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.     

RAPD variation in Solanum anguivi Lam. and S. aethiopicum L. (Solanaceae) in Uganda

Variation in 39 polymorphic RAPD markerswas studied for 18 populations of Solanum anguivi, S. aethiopicum Gilogroup and S. aethiopicum Shum groupin Uganda. UPGMA and PCO analyses of thedata revealed no clear groupingscorresponding to the species and groups. AnAMOVA analysis of the genetic variationshowed that the variation among species andgroups was less than 10%, whereas thevariation within species and groups wasmore than 90%. A Mantel test showed nocorrelation between genetic distance andgeographic distance. The data gives onlyvery weak support for the separation of themore morphologically distinct S.aethiopicum and S. anguivi. TheGilo- and Shum-groups of S.aethiopicum, which were previouslyregarded as two separate species can not beseparated on the basis of RAPD data, and itis most likely that they are keptmorphologically distinct by a strongman-made selection pressure.     

Genetic maps of RAPD, AFLP and ISSR markers in Ananas bracteatus and A. comosus using the pseudo-testcross strategy

Genetic maps of random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) and inter simple sequence repeats (ISSR) markers in pineapple (2n = 2x = 50) are reported for the first time. On the basis of a segregating population of 46 F1 individuals from a cross Ananas comosus x A. bracteatus, genetic maps of these two species were constructed using the two‐way pseudo‐testcross approach. The A. bracteatus map consists of 335 markers (60 RAPDs, 264 AFLPs and 11 ISSRs) assembled into 50 linkage groups, 26 of them with at least four markers. The A. comosus map consists of 157 markers (33 RAPDs, 115 AFLPs, eight ISSRs and the ‘piping’ trait locus) organized into 30 linkage groups, 18 of them with at least four markers. These maps cover, respectively, 57.2% of the A. bracteatus genome estimated as 3693 cM long, and 31.6% of the A. comosus genome calculated as 4146 cM. A rough estimate of 120 and 127 kbp/ cM on average was found for the relationship between physical and genetic distance for A. bracteatus and A. comosus, respectively.     

Isozyme diversity within African Manihot germplasm

Summary Isozyme diversity is described among a collection of 365 Manihot esculenta cultivars plus 109 accessions from wild relatives (M. glaziovii and spontaneous hybrids) from Africa. The study is based on 17 polymorphic loci. A natural hybrid swarm is detected between the two species. Although they were recently introduced, M. esculenta and M. glaziovii show high levels of polymorphism: heterozygosity estimates are 0.225 and 0.252 respectively. For the wild species, diversity is structured at the unilocus level, and the multilocus approach reveals a geographical pattern. The organization of the diversity is not so clear for the cultivated cassava, but a multilocus approach, based on both common and rare alleles, led us to identify different groups of clones with many intermediate genotypes between them. Elements of the secondary diversification process of Manihot in Ivory Coast are discussed.     

Isozyme variation in Helianthus petiolaris and sunflower,H. annuus

Summary Helianthus petiolaris is a wild species used in genetic improvement of sunflower, as a donor of cytoplasmic male sterility and of genetic resistance to diseases. Isozyme variation for ADH, ACP, EST, GDH, LAP, PGI, PGD and SKDH in this species was studied using starch gel electrophoresis. The patterns thus obtained were compared with zymograms of inbred lines, hybrids and open pollinated varieties of H. annuus. The same alleles for EST and SKDH isozymes were found in both species, while ACP showed an allele that has not been found in sunflower. The rest of the isozyme systems showed both common alleles and characteristic ones for each species. ACP, GDH and PGD were monomorphic in H. petiolaris, while ADH and LAP were monomorphic in H. annuus. The isozyme markers obtained here could be useful in breeding programs involving interspecific crosses, and studies on introgression and on genetic variation in other populations of this wild species.     

Genetic diversity and phylogenetic relationships among the annual Cicer species as revealed by isozyme polymorphism

Summary There are few estimates of genetic variability within and among populations of the nine annual Cicer species and for the wild species this information is based on few accessions. The present study was undertaken to examine genetic variation within and between annual Cicer species. One hundred and thirty-nine accessions of nine annual Cicer species were used for electrophoretic analysis at ICARDA. High levels of polymorphism in all eight wild annual Cicer species was found. This is in contrast to earlier research which had shown high polymorphism only in C. reticulatum. Cicer reticulatum had the highest proportion of polymorphic loci. However, for the cultigen, among 14 loci assayed, only two were polymorphic, ADH and PGD2. The nine species formed four phylogenetic groups based on the neighbor-joining method. The first group comprised C. arietinum, C. Reticulatum and C. echinospermum, the second C. bijugum, C. judaicum and C. pinnatifidum, the third C. chorassanicum and C. yamashitae; and the fourth group consisted of one species, C. cuneatum. The phylogenetic tree developed from the neighbor-joining technique illustrated that C. reticulatum is the probable progenitor of C. arietinum and that C. echinospermum split off from a common ancestor at an earlier stage in the evolutionary history of Cicer. Genetic diversity data showed that the greatest diversity was within C. reticulatum and the lowest with the cultigen, C. arientinum. With the exception of C. reticulatum, genetic diversity increased with genetic distance from the cultigen. Little geographic variation in genetic diversity was found.     

