Evaluation of Introduced and Naturalised Populations of Red Clover(<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) at Pergamino EEA-INTA,Argentina

Red clover (Trifolium pratense L.) is a temperate forage species that is extensively used in Argentina, but little effort in breeding has been made so far. Meanwhile, red clover breeding is strong in Europe and North America. In order to contribute to the utilisation of red clover genetic resources, 39 accessions of different origins were evaluated at Pergamino Inta Experimental Station. A broad phenotypic variation was detected for many of the 14 attributes considered. Variables that discriminate populations are related to seed production and winter yield. Two different groups clustered according to morphologic and productive mean attributes, and these groups were related to geographic origin of accessions.     

Discrimination among subterranean clover (<Emphasis Type="Italic">Trifolium subterraneum</Emphasis> L. complex) genotypes using RAPD markers

RAPD markers were applied to subterranean clover aiming at: (i) assessing the genetic relationships among the subspecies subterraneum L., brachycalycinum Katzn. et Morley, yanninicum Katzn. et Morley, as their taxonomic status is still debated; (ii) verifying the adoption of RAPDs to supplement the common morphological markers used for cultivar identification and protection; (iii) assessing the possible genetic diversity in relation to the geographic origin. Eight primers were selected for genetic analysis of 18 genotypes: 10 subsp. yanninicum (five from Greece and five from Sardinia), six subsp. subterraneum (forming three pairs, each one difficult to distinguish by morphological markers), and two subsp. brachycalycinum. Cluster analysis, performed on the Jaccards coefficients of association computed across the eight primers, formed three groups of genotypes, corresponding to the three subspecies. The results supported at the DNA level previous inferences, made at cytological, karyological, and isoenzymatic levels, on the ongoing speciation process within the subterranean clover complex, although not warranting yet the full species rank to the three forms. The genotypes of subsp. yanninicum were genetically closer to those of subsp. subterraneum than either group was to the subsp. brachycalycinum genotypes. Within the subsp. yanninicum cluster, the Sardinian genotypes appeared fairly distinct from those from Greece, suggesting a possible, independent evolution going on in different centres of diversity of this subspecies. In two pairs of subsp. subterraneum genotypes, the members could be unequivocally distinguished, thus supporting the role of RAPD fingerprinting in cultivar identification. In the third pair, the two genotypes appeared to be the same, inadvertently duplicated within the germplasm collection.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Origin and ancestry of Egyptian clover (<Emphasis Type="Italic">Trifolium alexandrinum</Emphasis> L.) As revealed by AFLP markers

The origin and ancestry for Egyptian clover, Trifolium alexandrinum, was examined using AFLP data. The data support a close relationship of T. alexandrinum accessions from Syria and Egypt to T. apertum, T. berytheum, and T. salmoneum. However, crossability and geographic distributions suggest that T. apertum is an unlikely progenitor. In contrast, T. salmoneum appears to be the most probable progenitor for Syrian material of Egyptian clover, although a close relationship to T. berytheum was also revealed. The ability of these species to cross freely indicates that T. salmoneum and T. berytheum may be regarded as the primary ancestors from, which man domesticated Egyptian clover through artificial selection in Syria. Following domestication, the earlier forms of the crop species could have been taken into rain-fed cultivation in Palestine and irrigated cultivation in Egypt. In this regard, the domestication of Egyptian clover may be analogous to other crops, such as barley and wheat, which were also domesticated in the Fertile Crescent and taken into cultivation in the Nile Valley. It appears that genetic improvement of the crop occurred in Egypt after cultivation, and that the varieties that were developed in Egypt were later distributed worldwide.     

Diversity analysis of Central Asia and Caucasian lentil (<Emphasis Type="Italic">Lens</Emphasis><Emphasis Type="Italic">culinaris</Emphasis> Medik.) germplasm using SSR fingerprinting

Diversity analysis was performed among 39 cultivated lentil (Lens culinaris Medik.) accessions of Central Asia and Caucasian origin using five highly polymorphic microsatellite markers. A total of 33 alleles determined ranging from 3 to 8 per locus. Estimated gene diversity value for 33 loci was 0.66. Genetic similarity indices among 39 accessions ranged from 0.24 to 1.0. Cluster analysis using the unweighted pair group method with arithmetic mean method classified accessions into six major groups at 0.5 similarity coefficient. More than half accessions from Tajikistan formed large cluster. On the other hand, a few accessions from each country showed unique genotypes. Overall, most of the accessions, except ones with closely related origin, were distinguished by the present high quality DNA fingerprinting. This molecular diversity information gives important basis for conservation strategy in gene bank and exotic germplasm introduction in breeding programs in Central Asia and Caucasian countries.     

