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.     

Polymorphisms Among Chloroplast and Mitochondrial Genomes of <Emphasis Type="Italic">Citrullus</Emphasis> Species and Subspecies

Twelve and six DNA clones representing various parts of chloroplast and mitochondrial genomes, respectively, were used to detect polymorphism among five watermelon cultivars and 21 U.S. Plant Introductions (PIs) collected from diverse geographical locations and representing major groups of Citrullus species. Cluster analysis based on 20 chloroplast DNA (cpDNA) and 10 mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) markers differentiated the accessions into three major phenetic groups: PIs and watermelon cultivars of Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa (also designated as C. lanatus var. lanatus) (group I), PIs of C. lanatus var. citroides (of C. lanatus subsp. lanatus Schrad. ex Eckl. et Zeyh.)(group II), and C. colocynthis (L.) Schrad. PIs (group III). The chloroplast and mitochondrial genomes of watermelon cultivars are distinct, but closely related to those of the C. lanatus var. lanatus PIs. On the other hand, the chloroplast and mitochondrial genomes of the wild species C. colocynthis are more similar to those of C. lanatus var. citroides. Polymorphic cpDNA and mtDNA markers identified in this study can complement isozyme and nuclear DNA data used in earlier phylogenetic and phenetic classifications of Citrullus PIs. These cpDNA and mtDNA markers are being used in experiments designed to enhance watermelon cultivars by replacing the chloroplast and mitochondrial genome of cultivated watermelon with those of the wild species C. colocynthis.     

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.     

Divergent evolution of wild and cultivated subspecies of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> as revealed by the study of <Emphasis Type="Italic">PolA1</Emphasis> gene

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241–243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively.     

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.     

Morphological and molecular diversity analysis among the Indian clones of <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> L.

Sesuvium portulacastrum L. (seapurslane) is a halophyte used as pioneer species in sand dune fixation and stabilization of saline soil. Studies on the morphological and molecular diversity were carried out for the 14 clones of Sesuvium collected from the different coastal regions of India. Significant differences were observed for morphological traits viz., length, width, diameter and area of leaf, internodal distance and stem diameter for different clones when compared with the clone from Gujarat state (GJ1). A UPGMA dendrogram for morphological traits based on the Pearson’s similarity coefficient clustered the clones into three groups considering 80% polymorphism as criteria. Molecular diversity among the clones was studied using Randomly Amplified Polymorphic DNA (RAPD), Internal Transcribed Spacer (ITS) and markers specific to Ac homologous region. Of the total 749 RAPD loci amplified with 70 random primers, 294 were polymorphic with 39.25% diversity. A phylogenetic tree constructed with UPGMA and SHAN, grouped the clones into three major clades based on RAPD data. The molecular diversity studied with ITS and markers specific to Ac homologous region revealed 37.50% and 66.66% polymorphism and clustered the clones into three and four clades, respectively. The genetic diversity analysis revealed wide variations among the S. portulacastrum clones, reflecting a high level of diversity within the species which might be due to anthropogenic impact and geographic environmental conditions. Further, the various clones from the different eco-geographic coastal localities might have originated from native places of wild abundance. To the best of our knowledge, this is the first attempt to evaluate both morphological and genetic diversity among the Sesuvium clones collected from the distant habitats of the coastal regions of the India.     

Comparison of the genetic diversity between <Emphasis Type="Italic">Triticum aestivum</Emphasis> ssp. <Emphasis Type="Italic">tibetanum</Emphasis> Shao and Tibetan wheat landraces (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) by using intron-splice junction primers

In order to evaluate and compare the germplasm resources of wheat in Tibet, we analyzed the genetic diversity of 136 Triticum aestivum ssp. tibetanum Shao and 119 Tibetan wheat landraces (Triticum aestivum L.) by using Intron-Splice Junction (ISJ) primers. The results showed that polymorphism of PCR products were obtained by 33 primer combinations, which accounted for 11% of the 300 primer combinations produced by 26 ISJ primers. A total of 333 stable bands can be amplified from the T. aestivum ssp. tibetanum Shao and 243 bands were polymorphic, which accounted for 72.9% of the total bands. Tibetan wheat Landraces produced 316 stable bands, of which 197 bands were polymorphic. The polymorphic bands accounted for 62.34% of the total bands produced from Tibetan wheat landraces. The genetic diversity of T. aestivum ssp. tibetanum Shao was higher than that of Tibetan wheat landraces in Tibet, suggesting that T. aestivum ssp. tibetanum Shao can be used as important genetic resource for the breeding and genetic improvement of wheat in Tibet. Matrix (1, 0) was generated according to the presence or absence of the bands produced from a particular wheat accession. Clustering and principle coordinates analysis showed that T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces were divided into two groups. We conclude that high polymorphisms produced by ISJ primers can reflect the genetic diversity between T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces.     

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.     

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.     

Development and utilization of diagnostic DAMD-PCR markers for <Emphasis Type="Italic">Capsicum</Emphasis> accessions

A polymerase chain reaction (PCR) based approach involving the directed amplification of minisatellite DNA region (DAMD-PCR) was used to identify accession specific DNA markers and study genetic relationships between and within 15 accessions corresponding to 11 species in genus Capsicum. A touch down PCR profile and unique chemical concentration of ingredients resulted in reproducible and reliable DNA amplifications. The number of amplified products varied from 1 to 12 fragments depending on the template DNA and the primers. The DAMD-PCR technique provided a total of 38 accession specific DNA markers (diagnostic DAMD-PCR) which can be utilized in accession identification, preservation and genetic studies of Capsicum germplasm. Based on 1,292 polymorphic and monomorphic DNA markers directed with 22 minisatellite specific primers, accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Capsicum chacoense was found to be the most distinct species.     

