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.     

Stability and potential use of RAPD markers in a sugarcane genealogy

Summary A complete ancestral history of the recently developed and closely related South African commercial sugarcane varieties N11 and NCo376, which differ markedly in their response to sugarcane mosaic virus (SCMV), was elucidated from archival records. The genealogy spans seven generations, starting with early intraspecific crosses between varieties of Saccharum officinarum and interspecific crosses between S. officinarum and either S. spontaneum or S. barberi. In total, the genealogy comprises 38 different varieties. Genomic DNA samples from N11 and NCo376 respectively were screened for polymorphisms using the PCR-RAPD technique. Ten polymorphic fragments ranging in molecular size from 317 to 1263bp were identified from a total of 1159 loci amplified with 100 random decamer primers. Two of the 10 polymorphic fragments were shown to be consistently present in N11 (resistant) and absent in NCo376 (susceptible), while 8 showed the reverse occurrence. The primers producing the polymorphisms were used to screen genomic DNA samples from all 19 varieties representing the genealogy. Results have indicated that (1) specific PCR-RAPD generated polymorphic fragments can indeed be identified across the seven generations; (2) certain fragments are sufficiently definitive to be used as markers to trace parentage; (3) the validity of documented crosses and/or the authenticity of germplasm material may be questioned using this technique, and (4) there is the potential to subject the markers to linkage analysis once a full and accurate assessment of the SCMV resistance phenotype is obtained.     

Assessment of genetic variation in a working collection of Vigna vexillata (L.) A. Rich. by isozyme and RAPD analyses

Genetic variation based on isozyme and RAPD analyses was investigated in 47 and 34 accessions respectively of Vigna vexillata from different geographical origins and belonging to three botanical varieties. A total of 9 enzyme systems were studied, accounting for 14 putative loci, 8 of which were polymorphic. The analysis of genetic diversity revealed a low level of within accession variation (H_S=0.013), while between accession diversity (D_ST) was 0.120. Coefficient of gene differentiation (G_ST) was 0.905, indicating that most variation was among accessions. Nei's genetic distances were calculated on the basis of allelic frequencies and a UPGMA dendrogram was constructed. Twenty arbitrary 10-mer oligonucleotides were used in RAPD analysis. Amplification profiles disclosed a higher level of polymorphism than isozymes. Based on amplification patterns, the similarity index of Jaccard was calculated and a dendrogram constructed on the basis of the similarity matrix. The final clustering based on RAPD data was similar to the one obtained using isozyme allelic frequencies. The classification in botanical varieties did not reflect the allelic constitution of the different samples. On the other hand, referring to geographical origin, most accessions from Africa and from Latin America were distributed respectively in two distinct clusters in the dendrogram. This grouping might also reflect the differences observed in the germination behaviour of V. vexillata from the two continents.     

Low level of selfing found in a sample of crosses in Australian sugarcane breeding programs

In sugarcane breeding programs, parents used in crosses are classified as male or female based on relative amounts of viable pollen produced. Usually the ‘female’ parent produces some pollen, albeit at a lower level than the ‘male’ parent. The possibility of selfing and associated problems have long been recognised by breeders. A high level of selfing may seriously bias estimates of breeding value of parents and affect the reliability of family means for identifying superior crosses. However, there is no reliable information reported indicating the level to which selfing is occurring in sugarcane crosses. The level of selfing was evaluated in eight Australian sugarcane crosses using RAPD markers. The crosses were selected as exhibiting variable levels of performance, as judged by measurements of commercial cane sugar (CCS), cane yield (TCH) and sugar yield (TSH) in plots consisting of random progeny clones. Five to eight male-specific RAPD bands were identified for each cross and used to screen twenty-eight to thirty-eight progeny from each cross. Selfed progeny were identified in each cross on the basis of absence of any male-specific RAPD bands after screening with at least 5 such RAPDs. Levels of selfing ranged from 0 to17.6%, with most of the crosses exhibiting zero or low levels of selfing. Although only a limited number of sugarcane crosses have been studied, this survey suggests that unwanted selfing is not a significant problem in the Australian sugarcane breeding program. This revised version was published online in July 2006 with corrections to the Cover Date.     

Linkage groups of isozymes,RFLP and RAPD markers in carrot (Daucus carota L. sativus)

Summary Genetic and linkage analysis of marker loci were performed with 4 selfed progenies, derived from single plant (I_0/1 lines) of carrot (Daucus carota L. sativus). The analysis of 58 markers included 1 morphological marker, 10 isozyme loci, 14 RFLPs, 28 RAPD markers, and 6 isolated PCR fragments used as RFLP probes. Linkage analysis was carried out with the MAPMAKER program and resulted in the construction of 8 linkage groups containing 55 markers with an average distance of 13.1 cM, 3 marker loci remained unlinked. 24% of the markers deviated significantly from the expected Mendelian ratios (1:2:1 or 3:1) due to gametic or zygotic selection. It was shown that isolated PCR amplification products can be used as RFLP probes to detect polymorphisms for a certain locus in progenies where the corresponding RAPD pattern is monomorphic or no amplification product is observed. Since carrot has a relative small genome the probability of amplifying repetitive DNA sequences is comparatively low. Thus PCR amplification products represent an additional useful source of RFLP probes.     

