Isozyme electrophoregrams of sixteen enzymes in five tissues of cassava (Manihot esculenta Crantz) varieties

Summary Methodology, based on starch and polyacrylamide gel electrophoresis, was developed for determining isozyme electrophoregrams (patterns) of 16 enzymes of cassava (Manihot esculenta Grantz) varieties as potential genotype markers. Extracts of five different tissues (root, stem, leaf, petiole and bud) were examined. In general, the nodal portions of the shoots gave isozyme patterns with the largest number of bands. Petiole extracts gave similar results but bud extracts gave poor patterns. The limited number of varieties that were examined could be distinguished by sequential classification on the basis of the isozyme patterns of acid phosphatase, esterase, glutamate oxaloacetase transaminase and phosphoglucoisomerase.Joint publication of the Centro Internacional de Agricultura Tropical and the Department of Plant Science (No. 716), University of Manitoba. Presented in part at the 2nd International Symposium on Biochemical Approaches to Identification of Cultivars and Evaluation of Their Properties, 5–9 May, 1985, Braunschweig, West Germany.     

PCR-RFLP analysis of chloroplast gene regions in Cajanus (Leguminosae) and allied genera

Twenty-eight species belonging to five genera of the sub tribe Cajaninae; viz., Cajanus(15 species), Rhynchosia (10 species), Dunbaria, Flemingia and Paracalyx were analysed for variations in four chloroplast generegions, rbcL, trnS-psbC, 16S and trnL-UAA. The four chloroplast gene regions were amplified with specific primers and subsequently digested with 15 restriction enzymes. Rhynchosia did not show any inter-specific differences in the four gene regions for any of the enzymes used. In Cajanus, the inter-specific PCR-RFLP profile of the four gene regions for all the enzymes were similar except for the Pst I digests of trnS-psbC gene region. At inter-generic level, rbcL gene region did not show polymorphism with any of the enzymes used; while in trnS-psbC gene region polymorphism was observed only with Pst I and Hae III digestions. Inter-generic PCR-RFLP of 16S did not reveal any variation. The trnL-UAA gene region had restriction site only forEcoR I in which Cajanus and Rhynchosia showed similar profiles while Dunbaria showed a different profile. The trnL-UAA gene region in Paracalyx and Flemingia did not have restriction site for this enzyme. Despite the analyses of four gene regions using 15 restriction enzymes, differentiation at genus level could not be obtained. These observations indicated limited divergence of the chloroplast genome among the four genera of the sub-tribe Cajaninae suggesting close relationships of the taxa. This revised version was published online in July 2006 with corrections to the Cover Date.     

Correlation of isoenzyme polymorphism and Bayoud-disease resistance in date palm cultivars and progeny

Summary Seedlings of date palm (Phoenix dactylifera L.), obtained from seven cultivars crossed with two males, were analyzed by polyacrylamide gel electrophoresis for esterase (EST), glutamate oxaloacetate transaminase (GOT), endopeptidase (ENP) and alcohol dehydrogenase (ADH) polymorphisms. Eleven, eight, five and two phenotypes were revealed for the enzymes tested, respectively. Seedlings of F₁ populations derived from Bayoud (Fusarium)-resistant and low fruit quality cultivars were characterized by a high electrophoretic polymorphism, when compared with progenies of Bayoud-susceptible and high fruit quality cultivars. In almost all cases, the most frequent electrophoretic phenotypes scored for each enzyme in different F₁ populations, were similar to those of the corresponding parent cultivars. Heterozygous phenotypes have been found for, at least, 3 loci; Got-2, Est-1 and Enp, indicating that such loci could be used to screen for hybrid seedlings. The expected Mendelian segregation of allozymes has been observed for these 3 loci, in many F₁ populations. It seems that progenies of Bayoud-resistant cultivars are characterized by a high level of electrophoretic polymorphism. The estimation of this index and the search for genetic linkage with segregating allozymes, may be biochemical criteria useful as an aid in distinguishing date palm seedling populations derived from Bayoud-resistant cultivars and suitable for breeding programs.     

Phenotypic polymorphism of six enzymes in the grasspea (Lathyrus sativus L.)