Salt tolerance in <Emphasis Type="Italic">Zea mays</Emphasis> (L). following inoculation with <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis>

This study aimed to investigate the effect of inoculation with plant growth-promoting Rhizobium and Pseudomonas species on NaCl-affected maize. Two cultivars of maize (cv. Agaiti 2002 and cv. Av 4001) selected on the basis of their yield potential were grown in pots outdoors under natural conditions during July. Microorganisms were applied at seedling stage and salt stress was induced 21 days after sowing and maintained up to 50% flowering after 120 days of stress. The salt treatment caused a detrimental effect on growth and development of plants. Co-inoculation resulted in some positive adaptative responses of maize plants under salinity. The salt tolerance from inoculation was generally mediated by decreases in electrolyte leakage and in osmotic potential, an increase in osmoregulant (proline) production, maintenance of relative water content of leaves, and selective uptake of K ions. Generally, the microbial strain acted synergistically. However, under unstressed conditions, Rhizobium was more effective than Pseudomonas but under salt stress the favorable effect was observed even if some exceptions were also observed. The maize cv. Agaiti 2002 appeared to be more responsive to inoculation and was relatively less tolerant to salt compared to that of cv. Av 4001.     

<Emphasis Type="Italic">Ziziphus </Emphasis><Emphasis Type="Italic">spina-christi</Emphasis> (L.) Willd.: a multipurpose fruit tree

Neglected and underutilized species often play a vital role in securing food and livestock feed, income generation and energy needs of rural populations. In spite of their great potential little attention has been given to these species. This increases the possibility of genetic erosion which would further restrict the survival strategies of people in rural areas. Ziziphus spina-christi is a plant species that has edible fruits and a number of other beneficial applications that include the use of leaves as fodder, branches for fencing, wood as fuel, for construction and furniture making, and the utilization of different parts e.g. Fruits, leaves, roots and bark in folk medicine. Moreover, the plant is adapted to dry and hot climates which make it suitable for cultivation in an environment characterized by increasing degradation of land and water resources. Lack of research in Z. spina-christi hinders its successful improvement and promotion. Therefore, studies are needed to fully exploit this species. This article aims at summarizing information on different aspects of Z. spina-christi to stimulate interest in this crop which is of importance in Sudan and other countries of the semi-arid tropics.

Genetic diversity of the D-genome in <Emphasis Type="Italic">T. aestivum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species using SSR markers

Simple sequence repeats (SSRs), highly dispersed nucleotide sequences in genomes, were used for germplasm analysis and estimation of the genetic relationship of the D-genome among 52 accessions of T. aestivum (AABBDD), Ae. tauschii (D^tD^t), Ae. cylindrica (CCD^cD^c) and Ae. crassa (MMD^cr1D^cr1), collected from 13 different sites in Iran. A set of 21 microsatellite primers, from various locations on the seven D-genome chromosomes, revealed a high level of polymorphism. A total of 273 alleles were detected across all four species and the number of alleles per each microsatellite marker varied from 3 to 27. The highest genetic diversity occurred in Ae. tauschii followed by Ae. crassa, and the genetic distance was the smallest between Ae. tauschii and Ae. cylindrica. Data obtained in this study supports the view that genetic variability in the D-genome of hexaploid wheat is less than in Ae. tauschii. The highest number of unique alleles was observed within Ae. crassa accessions, indicating this species as a great potential source of novel genes for bread wheat improvement. Knowledge of genetic diversity in Aegilops species provides different levels of information which is important in the management of germplasm resources.     

Genetic Diversity of the Greater Yam (<Emphasis Type="Italic">Dioscorea alata</Emphasis> L.) and Relatedness to <Emphasis Type="Italic">D. nummularia</Emphasis> Lam. and <Emphasis Type="Italic">D. transversa</Emphasis> Br. as Revealed with AFLP Markers

