Red List assessment of nine <Emphasis Type="Italic">Aegilops</Emphasis> species in Armenia

The aims of this study are to determine the geographical and ecological distribution of nine Aegilops species in Republic of Armenia and to make an assessment of their IUCN Red List status, using the IUCN Red list categories and criteria, in order to develop an in situ conservation strategy for wild relatives of wheat in Armenia. Ecogeographic surveys of nine Aegilops species were undertaken over 2 years in Armenia. They included a herbarium survey followed by extensive ground-truthing field surveys where targeted Aegilops species occur. The study showed that of the nine Aegilops species studied, four are threatened and of these, Ae. mutica and Ae. crassa are critically endangered. The latter species may even be extinct in Armenia. Ae. neglecta and A. biuncialis are endangered. Additional studies are required to assess the threat status of Ae. umbellulata. Ae. columnaris was assessed as near threatened, while the remaining species (Ae. triuncialis, Ae. cylindrica and Ae. tauschii) are of least concern. There has been a dramatic decline in the genetic resources of Aegilops species during recent years in Armenia as a result of adverse human impacts such as expansion of agriculture, urbanization and uncontrolled grazing. Several species, especially Ae. mutica and Ae. crassa, should be prioritized in conservation activities in Armenia. Efforts should be made to conserve genetic diversity of crop wild relative species both in situ and ex situ, bearing in mind that their germplasm carries potentially valuable information (traits) that can improve adaptability and productivity of cultivated wheat varieties.     

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.     

Identification of <Emphasis Type="Italic">Aegilops</Emphasis> L. species and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. based on chloroplast DNA

The genus Aegilops L. is a very important genetic resource for the breeding of bread wheat Triticum aestivum. Therefore, an accurate and easy identification of Aegilops species is required. Traditionally, identification of Aegilops species has relied heavily on morphological characters. These characters, however, are either not variable enough among Aegilops species or too plastic to be used for identification at the species level. Molecular markers that are more stable within species, therefore, could be the alternative strategy towards an accurate identification. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among 21 Aegilops species (including Ae. mutica that is now known as Amblyopyrum muticum) and between the latter and T. aestivum to generate markers for the diagnosis of all targeted species. Cleaved amplified polymorphic sequence (CAPS) applied on 22 coding and non-coding chloroplast regions using 80 endonucleases and sequencing of two of those regions revealed little polymorphism between T. aestivum and the various Aegilops species examined and to a less extent was the variation among Aegilops species. Polymorphism observed among species analysed allowed the discrimination of T. aestivum and 12 Aegilops species.     

Evaluation and utilization of <Emphasis Type="Italic">Aegilops</Emphasis> and wild <Emphasis Type="Italic">Triticum</Emphasis> species for enhancing iron and zinc content in wheat

Grains of 80 accessions of nine species of wild Triticum and Aegilops along with 15 semi-dwarf cultivars of bread and durum wheat grown over 2 years at Indian Institute of Technology, Roorkee, were analyzed for grain iron and zinc content. The bread and durum cultivars had very low content and little variability for both of these micronutrients. The related non-progenitor wild species with S, U and M genomes showed up to 3–4 folds higher iron and zinc content in their grains as compared to bread and durum wheat. For confirmation, two Ae. kotschyi Boiss. accessions were analyzed after ashing and were found to have more than 30% higher grain ash content than the wheat cultivars containing more than 75% higher iron and 60% higher zinc than that of wheat. There were highly significant differences for iron and zinc contents among various cultivars and wild relatives over both the years with very high broad sense heritability. There was a significantly high positive correlation between flag leaf iron and grain iron (r = 0.82) and flag leaf zinc and grain zinc (r = 0.92) content of the selected donors suggesting that the leaf analysis could be used for early selection for high iron and zinc content. ‘Chinese Spring’ (Ph ^I ) was used for inducing homoeologous chromosome pairing between Aegilops and wheat genomes and transferring these useful traits from the wild species to the elite wheat cultivars. A majority of the interspecific hybrids had higher leaf iron and zinc content than their wheat parents and equivalent or higher content than their Aegilops parents suggesting that the parental Aegilops donors possess a more efficient system for uptake and translocation of the micronutrients which could ultimately be utilized for wheat grain biofortification. Partially fertile to sterile BC₁ derivatives with variable chromosomes of Aegilops species had also higher leaf iron and zinc content confirming the possibility of transfer of required variability. Some of the fertile BC₁F₃ and BC₂F₂ derivatives had as high grain ash and grain ash iron and zinc content as that of the donor Aegilops parent. Further work on backcrossing, selfing, selection of fertile derivatives, leaf and grain analyses for iron and zinc for developing biofortified bread and durum wheat cultivars is in progress. Nidhi Rawat, Vijay K. Tiwari, and Neelam Singh have contributed equally to the work.     