Summary The phenotype variation in six enzymes, 6-phosphogluconate dehydrogenase (6-PGD), malate dehydrogenase (MDH), peroxidase (PRX), isocitrate dehydrogenase (IDH), galactose dehydrogenase (GD) and glutamate oxaloacetate transaminase (GOT), was investigated using horizontal starch gel electrophoresis in 52 accessions of the grasspea, Lathyrus sativus. Phenotypic polymorphism was observed for all six enzymes. High phenotypic polymorphism (Pj) was observed for PRX and 6-PGD, while there was little polymorphism for GOT, with only two accessions showing variation. There was no correlation between phenotypic polymorphism and region of origin, or groupings of accessions made on the basis of flower colour. Tentative genetic interpretations of banding patterns are given for five of the enzyme systems. The level of apparent heterozygosity was higher than expected in this predominantly autogamous species. The level of variation in the grasspea is discussed in terms of its potential for exploitation through plant breeding.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

Isoenzyme and cotyledon protein variation for identification of black beans (Phaseolus vulgaris L.) with similar seed morphology

Summary Methods developed to identify genetically diverse varieties of beans (Phaseolus vulgaris L.) were applied to closely related lines that were difficult to distinguish on the basis of seed morphology. Seedling tissues and seeds of black beans, were examined electrophoretically for isoenzyme and cotyledon proteinn protein patterns. Seven enzymes, extracted from seeds or from seedling stem, root or leaf tissues, were compared for polymorphism. Peptidase, polyphenol oxidase, phosphoglucoisomerase and glutamate oxaloacetate transaminase patterns were the same for all lines. Some differences were observed for acid phosphatase, peroxidase and esterase patterns, but complete discrimination of the six selected lines was not possible on the basis of isoenzyme patterns alone. Using polyacrylamide gel electrophoresis (PAGE) of dissociated 0.1 M acetic acid soluble proteins at pH 3.1 (acid-PAGE), or sodium dodecyl sulphate PAGE (SDS-PAGE) of residual protein extracts, all but one pair of samples in each case could be distinguished from the other samples. Using both techniques all of the lines could be identified unequivocally.     

Mitochondrial DNA variation in finger millet (Eleusine coracana L. Gaertn)

Summary This study was conducted to classify 26 lines of finger millet from Africa and India into cytotype groups based on the Southern blot hybridization patterns obtained with maize and sorghum mitochondrial cloned gene probes. Five restriction endonuclease enzymes were used for single digestions on total cellular DNA, giving a total of 20 enzyme/probe combinations. There was a low level of polymorphism, with identical RFLP banding patterns in 23 of the 26 lines. However, mtDNA clone atp9 hybridized to a 3.6 Kb Xba1 fragment in ecotype SDFM-1143 from Malawi; but did not hybridize to a 3.0 Kb fragment present in all other ecotypes. Two Zimbabwean lines, SDFM 63 and SDFM 77, had an extra, low intensity 6.5 Kb Xba1 fragment hybridized by mtDNA clone cox1. This data enabled classification of the lines into 3 cytopype groups.     

Genetic diversity in tef [Eragrostis tef (Zucc) Trotter] and its relatives as revealed by Random Amplified Polymorphic DNAs

Tef is one of the staple cereal crops in Ethiopia. To evaluate genetic diversity of tef and its relatives, 47 accessions of tef, three accessions of E. pilosa, and six accessions of E. curvulawere analyzed using random amplified polymorphic DNA (RAPD) markers. The level of polymorphism among the wild species was extremely high, while low polymorphism was detected among tef accessions. All cultivars and wild species under study could be distinguished with the help of different primers, thereby indicating the potential of RAPD in the genetic fingerprinting of tef. Accessions from E. curvula and E. pilosa can be differentiated by a single selected primer. In spite of low polymorphism within tef, accessions under study could be distinguished by a combination of selected primers. Cluster analysis indicated that tef is a very closely related species to E. pilosa with 45%similarity, supporting the hypothesis that tef originated from E. pilosa based on morphological data. Given that RAPD are relatively quick, simple to use, and are not subjected to environmental influences, they provide a valuable new approach for the genetic fingerprinting and study of genetic diversity in tef. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Interspecific hybridisation between Hibiscus syriacus, Hibiscus sinosyriacus and Hibiscus paramutabilis