Amplified fragment length polymorphism markers were used to assess the genetic relatedness between Dioscorea alata and nine other edible Dioscorea. These species include D. abyssinica Hoch., D. bulbifera L., D. cayenensis-rotundata Lamk. et Poir., D. esculenta Burk., D. nummularia Lam., D. pentaphylla L., D. persimilis Prain. et Burk., D. transversa Br. and D. trifida L. Four successive studies were conducted with emphasis on the genetic relationship within D. alata and among species of the Enantiophyllum section from Vanuatu. Study 1 was carried out to select a set of polymorphic primer pairs using 11 combinations and eight species belonging to five distinct sections. The four most polymorphic primer pairs were used in study 2 among six species of the Enantiophyllum section. Study 3 focussed mainly on the genetic relationship among 83 accessions of D. alata, mostly from Vanuatu (78 acc.) but also from Benin, Guadeloupe, New Caledonia and Vietnam. The ploidy level of 53 accessions was determined and results indicated the presence of tetraploid, hexaploid and octoploid cultivars. Study 4, included 35 accessions of D. alata, D. nummularia and D. transversa and was conducted using two primer pairs to verify the taxonomical identity of the cultivars `langlang', `maro' and `netsar' from Vanuatu. The overall results indicated that each accession can be fingerprinted uniquely with AFLP. D. alata is an heterogeneous species which shares a common genetic background with D. nummularia and `langlang', `maro' and `netsar'. UPGMA cluster analysis revealed the existence of three major groups of genotypes within D. alata, each assembling accessions from distant geographical origins and different ploidy levels. The analysis also revealed that `langlang', `maro' and `netsar' clustered together with the cultivar `wael' (D. transversa) from New Caledonia. Results are discussed in the paper.     

<Emphasis Type="Italic">Calamintha nepeta</Emphasis> (L.) Savi and <Emphasis Type="Italic">Micromeria thymifolia</Emphasis> (Scop.) Fritsch cultivated in Italy

Calamintha nepeta and Micromeria thymifolia have been traditionally used in the Mediterranean area as condiments and medicinal plants for a long time. Whereas in parts of Italy C. nepeta (special recipes have been developed in Lazio and Tuscany) is also an established garden plant showing different evolutionary products and their interaction among each other and the wild progenitor, M. thymifolia is being developed into a new crop plant. Both plants and their uses are described with regard to Italy. There is a marked tendency to broaden the use of condiments and spices which results in new crop plants which have to be documented and elaborated in further studies. Many species of Labiatae are predisposed to use by man and new items can be found even in areas which have to be considered as well studied.     

The Distribution of <Emphasis Type="Italic">Perilla</Emphasis> Species

Perilla (Lamiaceae) contains one tetraploid species, P. frutescens (L.) Britt. and three diploid species, P. citriodora (Makino) Nakai, P. hirtella Nakai and P. setoyensis G. Honda. Tetraploid species have been traditionally cultivated in Asia for their seed oil and for their fragrant leaves that are used as medicine or as a garnish for fish. The center of diversity is still obscure. To conserve the genetic resources, it is important to know the diversity of the tetraploid species. The three diploid species, which are possible parents of the tetraploid species, are all believed to be indigenous to Japan. Their distribution in China and Korea was clarified on the basis of herbarium and field surveys. The tetraploid species is assumed to have originated somewhere around the mid-to downstream area of the Changjiang River. Though Perilla is not cultivated as often in these areas as in northern China, Korea, the Himalayan region, or Myanmar, these areas should also be important for the conservation of genetic resources of tetraploid Perilla crops because of the expected high genetic diversity.     

AFLP and RFLP linkage map in <Emphasis Type="Italic">Coix</Emphasis>

Coix is a genus in the grass family placed in the tribe Maydeae. It is closely related to maize and is also used as a crop plant. Since many valuable traits have been identified recently in Coix, it is considered to be a valuable genetic resource, particularly for maize improvement. In this study, a Coix genetic linkage map was constructed using an F2 population of 131 individuals. Eighty AFLP and 10 RFLP markers were mapped, covering a total length of 1339.5 cM with an average interval of 14.88 cM. The map consisted of 10 linkage groups, were consistent with the chromosome numbers observed cytogenetically. Both AFLP and RFLP markers were used first for genetic analysis in Coix. AFLP markers were generated by two restriction enzyme combinations, EcoRI/MseI and PstI/MseI. A total of 1349 bands were amplified, of which 140 were polymorphic. The polymorphism detection efficiency of the two enzyme combinations was compared, and utility of AFLP markers to construct the linkage map was discussed. Ten RFLP markers detected by three different probes were distributed on eight different linkage groups. The results provide a foundation to map and isolate important genes in Coix, and to investigate its genomic architecture, possible origins, and relationship with maize at the DNA level.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Genetic resources of <Emphasis Type="Italic">Prunus</Emphasis> (Rosaceae) in India

The genetic diversity in genus Prunus is mainly confined to temperate regions of Himalaya and to a lesser extent in the sub-montane and hilly regions of peninsular India. The cultivated and wild species of Prunus have tremendous potential for improvement and utilization. This paper includes the genetic resources of cultivated and wild useful species of Prunus in India with emphasis on their distribution, potential traits/ useful characteristics and utilization. The information on potential genetic resources of Prunus would be helpful in collection, evaluation, conservation and utilization of species.     