Diversity analysis of <Emphasis Type="Italic">Aegilops</Emphasis> species from Morocco using RAPD markers

The diversity of 51 representative populations of the 5 Aegilops species from Moroccan collection was analyzed using 22 RAPD primers. We investigated the associations among these 5 Aegilops species (A. geniculata Roth (UUMM), A. triuncialis L. (UUCC), A. ventricosa Tausch (DDNN), A. peregrina (Hackel) Maire et Weiller (UUMM) and A. neglecta Req. ex Bert. subsp. recta (Zhuk.) K. Hammer (UUMMNN)); some diploid species considered as their ancestors; accessions of some neighboring countries and also accessions of Triticums. A total of 650 polymorphic RAPD fragments were amplified. A dendrogram was constructed using the un-weighed pair group method arithmetic average (UPGMA) and Jaccard`s similarity coefficient. The UPGMA clustering showed a regrouping to the level species with high level of the structuration of the diversity at A. geniculata. We confirm as reported by other authors, the proximity of N genome to U genome and C genome to M genome and also the difference between the genomes M and N. Thus, the phylogeny between the species and the different genomes were retracted.     

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.     

Phylogenetic relationships between <Emphasis Type="Italic">Ensete</Emphasis> and <Emphasis Type="Italic">Musa</Emphasis> species as revealed by the <Emphasis Type="Italic">trnT trnF</Emphasis> region of cpDNA

Complete sequences of transcribed spacers and introns from the trnT trnF region of chloroplast DNA (cp DNA) were generated from Musaceae species to establish the phylogenetic relationships among 3 species of Ensete including the economically important Ensete ventricosum (Welw.) Cheesman and 13 species of Musa. Parsimony analysis and pair wise distance data produced a single tree, with Ensete and Musa as clearly distinguished clades. Six Musa and three Ensete clades were generated. The topology of these clades did not change when the data were split into spacers and introns, although the split resulted in poor bootstrap support. Removing a hotspot from the entire data set improved clade support. The clades produced are discussed with reference to existing taxonomic and phylogenetic treatments. In contrast to previous suggestions, most of the Rhodochlamys species that we investigated clustered together with strong support establishing their distinctiveness from the Musa species studied. Ensete glaucum (Roxb.) Cheesman and Musa beccartii Simmonds appear to represent ancestral forms of Ensete and Musa, respectively for the presently studied species, and both genera have a common ancestor that is yet to be established. Our data also show that E. ventricosum cannot be reduced to E. glaucum, nor can E. gilleti (De Wild.) Cheesman be reduced to E. ventricosum, as some authorities have suggested. Ensete gilleti or a species very close to it appears to be the ancestral species of E. ventricosum.     

Production and cytogenetics of trigeneric hybrid involving <Emphasis Type="Italic">Triticum</Emphasis>, <Emphasis Type="Italic">Psathyrostachys</Emphasis> and <Emphasis Type="Italic">Secale</Emphasis>

Trigeneric hybrids may help establish evolutionary relationships among different genomes present in the same cellular-genetic background, and also offers the possibility to transfer different alien characters into cultivated wheat. In this study, a new trigeneric hybrid involving species from the Triticum, Psathyrostachys and Secale was synthesized by crossing wheat-P. huashanica amphiploid (PHW-SA) with wheat-S. cereale amphiploid (Zhongsi 828). The crossability of F₁ hybrid was high with 35.13%, and the fertility was 41.95%. The morphological characteristics of F₁ plants resembled the parent Zhongsi 828. The trigeneric hybrids pollen mother cells (PMCs) regularly revealed averagely 19.88 univalents, 9.63 ring bivalents, 3.97 rod bivalents, 0.60 trivalents and 0.03 tetravalents per cell. Multivalents consisted of trivalents and tetravalents can be observed in 52.7% of cells. A variation of abnormal lagging chromosome, micronuclei and chromosome bridge were formed at anaphase I and telophase II. The mean chromosomes number of F₂ progenies was 2n = 46.13, and the distribution range was 42–53. GISH results revealed that most F₂ plants had 6–12 S. cereale chromosomes, and only 0–2 P. huashanica chromosomes were detected. The results indicated that S. cereale chromosomes can be preferentially transmitted in the F₂ progenies of trigeneric hybrid than P. huashanica chromosomes. A survey of disease resistances revealed that the stripe rust resistance from the PHW-SA were completely expressed in the F₁ and some F₂ plants. The trigeneric hybrid could be a useful bridge for the transference of P. huashanica and S. cereale chromatins to common wheat.     