Interspecific hybrids from crosses between H. syriacus × H. paramutabilis and H. syriacus × H. sinosyriacus were obtained. In both cases unilateral incongruity was observed and reciprocal crosses yielded no fruits. In vitro embryo rescue, 11 weeks after pollination, increased the germination capacity of H. syriacus × H. sinosyriacus embryos, while this was not the case for H. syriacus × H. paramutabilis embryos. However, a lot of the generated H. syriacus × H. sinosyriacus seedlings were lost due to variegated and total albinism. In the progeny of H. syriacus ‘Oiseau Bleu’ × H. paramutabilis about 95% of the seedlings had an intermediate flower and leaf morphology compared to both parent plants. Leaves on the adult F1 hybrids showed a polymorphism. In total four different leaf types could be observed on the same plant. This leaf polymorphism also was seen in the progeny of H. syriacus ‘Melwhite’ × H. sinosyriacus ‘Lilac Queen’. In this progeny about 50% of the seedlings had an intermediate flower and leaf morphology compared with the parent plants. The hybrid nature of the seedlings of both progenies was also confirmed by AFLP analysis. Despite the low germination vigour of the pollen of the hybrids, a small F2 generation was obtained from H. syriacus ‘Oiseau Bleu’ × H. paramutabilis.     

Isozyme polymorphism in three gene pools of cultivated chicory (Cichorium intybus L.)

Summary Polymorphism of ten enzymes, acid phosphatase (APH), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), phosphorylase (PP), superoxide dismutase (SOD), malic enzyme (ME), glutamate oxaloacetate transaminase 1 and 2 (GOT-1, GOT-2) and phosphoglucomutase 1 and 2 (PGM-1 and PGM-2), was investigated in three gene pools of cultivated chicory, including six cultivated wild chccory, eight industrial chicory and eight Brussels chicory varieties. LAP, APH, PP and PGM-2 showed high phenotypic polymorphism whilst GOT-1 and ME had poor polymorphism. For three enzyme coding loci Lap, Pgm-1 and Got-2, allele frequencies were determined. Isozyme composition in the three chicory gene pools was significantly different, showing, respectively, high, intermediate and poor average amount of phenotypic polymorphism in cultivated wild chicory, industrial chicory and Brussels chicory. Isozyme variation within and between varieties of the three gene pools is discussed in relation to breeding practices.     

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.     

Correlated resistance of cassava to mosaic and bacterial blight diseases

Summary The two most serious diseases of cassava (Manihot esculenta Crantz) are cassava mosaic disease (CMD) and cassava bacterial blight (CBB) (Xanthomonas manihotis Starr). Clone 58308, derived from the third backcross of the interspecific cross of cassava (M. esculenta) x ceara rubber (M. glaziovii), showed a high level of resistance to both diseases. Crosses of 58308 with several other clones which varied from susceptible to moderately susceptible to both diseases gave progenies with a significant genotypic correlation between resistance to both diseases (r=0.90), apparently due to linkage. The heritabilities of resistance to the diseases were estimated at 50–70% for CMD and 25–65% for CBB. Resistance to both diseases is assumed to be polygenic. The correlated response to selection for CMD and for CBB was estimated.     

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.     

Assessment of genetic relatedness of the genera Ananas and Pseudananas confirmed by RAPD markers

Genetic relationships among 18 accessions, including 16 of Ananas and two of Pseudananas, were investigated using RAPD molecular markers. The procedure for DNA extraction was adapted from the method of Dellaporta et al. (1983) where an incubation in proteinase K and a purification step were included. From the total of 148 markers scored,132 (89.2%) were polymorphic. The similarity matrix was used for cluster analysis. The phenogram developed from the RAPD bands showed that for most of the cases, the accessions within a species grouped together. Nevertheless, a moderate infraspecific genetic variation was observed. For example, DNA data grouped all A. comosus accessions with a mean similarity coefficient of 0.85. Comparable results were obtained with all other species investigated. The highest genetic divergence was found withinA. lucidus where the mean similarity coefficient among accessions was0.75. A similar level of genetic polymorphism was observed among species,therefore, a definition about which species were involved in the constitution of A. comosus genotypes was not possible. These results agree with the breeders standpoint suggesting that all Ananas species belong to the primary gene pool of pineapple. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogenetic relationships as in Ocimum revealed by RAPD markers