On the occurrence,distribution and taxonomy of <Emphasis Type="Italic">Momordica subangulata</Emphasis> Blume subsp. <Emphasis Type="Italic">renigera</Emphasis> (G. Don) de Wilde in India

Different authors in India and South East Asia treat dioecious species in the genus Momordica differently. The cultivated ‘bhat karela’ of East and North East India is referred to as Momordica dioica Roxb. by botanists and herbarium curators whereas agricultural scientists, in general, designate it as Momordica cochinchinensis Spreng. A critical study of 266 herbarium sheets housed at CAL and BSISH and in situ field studies at specific pockets in the North East India followed by preliminary characterization revealed its correct identity as Momordica subangulata Blume subsp. renigera (G. Don) de Wilde. First hand information on its occurrence, taxonomy and distribution in India is given. The species was found in wild as well as in homestead cultivation in North Eastern India and exemplify direct utilization of biodiversity by indigenous people.

PCR Marker-based Analysis of Wild and Cultivated Yams (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.) in Nigeria: Genetic Relationships and Implications for <Emphasis Type="Italic">ex situ</Emphasis> Conservation

Reliable characterization of the variation among wild and cultivated yams in Nigeria is essential for improved management and efficient utilization of yam genetic resources. RAPD and double stringency PCR (DS-PCR) analyses were used to investigate genetic relationships and the extent of redundancy among 30 accessions of two cultivated, and 35 accessions of four wild yam species collected from Nigeria. Twenty-five selected random decamer and two microsatellite primers were used individually and in combination to generate DNA profiles for each accession of the six Dioscorea species. The number of amplified fragments varied from 7 to 18 fragments per primer/primer combination. Different levels of intraspecific genetic diversity were found, with Dioscorea rotundata Poir. being the most variable. Based on identical profiles for the RAPD and DS-PCR primers, 12 duplication groups consisting of a total number of 37 accessions were observed in the present study. An UPGMA analysis grouped the majority of plants according to the species. Cultivated yams belonging to the D. cayenensis–rotundata species complex, which were classified into seven morphotypes/varietal groups, could be clearly separated into two major groups corresponding to D. rotundata Poir. and D. cayenensis Lam. D. cayenensis cultivars exhibited a low level of intraspecific variation and were genetically close to the wild species Dioscorea burkilliana J. Miège. D. rotundata cultivars classified into six varietal groups showed a high degree of DNA polymorphism and were separated into two major groups that appeared most closely related to Dioscorea praehensilis Benth. and Dioscorea liebrechtsiana de Wild. We propose, based on these results, that cultivars classified into D. cayenensis should be considered as a taxon separate from D. rotundata. The implications of intraspecific variability for the ex situ conservation of wild and cultivated yam germplasm in Nigeria are discussed.     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Mitigation of salt stress in wheat seedlings by a <Emphasis Type="Italic">gfp</Emphasis>-tagged <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis>

Root colonization and mitigation of NaCl stress on wheat seedlings were studied by inoculating seeds with Azospirillum lipoferum JA4ngfp15 tagged with the green fluorescent protein gene (gfp). Colonization of wheat roots under 80 and 160 mM NaCl stress was similar to root colonization with this bacterial species under non-saline conditions, that is, single cells and small aggregates were mainly located in the root hair zone. These salt concentrations had significant inhibitory effects on development of seedlings, but not on growth in culture of gfp-A. lipoferum JA4ngfp15. Reduced plant growth (height and dry weight of leaves and roots) under continuous irrigation with 160 mM NaCl was ameliorated by bacterial inoculation with gfp-A. lipoferum JA4ngfp15. Inoculation of plants subjected to continuous irrigation with 80 mM NaCl or to a single application of either NaCl concentration (80 or 160 mM NaCl) did not mitigate salt stress. This study indicates that, under high NaCl concentration, inoculation with modified A. lipoferum reduced the deleterious effects of NaCl; colonization patterns on roots were unaffected and the genetic marker did not induce undesirable effects on the interaction between the bacterium and the plants.     

<Emphasis Type="Italic">Cistanche deserticola</Emphasis> Ma cultivated as a new crop in China

Cistanche deserticola Ma (Orobanchaceae) is known in Chinese as Rou cong rong, considered as an endangered wild species and adapted to arid or semi-arid areas. This species is traditionally used in China and Japan as a tonic. It has been found as a new cultivated plant in several plantations in northwestern China over the past decade. The objective of this study was to introduce the new crop plant to the world. Information on taxonomy, distribution, cultivation, genetic diversity etc. is reported in this paper. The crop may be of interest for regions, where the rainfall is low and the soil desertification is serious.