Genetic diversity and taxonomic relationships within the genus Lens as revealed by allozyme polymorphism

Summary A survey of allozyme polymorphism at 11 loci was carried out on 439 accessions from the genus Lens. This comprised 153 Lens culinaris subsp. orientalis, 35 L. odemensis, 117 L. ervoides, 32 L. nigricans, 2 of a differentiated cytotype of L. nigricans and 100 landrace accessions of the cultivated lentil (L. culinaris subsp. culinaris), from 10 different countries. The aim of the survey was to determine intra-specific genetic diversity and species relationships, based on phylogenetic and phenetic analyses, particularly regarding the position of L. odemensis and the differentiated cytotype of L. nigricans. Diversity was described by three statistics. The level of diversity in the cultivated taxon was lower than in any of the wild species according to two of these statistics, the percentage of polymorphic loci and mean number of alleles per locus. For the third measure (Nei's mean genetic diversity) it was only greater than L. ervoides. Genetic diversity statistics of the wild species indicated differences in the nature of between-population genetic diversity within the different taxa. Phylogenetic analysis suggests that L. odemensis and L. ervoides evolved from a common ancestor, and L. culinaris subsp. orientalis subsequently evolved from L. odemensis. Phenetic analysis, however, places L. odemensis closer to L. culinaris subsp. orientalis than to L. ervoides. Nei's mean genetic distance of L. odemensis from both L. culinaris subsp. culinaris (0.204) and L. culinaris subsp. orientalis (0.110) was greater than the distance between them (0.062). This evidence is not conclusive in determining whether L. odemensis should retain its specific status. Further crossability studies should be carried out on a range of genotypes to assess the potential for gene flow. The evidence presented shows the differentiated cytotype of L. nigricans to be quite distinct from other L. nigricans accessions, both phenetically and phylogenetically. This indicates that the differentiated cytotype of L. nigricans may constitute a new taxon. Discriminant function analysis reveals that isozymes may be useful in validating species classification.     

Genetic diversity within commercial populations of watercress (Rorippa nasturtium-aquaticum), and between allied Brassicaceae inferred from RAPD-PCR

Commercial productivity of watercress (Rorippa nasturtium-aquaticum) can be adversely affected by the pathogenic crook-root fungus, Spongospora subterranea f.sp. nasturti, and watercress viruses. As there are no effective control measures for these diseases, attempts have been made to breed varieties resistant to the crook-root pathogen. This work has been hindered by a lack of knowledge of the genetic base of commercial watercress, and the genetic distance between watercress and allied Brassicaceae which have been identified as candidates for hybridisation programmes. We measured the diversity within these two groups using the RAPD-PCR fingerprinting technique and analysed the data by both distance methods and principal co-ordinate analysis. Little genetic diversity was found within commercial watercress populations. However, watercress formed a unique cluster genetically distinct from other Rorippa species, but equidistant to Cardamine species. It was placed closer to Barbarea verna. This revised version was published online in August 2006 with corrections to the Cover Date.     

Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis

Summary The genetic variation among 23 accessions of 5 species in the subgenus Ceratotropis, genus Vigna, were investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 404 fragments amplified with 24 primers were scored and analyzed by cluster analysis. The accessions used were separated into two main groups with an average of 70% differences. Within the main groups, five subgroups were recognized, which are in complete agreement with taxonomic species. Wild forms were always grouped with their most closely related cultivated forms and they showed variation in each species. The largest intraspecific variation was found in V. radiata (mungbean), in which wild forms (V. radiata var. sublobata) were highly different from each other and from cultivated forms. V. angularis (adzuki bean) showed the least variation and thus, was probably differentiated in relatively recent times.     