Putative Role of <Emphasis Type="Italic">Flavobacterium</Emphasis>, <Emphasis Type="Italic">Dokdonella</Emphasis> and <Emphasis Type="Italic">Methylophilus</Emphasis> Strains in Paracetamol Biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time.

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Graphical Abstract ?

     

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.     

<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.     

<Emphasis Type="Italic">Achromobacter insolitus</Emphasis> and <Emphasis Type="Italic">Zoogloea ramigera</Emphasis> associated with wheat plants (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.     

<Emphasis Type="Italic">Trichoderma</Emphasis> improves the growth of <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Bio-fertilizer application has been proposed as a strategy for enhancing soil fertility, regulating soil microflora composition, and improving crop yields, and it has been widely applied in the agricultural yields. However, the application of bio-fertilizer in grassland has been poorly studied. We conducted in situ and pot experiments to investigate the practical effects of different fertilization regimes on Leymus chinensis growth, with a focus on the potential microecological mechanisms underlying the responses of soil microbial composition. L. chinensis biomass was significantly (P?<?0.05) increased by treatment with 6000 kg ha^?1 of Trichoderma bio-fertilizer compared with other treatments. We found a positive (R² =?0.6274, P <?0.001) correlation between bacterial alpha diversity and L. chinensis biomass. Hierarchical cluster analysis and nonmetric multidimensional scaling (NMDS) revealed that soil bacterial and fungal community compositions were all separated according to the fertilization regime used. The relative abundance of the most beneficial genera in bio-fertilizer (BOF) (6000 kg ha^?1Trichoderma bio-fertilizer) was significantly higher than in organic fertilizer (OF) (6000 kg ha^?1 organic fertilizer) or in CK (non-amend fertilizer), there the potential pathogenic genera were reduced. There were significant negative (P?<?0.05) correlations between L. chinensis biomass and the relative abundance of several potential pathogenic genera. However, the relative abundance of most beneficial genera were significantly (P?<?0.05) positively correlated with L. chinensis biomass. Soil properties had different effects on these beneficial and on these pathogenic genera, further influencing L. chinensis biomass.     

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.     

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.     

DNA markers and pollen morphology reveal that <Emphasis Type="Italic">Praecitrullus fistulosus</Emphasis> is more closely related to <Emphasis Type="Italic">Benincasa hispida</Emphasis> than to <Emphasis Type="Italic">Citrullus</Emphasis> spp.

The round melon Praecitrullus fistulosus (Stocks) Pangalo has been cultivated in Asia since ancient times and has been considered an underexploited crop in the western world. In the USA, there is an increased interest in using P. fistulosus as a commercial vegetable, and possibly as a rootstock for grafting watermelon, melon, or cucumber. However, the taxonomic classification of P. fistulosus is incomplete and for many years it has been considered a close relative of watermelon [Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa] and was previously classified as Citrullus lanatus subsp. fistulosus (Stocks) Duthie et J.B. Fuller. Here, we used two sets of DNA markers to assess the genetic similarity of P. fistulosus in relation to Citrullus spp. {including Citrullus lanatus subsp. vulgaris, C. lanatus subsp. lanatus, Citroides group [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.], and C. colocynthis (L.) Schrad.}, Cucumis spp. (including C. melo, C. sativus, C. anguria, C. meeusei, C. zeyheri), Benincasa hispida (Thunb.) Cogn., Lagenaria siceraria (Mol.) Standl. and Cucurbita spp. (including C. moschata Duchesne and the winter squash C. maxima Duchesne). The first marker set comprised 501 markers that were produced by 38 primer pairs derived from watermelon expressed sequenced tags (ESTs) containing simple sequence repeat (SSR) motifs (designated as EST-SSR primers; produced 311 markers), and by 18 primer pairs derived from ESTs that do not contain SSR motives (designated here as EST-PCR primers; produced 190 markers). The second marker set comprised 628 markers that were produced by 18 sequence related amplified polymorphism (SRAP) primer pairs. The phylogenetic data indicated that among these cucurbit species, the wax gourd B. hispida is the closest to the P. fistulosus. Pollen observations, using light microscopy, indicated that each of the cucurbit genera examined here has unique pollen morphology. The Cucurbita spp. have globular pollen grains with a stigmatic surface. The L. siceraria has polygonal pollen grains with symmetrical boundaries, while the Citrullus spp. and Cucumis spp. have ovular (conical) and triangular shaped pollen grains (respectively). The B. hispida and P. fistulosus have spherical or semispherical pollen grains. These pollen features appear to be in agreement with the phylogenetic relationships of these two species based on DNA markers. Analysis with 12 SRAP primer pairs revealed low genetic diversity among 18 United States Plant Introductions (PIs) of P. fistulosus, indicating the need to expand the germplasm collection of this cucurbit crop.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

