Genetic variation for phenolic acids concentration and composition in a tetraploid wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L.) collection

Phenolic acid intake through the consumption of whole-wheat foods provides important health benefits associated with reduced risks of cardiovascular diseases and colon cancer. The genetic variation for phenolic acids was extensively studied in common wheat, but a comprehensive survey in tetraploid wheat is lacking. In this study we evaluated the genetic variability for individual and total phenolic acids concentration existing in a large collection of tetraploid wheat (Triticum turgidum L.). A 2-year evaluation was undertaken on the whole-meal flour of 111 genotypes belonging to seven T. turgidum subspecies including cultivars, landraces and wild accessions. Durum cultivars [T. turgidum subsp. durum (Desf.) MacKey], had the highest average concentration of total phenolic acids (828.7 μg g^?1 dm in 2012; 834.5 μg g^?1 dm in 2013) with amounts varying from 550.9 μg g^?1 dm to 1701.2 μg g^?1 dm, indicating a variation of greater than threefold fold. The lowest concentration of phenolic acids was found in T. turgidum subsp. dicoccum (Schrank ex Schübler) Thell. Rivet wheat (T. turgidum L. subsp. turgidum) had phenolic acid concentrations similar to those in durum, but less variation was noted among the accessions. On the other hand, the accessions of the four remaining subspecies showed lower phenolic acid concentrations and variation among the accessions as compared to durum. A total of six phenolic acids were identified across the wheat genotypes. The effects of genotype, year and year × genotype were estimated by ANOVA and resulted significant for all phenolic acids. The ratio of genotypic variance to total variance suggested the possibility of improving phenolic acid content in elite wheat germplasm through appropriate breeding programs. Moreover, significant correlations between phenolic acids and other quality characteristics of the grain were detected.     

Remarks on genetic diversity and relationship of <Emphasis Type="Italic">Punica protopunica</Emphasis> and <Emphasis Type="Italic">P. granatum</Emphasis> assessed by molecular analyses

Here, two Punica species, viz., P. protopunica Balf. fil., reported as native to Socotra, and P. granatum L., were compared for the first time. Analysis of one P. protopunica and eleven P. granatum accessions was performed using three molecular markers, i.e., sequence related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), and intron targeted amplified polymorphism (ITAP), along with analysis of pgWD40 sequences, a gene involved in anthocyanin biosynthesis. All markers revealed the relationship between the two species and placed them at 33% similarity. SRAP, TRAP, and ITAP generated a total of 299, 260, and 160 bands, respectively. Of these, 78, 74, and 41 bands were specific for P. protopunica, and 92, 85, and 57 bands, respectively, were shared between both species. Sequence analysis of pgWD40~870 bp amplicons exhibited 100% identity among P. granatum accessions and 98% identity to that of P. protopunica. Phylogenetic analysis of WD40 sequences from monocot and dicot species, including both Punica species confirmed the relation between P. protopunica and P. granatum, supporting earlier reports that P. protopunica could be an ancestral species of P. granatum. Furthermore, the genetic diversity among and within P. granatum accessions from Egypt (3), Mexico (5), and Yemen (3) was assessed. Molecular marker-based relationships among region-bulked accessions was approximately the same (~90% similarity), whereas the degree of genetic variation was altered within each region. Specific bands (alleles) for accessions of each region along with those shared among them were identified. Thus, these bands could be used for pomegranate genotyping and breeding programs.     

Genetic diversity and relationships of wild and cultivated <Emphasis Type="Italic">Zanthoxylum</Emphasis> germplasms based on sequence-related amplified polymorphism (SRAP) markers