Genetic relationships among cultivated bananas and plantains from Asia and the Pacific

Summary Isozyme variation was studied to determine genetic relationships among 563 accessions of Musa, including diploid (AA and BB), triploid (AAA, AAB, and ABB), and a few tetraploid (ABBB) clones from Asia and the Pacific. Several open-pollinated seedling progenies of wild, diploid M. acuminata and M. balbisiana were also studied. Cryogenic preservation of leaf tissue in liquid nitrogen allowed sampling of a wide array of germplasm from Papua New Guinea and several Pacific Islands without transporting propagules which are subjected to quarantine regulations. Electrophoretic variation was recorded in three enzyme systems, MDH, PGI and PGM. In total, 52 distinct electromorphs were identified among 192 different isozyme phenotypes (zymotypes). Multivariate analyses of the data clearly differentiated the major genome groups and revealed patterns of association within groups. The isozyme data suggest that the genes contributed by the M. acuminata genome to the triploid Pacific plantain AAB subgroup are similar to those of the acuminata/banksii complex of Papua New Guinea. It is likely that the Pacific plantain subgroup, including the Hawaiian Maoli, Pp'ulu and Iholena cultivars, originated in Papua New Guinea/Melanesia, rather than in Asia or the Malay Archipelago.Journal Series no. 3783 of the Hawaii Institute of Tropical Agriculture and Human Resources.     

Interspecific hybrids and possible phylogenetic relations in grain amaranths

Summary Phylogenetic relations among the three species in grain amaranth need investigation to provide information for breeding experiments germplasm conservation efforts, and decision on evolutionary patterns in the grain types. Hybrid development from crosses between species was studied to find out genetic relationship between them. Interspecific crosses were made among Amaranthus hypochondriacus, A. caudatus and A. cruentus in the glasshouse. The F₁ plants were relatively easy to obtain but had low pollen fertility (10.3–15.1%) and low seed set. A few of these hybrids did not produce seeds. Only a few F₁ seeds obtained in crosses between A. cruentus and A. caudatus. All the F₁ plants from these crosses died at the seedling stage. Crosses between A. cruentus and A. hypochondriacus produced few seeds. Most of the F₁ plants obtained from the seeds died at the seedling stage with only four plants growing to maturity but were sterile. Based on hybrid development, it was suggested that A. hypochondriacus and A. caudatus were genetically closer than the other two combinations of species studied. A. cruentus seemed to be genetically closer to A. hypochondriacus than it was to A. caudatus.Research was supported by Grand No. AMA-KE-4-83-22. (CRG GRANT) from the National Academy of Science, U.S.A.     

Interfering with regular meiotic behaviour in Avena sativa as a method of incorporating the gene for mildew resistance from A. barbata

Summary The regular meiotic behaviour of the cultivated oat Avena sativa (2n=6x=42) is genetically controlled. The factors which control the diploid-like meiotic behaviour also restrict the amount of pairing that occurs between alien chromosomes and their homoeologues in A.sativa, and hence increases the difficulties of introducing desirable variation from wild species into the cultivated oat. A genotype of the diploid species A.longiglumis which interferes with the regular meiotic behaviour of A. sativa can be used to induce pairing between alien chromosomes and their corresponding chromosomes in A. sativa. Using this procedure the dominant gene conferring mildew resistance has been transferred from the tetraploid weed species A. barbata into the cultivated oat.     

Characterization of isozymes in Salvia columbariae

Summary Salvia columbariae is a herbaceous annual species which has potential for domestication as a new source of industrial oil. Isozyme markers provide a mean by which this process may be facilitated. Isozyme survey of field grown Salvia columbariae plants showed variation for malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphogllucomutase (PGM). Selfed seed was obtained from the field and was grown in the greenhouse for segregation analyses. Electrophoretic results indicated that MDH was variable at zone 1, showing presence or absence of a band. The observed segregation ratio was not significantly different from expected ratio for Pgi-4 and Pgm-3 isozymes, indicating monogenic control of the two loci. Pgi-4 locus was heterozygous for a null allele. Cross dimerization between the allozyme Pgi-3 and Pgi-4 loci resulted in an intergenic band for this isozyme system.Abbreviations ACO aconitase - ADH alcohol dehydrogenase - DTT-DL dithiothreitol - FDH formate dehydrogenase - GOT glutamate-oxalacetate transaminase - IDH isocitric dehydrogenase - MDH malate dehydrogenase - MNR menadione reductase - 6PGD 6-phosphogluconate dehydrogenase - PGI phosphoglucose isomerase - PGM phosphoglucomutase - PVP-40 polyvinylpyrrolidone - SKDH shikimate dehydrogenase - TPI triosephosphate isomerase     

Segregation of isozymes in selfed progenies of a synthetic amphidiploid between Solanum integrifolium and S. melongena