Karyotypic variation in <Emphasis Type="Italic">Nicotiana</Emphasis> section Suaveolentes

Nicotiana section Suaveolentes (Solanaceae) currently includes 28 species and subspecies that are endemic to Australasia and the South Pacific and one African species, N. africana. The section is monophyletic and of allotetraploid origin, but relationships among the species in it and its diploid progenitors are poorly understood. Here we report chromosome numbers for 20 of the 29 taxa from the Suaveolentes, including a count for one recently proposed species for which no number has previously been available. Many of the published chromosome numbers for the Suaveolentes are confirmed in this study. However, six counts were different from the published numbers including n = 15 for N. maritima and N. suaveolens, which is a new chromosome number for the genus. Nicotiana goodspeedii and N. rotundifolia were n = 16, and the same number was found in the suggested species N. sp. ‘Corunna’. Nicotiana suaveolens contains polyploid races of n = 32 and here we report the probable existence of an n = 31 race as well. Karyotypic variation within species and within the section is apparently much greater than previously thought and further investigation is warranted.     

Size homoplasy and mutational behavior of chloroplast simple sequence repeats (cpSSRs) inferred from intra- and interspecific variations in four chloroplast regions of diploid and polyploid <Emphasis Type="Italic">Triticum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species

Chloroplast simple sequence repeats (cpSSRs) are widely distributed in the chloroplast genomes of all plant species, and are frequently employed for genotypic and phylogenetic analysis. However, information on intra- and interspecies variation in cpSSRs is lacking. In this study, we sequenced four intergenic (non-coding) chloroplast DNA regions in 57 accessions of 12 tetraploid, and 16 accessions of 4 hexaploid species of Triticum and Aegilops. These sequence data added to our previous data for diploid species in the same chloroplast regions. Intra- and interspecific genetic variation was analyzed for a total of 189 accessions of 13 diploid, 12 tetraploid, and 4 hexaploid species of Triticum and Aegilops, such that all species were represented by multiple accessions. The data were used to infer phylogenetic relationships within and among Triticum and Aegilops species. Based on this robust phylogenetic tree, seven of eight cpSSR loci clearly exhibited “size homoplasy,” referring to the fact that cpSSRs of identical size and DNA sequence can arise even if the alleles are not descended from a common ancestor. These data indicate that cpSSRs should be used with caution in phylogenetic analyzes. Interestingly, as observed from several previous studies, our data also suggest that observed mutation rates may increase significantly when mononucleotide (homopolymer) repeat numbers reach or exceed 9 bp. In the present report, using this sequence data set involving cpSSRs, 81 unique haplotypes among 189 accessions were detected, and five tetraploid Triticum and Aegilops species were successfully identified and genotyped. Our results indicate that combinations of nucleotide substitutions, indels and SSRs of chloroplast nucleotide sequences are available for genotyping at the species accession level.     

On evolutionary pathways of <Emphasis Type="Italic">Avena</Emphasis> species

This article presents literary review and results analysis of evaluation of representative set of oat accessions of all Avena L. species. Results of complex study of major morphological characters and utilization of the karyotype structure data confirmed by the results of RAPD and avenin spectrum analysis are presented. Relationships of genomes of different Avena species at each ploidy level are discussed. Two genomes form the base of all Avena species, namely the A and C genomes. Results of the evaluation of several characteristics of the oat species and their geographical distribution are analysed. Probable evolutionary pathway of Avena species are suggested. Most likely the centres of origin of genus Avena L. are determined.