Polymorphism of <Emphasis Type="Italic">Got2</Emphasis> DNA sequences sheds light on <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss. intraspecies divergence and origin of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

DNA sequences of nuclear gene Got2 was studied in 60 accessions of Aegilops tauschii, 29 of subsp. tauschii and 31 of subsp. strangulata. It was found that Got2 allozyme polymorphism in Ae. tauschii is due to a single, unique, mutation which led to replacement of glutamic acid by isoleucine in residue 256 of the enzyme molecule, encoded by Got2. As revealed by Got2 DNA sequences variation, initially in its history Ae. tauschii was presented by subsp. strangulata, and among phylogenetic lineages of subsp. strangulata, the lineage “t-9¹s” (TauL3) is the most ancient, a relict one. Subspecies tauschii is relatively “young”. Initially it was presented by the lineage marked by combination of allozyme alleles Got2 ¹⁰⁵ and Acph1 ¹⁰⁰. In the past it inhabited the Continental area from Caucasia to Pakistan, but later on it was forced out by newly originated, now—a major lineage of subsp. tauschii, marked by Got2 ¹⁰⁰. This lineage extended the Continental area of the species up to Kirgizstan, but actually failed to penetrate into pre-Caspian area, occupied by subsp. strangulata. These results essentially differ from those obtained previously, using chloroplast DNA (cpDNA) sequences polymorphism. As revealed by cpDNA, the major, “usual”, subsp. strangulata (TauL2) is “younger” than subsp. tauschii, which resided on phylogenetic tree between relict lineage “t-9¹s”of subsp. strangulata—and major subsp. strangulata. But both cpDNA and Got2 DNA sequences indicate that the level of genetic variation in subsp. tauschii is much lower than in subsp. strangulata. According to Got2 DNA sequences variation, it was Ae. tauschii subsp. strangulata lineage “k-109″ which donated genome D to Triticum aestivum L. This lineage includes accessions: k-109 from South-Eastern Precaspian Azerbaijan; KU-2105, KU-2159 from Western Precaspian Iran; KU-2080 from Eastern Precaspian Iran.     

Differentiation between two sub-species of <Emphasis Type="Italic">Acacia senegal</Emphasis> complex: <Emphasis Type="Italic">Acacia senegal</Emphasis> (L.) Willd. and <Emphasis Type="Italic">Acacia dudgeoni</Emphasis> Craib ex Holland using morphological traits and molecular markers

The Acacia senegal complex is formed by closely related species of Acacia senegal (L.) Willd. These species share several botanical characters, so from a morphological point of view, there is no clear discontinuity between some of them. A. dudgeoni Craib ex Holland is one species of the A. senegal complex that was formerly described as A. senegal ssp. senegalensis var. samoryana (A. Chev.) Rob. In order to differentiate Acacia senegal from A. dudgeoni, we analyzed a range of morphological traits such as tree height and diameter in natural stands, and, at the nursery stage, seedling height, number of branches, main root depth, biomass dry weight and leaf characteristics. Within addition, molecular polymorphism analyses were conducted using 11 microsatellite markers. Leaf characteristics and molecular markers appear to be the most effective tools to distinguish A. senegal from A. dudgeoni. These tools can improve our understanding of the relationship between two species belonging to the same species complex.     

Genetic diversity among <Emphasis Type="Italic">Jatropha</Emphasis> species as revealed by RAPD markers

The genus Jatropha is native of tropical America with more than 200 species that are widely distributed in tropics with a promise for use as an oil crop for biodiesel. This investigation was carried out to assess the genetic diversity of 12 Jatropha species based on random amplified polymorphic DNA markers. From 26 random primers used, 18 primers gave reproducible amplification banding patterns of 112 polymorphic bands out of 134 bands scored accounting for 80.2% polymorphism across the genotypes. Three primers viz., OPA 4, OPF 11, and OPD 14 generated 100% polymorphic patterns. The polymorphic information content was highest for the primer OPD 14 (0.50) followed by the primers OPF 11 and OPAD 11 (0.48). Jaccard’s coefficient of similarity varied from 0.00 to 0.85, indicative of high level of genetic variation among the genotypes studied. UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L., while second included six species viz., J. ramanadensis Ramam., J. gossypiifolia L., J. podagrica Hook., J. tanjorensis J. L. Ellis et Saroja J. villosa Wight and J. integerrima Jacq. J. glandulifera Roxb. remained distinct and formed third cluster indicating its higher genetic distinctness from other species. The overall grouping pattern of clustering corresponds well with principal component analysis confirming patterns of genetic diversity observed among the species. The result provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources.     

Analysis of genetic variability in <Emphasis Type="Italic">Couepia</Emphasis> accessions using AFLP markers

Couepia is a genus distributed in tropical regions of America. The nut of some Couepia species is used as fresh fruit and oil source for local communities. Despite its critical situation of conservation and its economic potential, there is a lack of information on the genetic variability of Couepia species. This study examines AFLP variation among 40 accessions of Couepia collected in the Colombian Amazonian region, representing two species: Couepia dolichopoda and Couepia subcordata. The individuals were examined for 96 markers generated from four EcoRI/MseI primer pairs, with 80% polymorphism across all accessions studied. According to cluster analysis, 40 accessions were grouped into two major clusters, corresponding to the two species analyzed, except one case whose situation is discussed. In C. dolichopoda accessions, significant correlation between the clustering pattern and the geographical origin was detected; the extent of variation within and among its collect sites was examined by AMOVA. The knowledge about the genetic variability of the accessions examined contributes to development of Couepia conservational efforts.     

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.     

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.     

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.     

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.     

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.