaploid group, tetraploid group and octoploid group on the south coast are respectively clustered together. It is concluded that RAPDs can be used to assess the genetic variation at an intra-specific level in Lachenalia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic instability of bermudagrass (Cynodon) cultivars 'Tifgreen' and 'Tifdwarf' detected by DAF and ASAP analysis of accessions and off-types

DNA amplification fingerprinting (DAF) and arbitrary signatures from amplification profiles (ASAP) were used to evaluate the genetic stability of two important bermudagrass (Cynodon) cultivars, the interspecific cross Tifgreen and its somatic mutant Tifdwarf, and study genetic diversity and origin of derived bermudagrass off-types that exhibit patches of contrasting morphology and performance. Mini-hairpin primers produced complex and reproducible DAF and ASAP profiles with high levels of polymorphic DNA, and established genetic relationships between 11 Tifgreen and 8 Tifdwarf turf plot accessions and 16 off-types. DAF analysis revealed an average 14.1 ± 5.6 (SE) polymorphic bands/primer within cultivar accessions. In contrast, the higher resolving power of ASAP detected 24.5 ± 2.1 polymorphic bands/primer. Similarly, DAF and ASAP produced 13.0 ± 5.5 (SE) and 20 ± 2.8 polymorphic bands/primer within off-types, respectively. Phenetic analysis using cluster (UPGMA) and ordination (PCO) techniques showed that both Tifdwarf and Tifgreen were genetically unstable. The analysis showed that almost all cultivar accessions and one-half of the off-types studied were genetically distinct, but very close to each other. In this case, genetic variation was probably the result of somatic mutations. The other off-types and some Tifgreen accessions represented a genetically distant and diverse bermudagrass group of interspecific hybrid (n=27) origin. Off-types were probably the result of sod contamination. Results complement a previous study that established that the interspecific Tifway bermudagrass was genetically stable whereas derived off-types were contaminants rather than somatic mutants. Tifgreen and Tifdwarf showed genetic instabilities that were readily detected by DNA amplification with mini-hairpin primers. The present study offers a direct molecular alternative capable of evaluating the genetic stability of selected cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening fruit tree genetic resources in Belgium for disease resistance and other desirable characters

Summary The wide diversity of old fruit-tree cultivars originating or introduced into Belgium during the 18 ^th and 19 ^th centuries was collected as far as feasible over the last fifteen years at the State Plant Pathology Station in Gembloux. Out of the 2400 accessions now collected, one quarter was recovered from old public collections, and three quarters came from farms or gardens. The initial intention was to screen the material for disease resistance and other characters of agronomic interest with a view to using the best cultivars as breeding parents. However, as the collection developed, genetic resources conservation also became an objectiveper se. The collection presently contains 1150 apple, 850 pear and 300 plum accessions, and smaller numbers of other fruit species. Each accession is evaluated in an experimental orchard for at least ten years. In view of the growing public interest in old fruit-tree cultivars, the Plant Pathology Station has for several years been releasing to the nursery trade the better cultivars emerging from the evaluation, namely nine apple and four plum cultivars, and one peach cultivar. The principal features of the apple cultivars are presented in this paper. Since 1988, old apple and plum cultivars have been being used at the Station as parents in a breeding programme, with both controlled and open pollination. In some instances, old apple cultivars have also been crossed with a modern parent carrying the Vf gene for scab resistance. The preliminary observations on some of these seedlings are presented.     

Nematode resistance in bananas: screening results on some wild and cultivated accessions of Musa spp.

Bananas cultivated for export all belong to Cavendish cultivars and are all recognized as very susceptible to nematodes, particularly to the burrowing nematode Radopholus similis and the lesion nematode Pratylenchus coffeae. Even if there have been many changes in the management of banana nematodes in large commercial banana plantations, chemical control still remains most often the last resort method to manage the nematodes, although the number of registered products is definitely declining. Therefore, nematode control though genetic improvement is gaining new interest worldwide. In this study, 55 banana accessions mostly diploids from the Musa acuminata genome group (AA) but including some triploid accessions (AAA), some diploids of the Musa balbisiana genome group (BB) and some interspecific hybrids (AAB, AB) were evaluated for resistance to four nematode species R. similis, P. coffeae, Meloidogyne incognita and M. arenaria. These experiments were conducted in a growth chamber under controlled conditions. All banana accessions were susceptible to nematode species, although many different levels of susceptibility were detected. This study confirmed the good resistance status to R. similis of some cultivars from the Pisang jari buaya and Pisang batuau subgroups and the partial resistance of 17 diploid accessions significantly different from the susceptible reference cv. Grande Naine. This study also showed that 12 diploid accessions exhibited a partial resistance to P. coffeae, including some usual or potential genitors belonging to the wild diploids subspecies burmannica (cvs. Long Tavoy 1 and 2) and burmannicoides (cv. Calcutta 4). No source of resistance to Meloidogyne spp. was found. These screening results, combining for the first time four nematode species, are discussed within the scope of banana breeding in order to produce parental diploid lines with single or combined nematode resistances and further develop triploids that can substitute existing susceptible commercial cultivars.