PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Diversity of seed storage protein patterns in wild peanut (Arachis,Fabaceae) species

55 accessions of wild peanuts (Arachis spp.) introduced from South America were analyzed for seed storage protein composition using SDS-PAGE electrophoresis. The objectives of the study were to evaluate variability within sect.Arachis and to classify taxa based on protein composition. 25 different band positions were resolved. Individual accessions had 11 to 18 bands which included the conarachin region (MW > 50 kD), two to five bands in the acidic arachin region (MW 38–49.9 kD), three to seven in the intermediate MW region (23 to 37.9 kD), two to five bands in the basic arachin region (18–22.9 kD), and one to three bands in the low MW protein region (14–17.9 kD). These data were utilized in a principal coordinate analysis based on the matrix of genetic distances between all pairs of the 55 accessions. Several groups of accessions conformed to expected species classification includingA. batizocoi, A. stenosperma, andA. monticola; whileA. duranensis, A. cardenasii, A. helodes, andA. correntina did not form good groups. The study showed that great diversity exists for protein profiles and seed storage proteins have potential for aiding species classification and for serving as markers for interspecific hybridization studies.     

Molecular markers for wheat leaf rust resistance gene Lr41

Leaf rust, caused by Puccinia triticina Eriks., is an important foliar disease of common wheat (Triticum aestivum L.) worldwide. Pyramiding several major rust-resistance genes into one adapted cultivar is one strategy for obtaining more durable resistance. Molecular markers linked to these genes are essential tools for gene pyramiding. The rust-resistance gene Lr41 from T. tauschii has been introgressed into chromosome 2D of several wheat cultivars that are currently under commercial production. To discover molecular markers closely linked to Lr41, a set of near-isogenic lines (NILs) of the hard winter wheat cultivar Century were developed through backcrossing. A population of 95 BC₃F_2:6 NILs were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers using bulked segregant analysis. Four markers closely linked to Lr41 were identified on chromosome 2DS; the closest marker, Xbarc124, was about 1 cM from Lr41. Physical mapping using Chinese Spring nullitetrasomic and ditelosomic genetic stocks confirmed that markers linked to Lr41 were on chromosome arm 2DS. Marker analysis in a diverse set of wheat germplasm indicated that primers BARC124, GWM210, and GDM35 amplified polymorphic bands between most resistant and susceptible accessions and can be used for marker-assisted selection in breeding programs.     

Assessing genetic diversity in <Emphasis Type="Italic">Gossypium arboreum</Emphasis> L. cultivars using genomic and EST-derived microsatellites

The cultivated diploid, Gossypium arboreum L., (A genome) is an invaluable genetic resource for improving modern tetraploid cotton (G. hirsutum L. and G. barbadense L.) cultivars. The objective of this research is to select a set of informative and robust microsatellites for studying genetic relationships among accessions of geographically diverse G. arboreum cultivars. From more than 1,500 previously developed simple sequence repeat (SSR) markers, 115 genomic (BNL) and EST-derived (MUCS and MUSS) markers were used to evaluate the allelic diversity of a core panel of G. arboreum accessions. These SSR data enabled advanced genome analyses. A set of 25 SSRs were selected based both upon their high level of informativeness (PIC ≥ 0.50) and the production of clear PCR bands on agarose gels. Subsequently, 96 accessions representing a wide spectrum of diversity of G. arboreum cultivars were analyzed with these markers. The 25 SSR loci revealed 75 allelic variants (polymorphisms) ranging from 2 to 4 alleles per locus. The Neighborjoining (NJ) method, based on genetic dissimilarities, revealed that cultivars from geographically adjacent countries tend to cluster together. Outcomes of this research should be useful in decreasing redundancy of effort and in constructing a core collection of G. arboreum, important for efficient use of this genetic resource in cotton breeding.     

Genetic relatedness among dioecious Ficus carica L. cultivars by random amplified polymorphic DNA analysis,and evaluation of agronomic and morphological characters

A collection of 64 fig (Ficus carica L.) accessions was characterized through the use of RAPD markers, and results were evaluated in conjunction with morphological and agronomical characters, in order to determine the genetic relatedness of genotypes with diverse geographic origin. The results indicate that fig cultivars have a rather narrow genetic base. Nevertheless, RAPD markers could detect enough polymorphism to differentiate even closely related genotypes (i.e., clones of the same cultivar) and a unique fingerprint for each of the genotypes studied was obtained. No wasteful duplications were found in the collection. Cluster analysis allowed the identification of groups in accordance with geographic origin, phenotypic data and pedigree. Taking into account the limited information concerning fig cultivar development, the results of this study, which provide information on the genetic relationships of genetically distinct material, dramatically increase the fundamental and practical value of the collection and represent an invaluable tool for fig germplasm management.     