Genetic relationships were examined among thirty germplasm accessions belonging to five Ocimum species using RAPD markers. A very high degree of polymorphism (98.20%) was observed. UPGMA cluster analysis of genetic similarity indices grouped all the accessions into two major clusters corresponding to previously reported botanical sections. Intra-clustering within the two clusters precisely grouped the accessions belonging to one species in one sub-cluster as expected from their genetic background. Our results show that RAPD technique is a sensitive, precise and efficient tool for genomic analysis in Ocimum species, that may be useful in future studies, by assigning new unclassified germplasm accessions to specific taxonomic groups and reclassifying previously classified accessions of other Ocimum species by traditional criteria on a more objective basis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance of resistance to wildfire and angular leaf spot derived from Nicotiana rustica var. Brasilea

Summary The introgression of wildfire (races 0 and 1) and angular leaf spot (ALS) resistance from N. rustica var. Brasilea into N. tabacum has proved economically useful in Zimbabwe although the mode of inheritance of, and genetic relationships between the resistance are unknown. This study was undertaken to (1) examine the mode of inheritance of the resistance to races 0 and 1 of wildfire, and ALS, (2) determine the genetic relationship between the resistances and (3) establish whether the N. rustica-derived wildfire race 0 resistance is allelic to that obtained from N. longiflora. Inheritance was examined under greenhouse and field conditions by studying disease reactions in the parental, F1, F2 and backcross generations derived from crosses of three susceptible lines to a resistant line Nr-7. Three-point backcrosses to the susceptible parent were examined for linkage and segregating generations from a cross of Nr-7 to Burley 21 which carries the N. longiflora race 0 resistance were used to test for allelism. In general, we observed that all resistances are determined by a single dominant gene although some incosistent ratios were obtained likely due to misclassification of disease reactions and erratic transmission. All resistances showed linkage although pleiotropism cannot be ruled out. Allelism tests demonstrated that the N. rustica race 0 resistance is not allelic to that obtained from N. longiflora. Our findings are examined in relation to the efficacy of indirect selection for resistance.     

PCR-RFLP analysis of the whole chloroplast DNA from three cultivated species of cotton (Gossypium L.)

Variations of the chloroplast DNA (cpDNA) from three of the four cultivated species of cotton (Malvaceae); Gossypium barbadense L., Gossypium hirsutum L., Gossypium arboreum L. and its synonym Gossypium nanking Meyen., were analyzed. Using specific set of primers, the whole circular cpDNAs from the four test species were amplified. These were subsequently digested with the use of seven restriction enzymes. The amplified fragments of the whole cpDNAs of the diploid cultivated cotton G. arboreum and its synonym G. nanking did not show any differences. However, the allotetraploid cultivated cottons G. barbadense and G. hirsutum, showed some fragment length differences directly visible after amplification and two types of restriction fragment length polymorphism (RFLP), the first appeared as slightly lengthened bands and the other as gain or loss of a restriction site. The results also showed that the chloroplast genomes of the allotetraploid cultivated cottons are highly similar to the diploid cultivated cottons tested in terms of length and digestion patterns. The detected amplified length differences, RFLPs and the restriction sites can be considered as species specific markers for the allotetraploid cultivated cottons, which could be a useful tool for future studies of the cpDNA of the genus Gossypium L.     

Characterization of resistance of cassava genotypes to bacterial blight by evaluation of leaf and systemic symptoms in relation to yield in different ecozones

Twenty-two improved and local cassava genotypes were evaluated for their bacterial blight symptom types in reaction to infection by Xanthomonas axonopodis pv. manihotis under field conditions in the forest, forest savanna transition and wet savanna zones of Togo. High genotype × environment interactions in development of each symptom type were observed. Combining data on environments and genotypes, spot, blight and wilt symptoms were positively correlated. Analysing genotype reactions across environments, indications for independent mechanisms of resistance on leaf and stem level, varying by genotype, were found. Genotypes Main27 with resistance to spot and blight symptoms and TMS4(2)1425 with resistance to wilt symptoms are recommended to breeders to introgress their resistance characteristics. Significant negative correlations were generally observed between blight and wilt symptom development and root yield across ecozones, with blight being more important under lower, and wilt under higher inoculum pressure. Genotypes TMS30572, CVTM4, TMS92/0429 and TMS91/02316 showed low spot, blight and wilt symptoms combined with high root yield across ecozones.