The genus Zanthoxylum, belonging to Rutaceae, has a long history of cultivation both for economic and chemical values in China. To effectively conserve and sustainably utilize this genus resource, a study on genetic diversity and relationships of Zanthoxylum germplasms was carried out by employing SRAP markers. We used 16 primer combinations to assess genetic variations and relationships among 175 accessions from eight cultivated provenances, including Shandong, Henan, Shanxi, Shaanxi, Gansu, Sichuan, Guizhou and Yunnan. A total of 145 clear repetitive and intense bands were yielded, and the percentage of polymorphic bands was 100 % for per primer combination, indicating a relatively high diversity among Zanthoxylum germplasms. From a geographic perspective, the highest genetic diversity level was observed within Guizhou provenance (N _a  = 1.97, Ne = 1.52, H = 0.31, I = 0.46) while Henan provenance had the lowest genetic diversity (N _a  = 1.68, Ne = 1.45, H = 0.25, I = 0.37). Based on AMOVA results, the abundant genetic variation was mainly caused by variation of intra-provenances (84.96 %), rather than among provenances (15.038 %). The results indicated low genetic differentiation (G _st  = 0.133) and high gene flow (N _m  = 3.2605) among provenances. The neighbor-joining tree revealed that the 175 accessions could be divided into four groups, and groupings indicated a divergence between the cultivated accessions of Zanthoxylum bungeanum Maxim. and Z. armatum DC. Moreover, three accessions of Z. piperitum DC. var. inerme without prickles introduced from Japan gathered one cluster. Cluster IV is composed of accessions of different geographical origin, including 11 wild species and 10 cultivated accessions of Z. bungeanum. The cluster analysis also reflected a relatively close relationship between the geographical origins and the classification of accessions in cluster I. Structure analysis indicated that collected Zanthoxylum accessions could be divided into two major groups. The information obtained from our research would benefit to make use of Zanthoxylum germplasms and assist the management of a Zanthoxylum germplasms collection.     

Genetic diversity of dihydrochalcone content in <Emphasis Type="Italic">Malus</Emphasis> germplasm

Dihydrochalcones, beneficial phenolic compounds, are abundant in Malus Mill. species, particularly in vegetative tissues and seeds. Phloridzin (phloretin 2′-O-glucoside) is the primary dihydrochalcone in most Malus species including cultivated apple, Malus?×?domestica Borkh. A few species contain sieboldin (3-hydroxyphloretin 4′-O-glucoside) or trilobatin (phloretin 4′-O-glucoside) in place of phloridzin, and interspecific hybrids may contain combinations of phloridzin, sieboldin, and trilobatin. Proposed health benefits of phloridzin include anti-cancer, antioxidant, and anti-diabetic properties, suggesting the potential to breed apples for nutritional improvement. Sieboldin and trilobatin are being investigated for nutritional value and unique chemical properties. Although some of the biosynthetic steps of dihydrochalcones are known, little is known about the extent of variation within Malus germplasm. This research explores the genetic diversity of leaf dihydrochalcone content and composition in Malus germplasm. Dihydrochalcone content was measured using high performance liquid chromatography (HPLC) from leaf samples of 377 accessions, representing 50 species and interspecific hybrids from the USDA-Agricultural Research Service (ARS) National Plant Germplasm System Malus collection. Within the accessions sampled, 284 accessions contained phloridzin as the primary dihydrochalcone, one had only trilobatin, two had phloridzin and trilobatin, 36 had sieboldin and trilobatin, and 54 had all three. Leaf phloridzin content ranged from 17.3 to 113.7 mg/g with a heritability of 0.76 across all accessions. Beyond the potential of dihydrochalcones for breeding purposes, dihydrochalcone composition may be indicative of hybridization or species misclassification.     

Cross-transferability of SSR markers in <Emphasis Type="Italic">Osmanthus</Emphasis>

Developing a molecular tool kit for hybrid breeding of Osmanthus species and related genera is an important step in creating a systematic breeding program for this species. To date, molecular resources have been aimed solely at Osmanthus fragrans with little work to develop markers for other species and cultivars. The objectives of this study were to (1) determine cross-transferability of O. fragrans and Chionanthus retusus derived SSRs in diverse Osmanthus taxa, (2) quantify the influence of locus-specific factors on cross-transferability, and (3) determine the genetic relationships between accessions. We tested 70 SSR markers derived from O. fragrans and C. retusus in 24 accessions of Osmanthus. Sixty-seven markers showed transfer to at least one other Osmanthus species with an overall transfer rate of 84% of loci across taxa. Genotyping with 42 microsatellite markers yielded a total of 367 loci. Number of alleles per locus ranged from 2 to 17 with a mean of 8.7 ± 4.8. Mean observed and expected heterozygosities were 0.560 ± 0.225 and 0.688 ± 0.230, respectively. Percent of polymorphic loci ranged from 40% in Osmanthus delavayi to 100% in O. fragrans. Osmanthus fragrans had the highest mean number of alleles per locus (4.2) while O. delavayi had the lowest (1.1). A reduced suite of eight-markers can distinguish between accessions with non-exclusion probabilities of identity from 3.91E?⁰⁴ to 2.90E?⁰⁷. The SSR markers described herein will be immediately useful to characterize germplasm, identify hybrids, and aid in understanding the level of genetic diversity and relationships within the cultivated germplasm.     