Genetic relationships within Brassica rapa as inferred from AFLP fingerprints

Amplified fragment length polymorphism (AFLP) markers were employed to assess the genetic diversity amongst two large collections of Brassica rapa accessions. Collection A consisted of 161 B. rapa accessions representing different morphotypes among the cultivated B. rapa, including traditional and modern cultivars and breeding materials from geographical locations from all over the world and two Brassica napus accessions. Collection B consisted of 96 accessions, representing mainly leafy vegetable types cultivated in China. On the basis of the AFLP data obtained, we constructed phenetic trees using mega 2.1 software. The level of polymorphism was very high, and it was evident that the amount of genetic variation present within the groups was often comparable to the variation between the different cultivar groups. Cluster analysis revealed groups, often with low bootstrap values, which coincided with cultivar groups. The most interesting information revealed by the phenetic trees was that different morphotypes are often more related to other morphotypes from the same region (East Asia vs. Europe) than to similar morphotypes from different regions, suggesting either an independent origin and or a long and separate domestication and breeding history in both regions.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

An analysis of the B genome speciesArachis batizocoi (Fabaceae)

Arachis batizocoi Krap. & Greg. is a suggested B genome donor to the cultivated peanut,A. hypogaea L. Until recently, only one accession of this species was available in U.S.A. germplasm collections for analyses and species variability had not been documented. The objective of this study was to determine the intraspecific variability ofA. batizocoi to better understand phylogenetic relationships in sect.Arachis. Five accessions of the species were used for morphological and cytological studies and then F₁ intraspecific hybrids analyzed. Some variation was observed among accessions—for example, differences in seed size, plant height and branch length. The somatic chromosomes of accessions 9484, 30079, and 30082 were nearly identical, whereas, the karyotypes of accessions 30081 and 30097 have several distinct differences. For example, 30081 had significantly more asymmetrical chromosomes 2 and 6 and more median chromosomes 7 and 10, and 30097 had significantly more asymmetrical chromosomes 3 and 10 and more median chromosomes 1 and 5 than accessions 9484, 30079, and 30082. All F₁ hybrids among accessions were highly fertile. Meiotic observations indicated that hybrids among accessions 9484, 30079, or 30082 had mostly bivalents. However, quadrivalents were observed when either 30081 or 30097 was crossed with the above three accessions and 30081 × 30097 had quadrivalents, hexavalents and octavalents. The presence of translocations is the most likely cause of multivalent formation inA. batizocoi hybrids. Cytological evolution via translocations has apparently been an important mechanism for differentiation in the species.Paper No. 12382 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Molecular studies on genetic integrity of open-pollinating species rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) after long-term genebank maintenance

The genetic integrity of six accessions represented by 14 sub-populations of the open-pollinating species rye (Secale cereale L.) was investigated. Seeds available from a herbarium collection (first regeneration) and from the cold store (most recent regeneration) were multiplied two to fourteen times and fingerprinted using microsatellite markers. Four accessions had significantly different allele frequencies. These were multiplied seven to thirteen times. Nearly 50% of the alleles discovered in the original samples were not found in the material present in the cold store. However alleles were detected in the most recently propagated sub-populations, that were not observed in the investigated plants of the original one. The change in allele frequencies is a continuous process. Reasons for the occurrence of genetic changes and consequences for managing open pollinating species maintained in ex situ genebanks are discussed.Communicated by G. Wenzel     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.     

Genetic diversity in <Emphasis Type="Italic">in vitro</Emphasis>-conserved germplasm of <Emphasis Type="Italic">Curcuma</Emphasis> L. as revealed by RAPD markers

A set of 30 accessions of five Curcuma species-C. latifolia, C. malabarica, C. manga and C. raktakanta and 13 morphotypes (identified on the basis of morphological markers) of C. longa conserved in the In Vitro Genebank at National Bureau of Plant Genetic Resources, New Delhi, were subjected to RAPD analysis. Of the 200 RAPD primers screened, 21 polymorphic primers were selected for further study. Mean genetic similarities based on Jaccard’s similarity coefficient ranged from 0.18 to 0.86 in accessions of cultivated species, i.e., C. longa and from 0.25 to 0.86 in wild species. The dendrogram derived from the RAPD data corroborated the morphological classification of the morphotypes. The efficiency of individual RAPD primers was also compared; primers OPC-20, OPO-06, OPC-01 and OPL-03 were adjudged highly informative in discriminating the germplasm of Curcuma.     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

Stylosanthes sp. aff.S. scabra: a putative diploid progenitor ofStylosanthes scabra (Fabaceae)

Stylosanthes sp. aff.S. scabra is an undescribed taxon showing affinities with the allotetraploid speciesS. scabra, but distinct in a number of attibutes. Several collections show potential as forage for clay soils in northern Australia. Twelve accessions have been analysed using STS (sequence-tagged-sites) as genetic markers, and they all displayed STS phenotypes of typical diploid species. Taking into account their morphological similarities, the STS analysis provides strong evidence thatStylosanthes sp. aff.S. scabra might be a diploid progenitor of the allotetraploidS. scabra. This speculation was supported by cytological examinations. Somatic chromosome numbers of two of these accessions were counted and both were found to be diploid (2n = 20). The level of polymorphism among the 12Stylosanthes sp. aff.S. scabra accessions, estimated using randomly amplified polymorphic DNA (RAPD) as markers, was 7.8%, and the dissimilarity value betweenStylosanthes sp. aff.S. scabra andS. viscosa (the other putative progenitor ofS. scabra) was 89%.     