Isozyme and cytogenetic analyses were performed on selfed progenies of a synthetic amphidiploid between scarlet eggplant, Solanum integrifolium (= S. aethiopicum),and eggplant, Solanum melongena `DMP', for estimating genetic uniformity. Isozymes in the 379 examined seedlings segregated into five genotypes (phenotypes) each at the four loci examined, Pgd-2 of phosphogluconate dehydrogenase (E.C.1.1.1.43), Idh-2 of isocitrate dehydrogenase (E.C.1.1.1.41), Pgm-2 of phosphoglucomutase (E.C.2.7.5.1)and Skdh-1 of shikimate dehydrogenase (E.C.1.1.1.25), indicating that the selfed seedlings were not genetically uniform. Most of the examined 15 selfed seedlings exhibited a somatic chromosome number of 48, that is the same number of the synthetic amphidiploid, whereas isozyme genotypes among them were variable. It is suggested that the segregation of isozymes was not caused by variation of chromosome number but by genetic segregation of isozyme genes. The genome of the synthetic amphidiploid was indicated to be unstable. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

Isozyme variation in a number of populations of Theobroma cacao L. obtained through various sampling regimes

Summary Isozyme polymorphism in four enzyme systems was used to estimate the genetic diversity in nine populations of Theobroma cacao L. The distribution of allelic variation observed in this study was inconsistent with the currently held view that the Upper Amazon region of Peru is the centre of cacao genetic diversity. All the populations analysed from this area of Peru were genetically similar, and contained a low level of isozyme polymorphism. It is argued that this is a result of genuine low diversity in the region rather than a sampling artifact. If a centre of diversity of wild cacao truly exists, this work indicates that it lies further north in Ecuador and Colombia.Although they are morphologically highly variable, Trinitario cacao populations derived from cultivation are currently accorded low priority for conservation. This phenotypic variation was observed to be reflected in high levels of isozyme polymorphism. It is suggested that the present strategy for the conservation of cacao germplasm, which accords priority to the Upper Amazon region of Peru while neglecting the Carribbean should be reconsidered.     

Inter and intra-species Inter Simple Sequence Repeat (ISSR) variations in the genus Cicer

Intra and inter-species ISSR variation and use of ISSR markers in determination of genetic relationship were investigated in an accession collection representing twoperennial and six annual Cicerspecies. Screening of Ciceraccessions with SSR primers revealed highly reproducible amplicon profiles with relatively high multiplex ratios. Many of the primers generated amplicon profiles with which not only the differences among species can readily be identified, but also polymorphisms within species could be detected more efficiently. PCR products at 150 gel positions detected using six SSR primers in Cicer accessions were treated as dominant DNA markers and utilized to compute the distances among accessions and species. Cluster analysis of accessions and species revealed groupings that corroborate our previous studies of relationships based on allozyme and AFLP analysis. Consistent with the AFLP analysis carried out in the same accession collection, ISSR-based groupings indicated that perennial C. incisumis genetically close to the annuals of the second crossability group (C. pinnatifidum,C. bijugum, C. judaicum) while C. reticulatum is the closest wild species to the cultivated chickpea. ISSR-based variation estimates were relatively higher when compared to previous estimates computed from RAPD and AFLP data. Technically, ISSR analysis combines the PCR-based targeting of microsatellite-associated polymorphisms with no prior sequence requirement and stringent PCR conditions. Similarly, when compared to AFLP analysis, it is less technically demanding allowing to survey polymorphic loci in the genome. Thus, ISSR-PCR technology is a reliable, fast, and cost-effective marker system that can be used to study genetic variation and genetic relationships in the genusCicer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure and variation of subterranean clover populations from Sicily,Italy

Summary A collection of subterranean clover lines singled out from populations of the species Trifolium brachycalycinum and T. subterraneum collected in Sicily, Italy, was examined at two localities for flowering time and at one locality for oestrogen content and seed yield. The structure and variation of the populations of the two species were compared. The relationship between flowering time of the populations and some environmental features of their collection sites was examined to assess whether the maturity requirements of the two species were similar and to frame selection models focused on developing varieties of appropriate maturity. Populations of T. subterraneum were, on average, more complex than those of T. brachycalycinum, being characterized by higher number of lines per population and greater intra-population variation for flowering time, oestrogen content and seed yield. Furthermore, populations of T. subterraneum were, on average, about 15 days earlier than sympatric populations of T. brachycalycinum. Both the greater variation and the relative earliness of T. subterraneum occurred irrespective of the environments of origin of the populations. Inferences are drawn on the adaptive advantages that such features confer to T. subterraneum. Mean flowering time of the populations increased on increasing annual rainfall and altitude of the collection sites. However, the changes in maturity appeared almost exclusively related to variations in rainfall in T. subterraneum, while in T. brachycalycinum the effect of altitude was greater and that of rainfall less marked than in the former species.     

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

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

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.