Diversity analysis and evaluation of wild <Emphasis Type="Italic">Abelmoschus</Emphasis> species for agro-morphological traits and major biotic stresses under the north western agro-climatic condition of India

The wild species in general is considered to be the reservoir of genes especially for biotic and abiotic stresses. In okra, the predominant biotic stresses are yellow vein mosaic disease (YVMD), shoot and fruit borer and leaf hopper. Sixty eight (68) accessions belonging to four wild Abelmoschus species [Abelmoschus caillei (A. Chev.) Stevels, Abelmoschus manihot (L.) Medik., Abelmoschus moschatus (L.) Medik. and Abelmoschus tuberculatus Pal et Singh] and eight okra varieties were characterized and evaluated for phenological characters including biotic stresses under natural epiphytotic condition. The wild species examined consisted of 18 accessions (16 exotic and 2 indigenous) of A. caillei, 29 accessions of A. manihot, 16 accessions of A. moschatus and 5 accessions of A. tuberculatus. All the wild Abelmoschus species exhibited high diversity (as measured by Shannon Diversity Index) for 3 qualitative characters viz. intensity of stem colour, leaf shape, epicalyx shape, 13 quantitative characters and 3 biotic stress parameters. Among the wild species, A. caillei and A. tuberculatus showed maximum and minimum diversity for qualitative characters, respectively. There was significant variation for 19 out of 24 quantitative characters studied. Inter-species diversity pattern as estimated through Ward’s Minimum Variance Dendrogram and Principal Component Analysis revealed clear differentiation among the species with minimum overlapping indicating close association between geographical origins and clustering pattern. Intra-species diversity indicated role of specific adaptation in sub-clustering. Resistance to YVMD was found in accessions belonging to three wild species viz. A. caillei, A. manihot and A. moschatus while resistance to shoot and fruit borer and leaf hopper was found in accessions of all the four wild species. The resistant accessions can further be used for introgressing biotic stress resistance through pre-breeding into cultivated okra species.     

Genetic diversity in populations of the medicinal plant <Emphasis Type="Italic">Leonurus cardiaca</Emphasis> L. revealed by inter-primer binding site (iPBS) markers

Motherwort (Leonurus cardiaca L.) is a medicinal plant indigenous to the Mediterranean regions in Europe and Asia. The objective of this study is to apply inter-primer binding site (iPBS) markers to assess the molecular variation and genetic relationships of 89 genotypes of motherwort to assist the genetic improvement of this species. The genotypes comprised 79 from Iran and 10 collected in Australia and 15 additional accessions of two related species (L. heterophyllus Sweet and L. sibiricus L.) collected in Australia, were also included. PCR of 7 iPBS primers (dominant markers) produced a total of 191 bands ranging from 180 to 4000 bp and the mean PIC for primers ranged from 0.2213 to 0.3206 with a mean value 0.2921. The mean expected heterozygosity (0.134), the mean unbiased expected heterozygosity (0.140) and Shannon’s information index (0.213) indicated a high level of inbreeding among the accessions tested. Ordination and cluster analysis showed that the genetic relationships among all accessions could be separated into three major groups—L. cardiaca, L. heterophyllus and L. sibiricus. However, among the 89 accessions of L. cardiaca, genotypes collected from the same geographic region tended to cluster together thus indicating greater genetic similarity. The Motherwort accessions originating in Iran are highly divergent and possess abundant genetic diversity and clearly provide a basis for selection and breeding. The iPBS PCR-based genome fingerprinting technology used in this study is low-cost and effective in differentiating accessions of motherwort and their related species.     