Congruence between morphological and molecular markers inferred from the analysis of the intra-morphotype genetic diversity and the spatial structure of Oxalis tuberosa Mol.

Oxalis tuberosa is an important crop cultivated in the highest Andean zones. A germplasm collection is maintained ex situ by CIP, which has developed a morphological markers system to classify the accessions into morphotypes, i.e. groups of morphologically identical accessions. However, their genetic uniformity is currently unknown. The ISSR technique was used in two experiments to determine the relationships between both morphological and molecular markers systems. The intra-morphotype genetic diversity, the spatial structures of the diversity and the congruence between both markers systems were determined. In the first experience, 44 accessions representing five morphotypes, clearly distinct from each other, were analyzed. At the molecular level, the accessions exactly clustered according to their morphotypes. However, a genetic variability was observed inside each morphotype. In the second experiment, 34 accessions gradually differing from each other on morphological base were analyzed. The morphological clustering showed no geographical structure. On the opposite, the molecular analysis showed that the genetic structure was slightly related to the collection site. The correlation between both markers systems was weak but significant. The lack of perfect congruence between morphological and molecular data suggests that the morphological system may be useful for the morphotypes management but is not appropriate to study the genetic structure of the oca. The spatial structure of the genetic diversity can be related to the evolution of the species and the discordance between the morphological and molecular structures may result from similar selection pressures at different places leading to similar forms with a different genetic background.     

Identification of new sources of aluminum resistance in wheat

Aluminum (Al) toxicity is a major constraint for wheat production in acidic soils. An Al resistance gene on chromosome 4DL that traces to Brazilian wheat has been extensively studied, and can provide partial protection from Al damage. To identify potentially new sources of Al resistance, 590 wheat accessions, including elite wheat breeding lines from the United States and other American and European countries, landraces and commercial cultivars from East Asia, and synthetic wheat lines from CIMMYT, Mexico, were screened for Al resistance by measuring relative root elongation in culture with a nutrient solution containing Al, and by staining Al-stressed root tips with hematoxylin. Eighty-eight wheat accessions demonstrated at least moderate resistance to Al toxicity. Those selected lines were subjected to analysis of microsatellite markers linked to an Al resistance gene on 4DL and a gene marker for the Al-activated malate transporter (ALMT1) locus. Many of the selected Al-resistant accessions from East Asia did not have the Al-resistant marker alleles of ALMT1, although they showed Al resistance similar to the US Al-resistant cultivar, Atlas 66. Most of the cultivars derived from Jagger and Atlas 66 have the Al-resistant marker alleles of ALMT1. Cluster analysis separated the selected Al-resistant germplasm into two major clusters, labeled as Asian and American–European clusters. Potentially new germplasm of Al resistance different from those derived from Brazil were identified. Further investigation of Al resistance in those new germplasms may reveal alternative Al-resistance mechanisms in wheat. Electronic supplementary material The online version of this article (doi:contains supplementary material, which is available to authorized users. Responsible Editor: Thomas B. Kinraide.     

Evaluation of Lespedeza germplasm genetic diversity and its phylogenetic relationship with the genus Kummerowia

The genetic diversity of the genus Lespedeza is not well known and the phylogenetic relationship of Lespedeza with the genus Kummerowia is unclear. We report the first study in which polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from Medicago, cowpea and soybean were used to assess the genetic diversity of the USDA Lespedeza germplasm collection and clarify its phylogenetic relationship with the genus Kummerowia. Phylogenetic analysis partitioned 44 Lespedeza accessions into three main groups some of which were species-specific and eight subgroups. This data set revealed some misidentified accessions, and indicated that the two species in the genus Kummerowia are closely related to the genus Lespedeza. Morphological reexamination was used to correct the misidentified accessions within the genus Lespedeza. Our results demonstrated that phylogenetic analysis with morphological reexamination provides a more complete approach to classify accessions in plant germplasm collection and conservation.     

Brassica naponigra,a somatic hybrid resistant to Phoma lingam

Summary Brassica napus and B. nigra were combined via protoplast fusion into the novel hybrid Brassica naponigra. The heterokaryons were identified by fluorescent markers and selected by flow sorting. Thirty hybrid plants were confirmed by isozyme analysis to contain both B. nigra and B. napus chromosomes; of these, 20 plants had the sum of the parental chromosome numbers. A non-random segregation of the chloroplasts was found in the hybrids. Of 14 hybrid plants investigated, all had the B. napus type of chloroplast. The resistance to Phoma lingam found in the B. nigra cultivar used in the fusion experiments was expressed in 26 of the hybrid plants. The hybrids obtained in this study contain all of the three Brassica genomes (A, B and C) and have thus created unique possibilities for genetic exchanges between the genomes. Since most of the plants were fertile as well as resistant to P. lingam, they have been incorporated into conventional rapeseed breeding programs.