Analysis of genetic diversity in Agave tequilana var. Azul using RAPD markers

By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production of any tequila. Our objective was to assay levels of genetic variation in field populations of A. tequilana var. Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four different fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A. tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130 markers, of which 20 (15.4%) were polymorphic betweenA. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of124 RAPD products (0.8%) was polymorphic and 39 of 40 plants were completely isogenic. This is one of the lowest levels of polymorphism detected to date for the analysis of a crop species, and is proposed to be the result of the promotion of a single conserved genotype over many years due to an exclusive reliance on vegetative propagation for the production of new planting materials. The significance of these results is discussed in relation to breeding programs focused on the improvement of A. tequilana var. Azul. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tracing the origin of Spanish common bean cultivars using biochemical and molecular markers

Fifty-four accessions of Phaseolus vulgaris cultivated in Spain and representing a broad variability for this country, were studied together with 30 samples of wild forms of American origin. Two reference cultivars (from the Andes and Mesoamerica) plus two outgroups (P. coccineus and Vigna unguiculata) were also included. RAPD analysis of DNA leaf extracts were carried out with four selected primers. We also studied morphological characters of the seeds and the phaseolin electrophoretic patterns. Multivariate analysis with the UPGMA method using RAPD data clustered the samples in four groups and, comparing with morphological data and phaseolin types, showed that the Spanish cultivars were mainly of Andean origin. Nevertheless, occurrence of introgression in Spain and the consideration of the country as a second center of variability for beans can also explain the obtained results.     

Genetic variation in the <Emphasis Type="Italic">Crambe abyssinica - C. hispanica - C. glabrata</Emphasis> complex

Crambe abyssinica Hochst. ex R.E. Fries (n = 45) is an industrial oilseed crop that is high in erucic acid. It is most closely related to C. hispanica L. (n = 30) and C. glabrata DC. (n = 15), although the latter species is most often included in the synonymy of C. hispanica. The species complex extends throughout the Mediterranean region, Ethiopia and East Africa. Crambe abyssinica is endemic to Ethiopia, C. glabrata to Spain, Portugal and Morocco, and C. hispanica is distributed in the Mediterranean region and Middle East. The present study compared genetic relationships among C. abyssinica, C. hispanica and C. glabrata and attempted a taxonomic separation of them using traditional morphological traits, agronomic and seed quality data, chromosome number, and various molecular data sets including nuclear-DNA based RAPD data, chloroplast (cpDNA) restriction site data and ITS sequence data for the internal transcribed spacer region of the nuclear ribosomal DNA. The three species can be distinguished most reliably by chromosome number. Accessions could generally, but not always, be distinguished morphologically by plant branching pattern, fruit articulation and colour, leaf pubescence and leaf shape. cpDNA restriction site data and ITS sequence data, two relatively conserved DNA data sets, supported the recognition of C. glabrata as a distinct species separate from the C. hispanica/C. abyssinica accessions. Within the latter group, both RAPD data and field evaluation data revealed greater amounts of genetic variation in C. hispanica compared with accessions of C. abyssinica, with the latter included as a subset of C. hispanica. Crambe glabrata was genetically distinct for all data sets and warrants separate species status.     

Genetic similarities among Spanisch populations of Agropyron, Elymus and Thinopyrum, using PCR-based markers

Arbitrary oligonucleotides were used as primers to amplifygenomic DNA of 48 wild Spanish populations of Agropyroncristatum, Elymus hispanicus,E. caninus,E. repens,Thinopyrum curvifolium, Th.junceum and Th.intermedium. Genetic diversity was analysedusing nineteen primers. The number of amplified products ranged from8 to 18 per primer and a total of 240 markers were scored. Differentlevels of intraspecific genetic diversity were found, the allogamousspecies E. repens andTh. intermedium being themost variable. Jaccard's similarity coefficients for internalmeasure within and between populations were used to produce a clusterdiagram. The results demonstrate differences in the degree ofsimilarity between taxonomic units. Interpopulational variability andinterspecific genomic relationships of these species arediscussed.     

Comparison of isozyme and random amplified polymorphic DNA data for determining intraspecific variation in Cucumis

Variation at isozyme and random amplified polymorphic DNA (RAPD) loci in eight cucumber and seven melon cultivars, breeding lines, and plant introductions were used to determine the utility of these markers for assessing genetic variation among populations of each species. Although dendrograms derived from cluster analyses using species' variation at marker loci were dissimilar, these disparities were consistent with differences in the pedigrees and/or other information (e.g., morphological) known about each accession and species. Empirical estimations of variances associated with each marker type in the cucumber and melon accessions examined indicate that, per band, lower coefficients of variation can be attained in the estimation of genetic difference when using RAPDs compared to isozymes. The disparity between the marker analyses made may be related to the amount of genome coverage characteristic of a particular marker system in a species and its efficiency in sampling variation in a population.