Estimation of natural outcrossing rate and genetic diversity in Lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. var. <Emphasis Type="Italic">lunatus</Emphasis>) from Brazil using SSR markers: implications for conservation and breeding

Lima bean (Phaseolus lunatus L.) is an important food source in Brazil, especially in the northeast region, where its production and consumption are high. The goals of the present study were to estimate natural outcrossing rates and genetic diversity levels of Lima bean from Brazil, using ten microsatellite loci to obtain information for their conservation and breeding. Fourteen accessions were selected from an experiment in field with open-pollinated and with the presence of pollinating insects. Twelve seeds of each of the 14 selected accessions were grown in screenhouse for tissue harvest and DNA extraction. The multilocus model was used to determine the reproductive system. The outcrossing rate was 38.1 % (t_m = 0.381; t_s = 0.078), and the results indicated a mixed mating system with a predominance of selfing (1 ? t_m = 61.9 %). The biparental inbreeding rate was high (t _m  ? t _s  = 0.303) and the multilocus correlated paternity was quite high (r _p(m) = 0.889), indicating that the progeny was mostly composed of full sibs. The average effective number of pollen donors per maternal plant (N _ep ) was low (1.12), and the fixation index for maternal genotypes (F _m ) was 0.945, indicating that most genitors resulted from inbreeding. The studied families presented considerable genetic variability: A = 6.10;  %P = 30; H _e  = 0.60 and H _o  = 0.077. Total diversity was high (H _T = 0.596), and a portion was distributed within families (H _S = 0.058). In addition, diversity was higher between families (D _ST = 0.538), and genetic differentiation was high (G _ST = 0.902). The results presented here can be used in the implementation of Lima bean conservation and breeding programs in Brazil.     

The genetic relationship and structure of some natural interspecific hybrids in <Emphasis Type="Italic">Prunus</Emphasis> subgenus <Emphasis Type="Italic">Prunophora</Emphasis>, based on nuclear and chloroplast simple sequence repeats

In the subgenus Prunophora of the genus Prunus, many transitional traits presented in interspecific hybrids, the so-called ‘new species’, were frequently named due to the complicated botanical classification system. In this study, we used 16 nuclear simple sequence repeats (nSSRs) and 10 chloroplast simple sequence repeats (cpSSRs) to evaluate genetic relationships among 42 accessions, which included 15 putative interspecific hybrids, and then to reveal the speciation and differentiation in the subgenus Prunophora. In total, 231 and 27 alleles were observed in nSSRs and cpSSRs respectively; and with cpSSRs 20 haplotypes were revealed among the accessions. Furthermore, the haplotype and genetic structure analysis implied that (1) Prunus simonii Carr. might be a subspecies or a forma of Prunus salicina L., rather than an interspecific hybrid between P. salicina and Prunus armeniaca L., (2) Prunus limeixing Zhang et Wang was derived from a natural hybrid with P. salicina as its maternal progenitor and P. armeniaca as the female parent, and (3) Prunus cathayana Fu et al. (or kernel-using apricot) was an interspecific hybridization species of P. armeniaca (maternal parent) and Prunus sibirica L. (female parent). These results will be useful for clarifying the problems in the botanical classification, and facilitate the conservation and management of plum and apricot genetic resources in the Chinese National Germplasm Repository for Plums and Apricots.     

Phenetic and genetic diversity in Indian <Emphasis Type="Italic">Luffa</Emphasis> (Cucurbitaceae) inferred from morphometric,ISSR and DAMD markers

Genetic variations and relationships among cultivated and wild genotypes of five taxa of Indian Luffa were examined using inter simple sequence repeats (ISSR), directed amplification of minisatellite DNA (DAMD) markers and morphological characterization. Morphometric evaluation of 21 discrete characters in 51 representative accessions segregated the five taxa of Luffa in three main clusters: the two wild species (L. echinata, L. graveolens) in the first, and the cultivated L. aegyptiaca (smooth gourd) and L. acutangula (ridged gourd)/L. hermaphrodita (Hermaphrodite luffa) in the second and third clusters, respectively. Cumulative data analysis of 15 ISSR and seven DAMD markers revealed high percentage polymorphism (97.67 %), moderate genetic distance (0.06–0.72, avg. 0.51), and low heterozygosity and Shannon index values (H = 0.15; I = 0.22) across all the 76 genotypes assayed. A UPGMA dendrogram, based on the combined marker data, resolved the five taxa in two main clusters with high bootstrap support. The morphological and molecular trees showed incongruence in the number of main clusters resolved and in the disposition of the wild and cultivated taxa in different sub-clusters. The cluster analyses and PCoA plots revealed a nested grouping of the hermaphrodite luffa within the ridge gourd group. The Bayesian STRUCTURE analysis identified three genetic clusters for the five assumed taxa. Outcrossing test revealed a mixed mating system in Indian Luffa. This is the first ever report on the mating system in Luffa using molecular markers. The study also demonstrates the utility of using more than one DNA marker in the assessment of molecular diversity in a widely cultivated crop genus like Luffa with a narrow genetic base.     

Characteristics of the root system in the diploid genome donors of hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Wild crop relatives are of considerable interest in plant breeding and significant efforts have been made to transfer their genetic variation into modern crops. Of the three diploid progenitors of bread wheat (Triticum aestivum L.), only Aegilops tauschii Coss. has been explored and exploited and only for some above ground characteristics. The three wild progenitors (Aegilops speltoides Tausch., Triticum urartu Tumanian ex Gandilyan, and Aegilops tauschii) have never been assayed for root traits. Here we report such a root study, and include Triticum monococcum L. subsp. boeoticum (Boiss.) Hayek and T. turgidum L. subsp. dicoccoides (Koern. ex Asch. et Graebn.) Thell. Fifteen accessions were selected from the above wild species and tested in the presence of one bread wheat cultivar Pavon F76. Significant variation was observed between and within the taxa. Of all accessions tested, cv. Pavon F76 had the smallest root system at maturity while A. speltoides had the largest root system. Moreover, Aegilops spp. had larger mean values for root biomass when compared with Triticum spp. These results suggest there is significant unexplored potential for the use of wheat wild relatives in wheat breeding to improve the root system, or to develop synthetic mapping populations to study root traits.     

Screening species of <Emphasis Type="Italic">Pilocarpus</Emphasis> (Rutaceae) as sources of pilocarpine and other imidazole alkaloids

Plants of the Pilocarpus genus (Rutaceae) are popularly known as jaborandi and are the only source of pilocarpine, an imidazole alkaloid used in eye-drops for the treatment of glaucoma as well as for the stimulation of sweat and lachrymal glands. Alkaloid extracts from leaf samples of seven species of Pilocarpus, from the states of São Paulo and Maranhão in Brazil, were analyzed using HPLC–ESI–MS/MS. The samples contained between 0.88 ± 0.04 and 1.00 ± 0.14% of alkaloids in relation to the dry weight of their leaves, with significant differences in results (P ≤ 0.05) found only between Pilocarpus microphyllus planted in the state of Maranhão and Pilocarpus spicatus, Pilocarpus trachyllophus, Pilocarpus pennatifolius and Pilocarpus jaborandi; as well as between Pilocarpus spicatus and Pilocarpus racemosus. Pilocarpine was not found in P. spicatus, whereas in the other species it ranged from 2.6 ± 0.1 to 70.8 ± 1.2% of total alkaloids. P. microphyllus planted in the state of Maranhão for pilocarpine extraction had the highest total alkaloid content, but it had only 35% of pilocarpine in relation to total alkaloids. Three other species contained more pilocarpine in relation to total alkaloids: P. jaborandi (70.8%), P. racemosus (45.6%) and P. trachyllophus (38.7%); and could be candidates for pilocarpine extraction. Differences in alkaloid content were significant for all these samples (P ≤ 0.05). Imidazole alkaloids were observed and partially characterized based on their retention times and high resolution mass. The seven species analyzed had different imidazole alkaloid profiles, but only one did not present quantifiable pilocarpine contents in its leaves. The Pilocarpus genus shows potential for the prospection of novel alkaloids.     

Chromatographic fingerprinting of <Emphasis Type="Italic">Lupinus luteus</Emphasis> L. (Leguminosae) main secondary metabolites: a case of domestication affecting crop variability

Secondary metabolites (SMs), such as alkaloids and raffinose oligosaccharides (RFOs), play important roles in plant physiology. Although alkaloid and RFO phenotypic variation has been reported for yellow lupin (Lupinus luteus L.), most evaluations have used a reduced number of accessions; thus, limiting the understanding of accumulation patterns and variation ranges. The main goal of this research was to assess alkaloid and RFO content in a diverse L. luteus sample to understand possible SM accumulation patterns across this legume species. Eighteen yellow lupin accessions were analyzed using high performance thin layer chromatography to provide insights on seed and leaf RFO and alkaloid phenotypic variation. Co-dominant markers (170) were used to examine genetic relationships among L. luteus accessions and possible accumulation patterns across closely related genotypes. Significant differences were observed for seed and leaf RFOs. Total seed RFO accumulation ranged from 79.738 to 131.079 mg g^?1. Raffinose, stachyose, and verbascose were observed in all genotypes’ seeds, but at different RFO concentrations. Raffinose was the only RFO detected in leaves (2.793–0.4224 mg g^?1). Total alkaloid accumulation ranged from 0.22 to 15.12 and 0.00 to 8.007 mg g^?1 for seeds and leaves, respectively. Lupinine, sparteine, and gramine were observed in seeds and leaves, and showed a wide range of variation. Neighbor-Joining (NJ) analysis showed an apparent pattern of seed alkaloid accumulation, most likely due to domestication events. However, high RFO accumulating accessions were scattered across the NJ tree. Alkaloid and RFO significant phenotypic variation will not only help to understand the roles of these SMs in L. luteus, but also to uncover the genetic basis behind their accumulation.     

Nuclear and cytoplasmic contributions from <Emphasis Type="Italic">Erianthus arundinaceus</Emphasis> (Retz.) Jeswiet in a sugarcane hybrid clone confirmed through genomic in situ hybridization and cytoplasmic DNA polymorphism

A hybrid between Erianthus arundinaceus (Retz.) Jeswiet and Saccharum spontaneum L. which are wild related species of sugarcane (Saccharum L., Family Poaceae), was repeatedly crossed as female parent with sugarcane commercial varieties to develop near commercial sugarcane clones. The cytoplasm type of the hybrid derivatives were confirmed to be of E. arundinaceus through the mitochondrial and chloroplast DNA polymorphism of nad 4/3-4 intron segment and psbC–trnS segment, respectively. The E. arundinaceus × S. spontaneum hybrid with somatic chromosome number 2n = 62 was confirmed to have 30 chromosomes from E. arundinaceus through genomic in situ hybridization (GISH). The (E. arundinaceus × S. spontaneum) × sugarcane hybrid (2n = 118) had 24 chromosomes from E. arundinaceus whereas its next generation hybrid with sugarcane (2n = 108) had only 12 Erianthus chromosomes. The commercial sugarcane hybrid Co 15015, which is the third generation hybrid with 2n = 106 was confirmed to have two E. arundinaceus chromosomes through GISH. It is the first report of sugarcane with both alien cytoplasm and chromosome contributions from E. arundinaceus.     

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.     

Relationships among <Emphasis Type="Italic">Crataegus</Emphasis> accessions sampled from Hatay,Turkey, as assessed by fruit characteristics and RAPD

The genus Crataegus known as hawthorns, is the largest genus among the Maloideae, which comprises 265 species. Turkey is one of the genetic centers of Crataegus and there are more than 20 species found in Turkey. The fruits of Crataegus are used as food and have high flavonoid, vitamin C, glycoside, anthocyanidin, saponin, tannin, and antioxidant levels. In this study, we attempted to characterize 15 Crataegus accessions sampled from Hatay, located in Eastern Mediterranean region of Turkey. The accessions belonged to several species; C. aronia (L.) DC. var. aronia, C. aronia var. dentata Browicz, C. aronia var. minuta Browicz, C. monogyna Jacq. subsp. azarella (Griseb.) Franco, and C. orientalis Pall. ex M. Bieb. var. orientalis. Fruit characteristics of the accessions exhibited considerable variation. The multivariate, principle component and cluster analyses indicated that the accessions belonged to three groups: (1) C. aronia var. arona accessions; (2) C. aronia var. dentata accessions; and, (3) C. monogyna subsp. azarella and C. orientalis var. orientalis accessions. The principle component analysis results also revealed that the first three components explained 46, 21, and 14% of the variation, comprising a total of 81%. The fruit length and width, leaf area, and soluble solids contents were highly correlated characteristics for the first three components. The 19 RAPD primers generated a total of 107 bands, where 76 of these were polymorphic. The molecular data analyses by principle coordinate and clustering showed similar results to those of pomological characteristics. There were three groups, (1) C. aronia var. arona accessions; (2) C. aronia var. dentata accession; and, (3) C. monogyna subsp. azarella. C. orientalis var. orientalis accession grouped with C. aronia var. arona accessions. Therefore, it can be concluded that, overall, the diversity patterns of pomological and molecular data, generated by RAPD, for Crataegus are in good agreement and the accessions of C. aronia var. aronia, C. aronia var. minuta, C. monogyna subsp. azarella and C. orientalis var. orientalis accessions.     

C-banded karyotype of <Emphasis Type="Italic">Thinopyrum bessarabicum</Emphasis> and identification of its chromosomes in wheat background

C-banded pattern in two accessions of Thinopyrum bessarabicum (Save ex Rayss) A. Löve (2n = 2x = 14, E^bE^b) and their idiogram was established. C-banding analysis was further used to identify the chromosomes of Tritipyrum amphiploid (2n = 6x = 42, AABBE^bE^b) and a BC₁F₂ genotype from wheat and Tritipyrum. Two 18S-26S rDNA loci were detected on Th. bessarabicum chromosomes by in situ hybridization using an 18S-26S rDNA probe. E^b chromosomes in Tritipyrum generally were identified by their distinctive C-banding patterns which reflected heterochromatin regions. C-banding procedure resulted in sharp and distinct bands in one or both ends of E^b chromosomes without interval bands. Observed C-bands in E^b genome mainly reflected the telomeric and subtelomeric sequences which also showed more strong signals in genomic in situ hybridization. Results showed the importance of the C-banding technique as a screening tool in identification of addition and substitution lines in the progenies of wheat and Tritipyrum crosses during segregating generations.     

<Emphasis Type="Italic">Marsilea quadrifolia:</Emphasis> a New Bioagent for Treating Wastewater

We report for the first time the capability of four-leaf clover (Marsilea quadrifolia), a wetland plant which grows rooted in soil, in efficiently treating sewage. The use of M. quadrifolia was made possible because of the special attributes of the SHEFROL^® (SHEet Flow ROot Level) bioreactor in which it was employed. This bioreactor enables the use of free-floating aquatic plants as well as terrestrial and rooted-in-soil wetland plants by hydroponics. The plants are staked in narrow channels to enable them to support each other while sewage is made to flow rapidly as a sheet of wastewater at a level that covers only the plant roots (hence the name). It was seen that M. quadrifolia was able to treat sewage of strength varying in the chemical oxygen demand (COD) range of 600–1800 mg/L to the extent of >?80% at a hydraulic retention time (HRT) of just 4.5 h. There was a near total removal of biological oxygen demand and suspended solids while total Kjeldahl nitrogen, soluble phosphorous, and heavy metal zinc were also substantially removed. The macrophyte was equally effective when used indoors under artificial lighting, as well as when used outdoors.     

Raffinose family oligosaccharides in seeds of <Emphasis Type="Italic">Pisum</Emphasis> wild taxa,type lines for seed genes,domesticated and advanced breeding materials

The content of raffinose family oligosaccharides (RFOs) in pea seeds constrains their usage in feeding humans and animals. In our research, the content of soluble carbohydrates—particularly α-d-galactosides of sucrose (RFOs)—was analyzed. The materials were as follows: 248 accessions from the Polish Pisum Genebank including representatives of taxa (from species to convarietas), type lines for genes controlling seed characters, and breeding materials and cultivars. Accessions were divided into homogeneous groups considering content of total soluble carbohydrates, total RFOs and individual RFOs: raffinose, stachyose and verbascose. The highest content of total soluble carbohydrates and total RFOs were stated for accessions with wrinkled seeds (r and rb genes) and the lowest content for seeds of the wild species P. fulvum Sibth. et Sm. Accessions valuable for breeding (for further decreasing of anti-nutritional compounds) were found among domesticated taxa (P. sativum subsp. sativum convar. vulgare Alef. and speciosum (Dierb.) Alef.), breeding lines, and some wild taxa. Accessions with decreased content of a total RFOs and verbascose are particularly valuable. It was found that the content of total RFOs was the most highly, frequently, and positively correlated with a stachyose and verbascose content. However, in P. fulvum seeds with the lowest content of RFOs and verbascose, a high correlation between the content of total RFOs and stachyose was revealed. Contents of all oligosaccharides were substantially lower in lines with dominant alleles of main pea seed genes (R, A, and I). It can be assumed that wild, primitive peas were characterized by not-all-to-high (rather not high) content of oligosaccharides; then recessive mutations in mentioned genes resulted in an increased content of RFOs. It seems to be an interesting observation from an evolutionary point of view.