A hypervariable intron of the STAYGREEN locus provides excellent discrimination among Pisum fulvum accessions and reveals evidence for a relatively recent hybridization event with Pisum sativum.

An analysis of 82 non-synonymous Pisum fulvum accessions for sequence variation in a fragment of the STAYGREEN (SGR) locus revealed 57 alleles, most of which differed in indel structure. Eight additional P. fulvum accessions, each supposedly synonymous with a different accession of the initial group, were also analyzed. In every case the paired synonymous accessions possessed the same SGR sequence but varied slightly for a 6-trait morphological phenotype, indicating that SGR sequence is a much more reliable indicator of accession identity than is a morphological characterization. SGR sequence analysis confirmed our previous finding that P. fulvum accessions separate into two allele groups. This division was not supported by results of previous studies that were based on sequences distributed across the entire genome, suggesting that the division may have been produced by selection at a nearby locus and that the SGR phylogeny may not be good indicator of overall relationships within the species. One P. fulvum accession, PI 595941 (=JI1796), displayed an SGR sequence outside the variation typical of the species. Instead, its allele resembled alleles limited to a set of Pisum sativum landraces from the Middle East, suggesting hybridization between ancestors of PI 595941 and some primitive form of domesticated P. sativum. With one exception from the extreme northwest corner of Israel, P. fulvum accessions collected north of latitude 35.5° N were fixed for alleles from group A. These northern accessions also displayed greatly reduced SGR sequence diversity compared to group A accessions collected from other regions, suggesting that the northern populations may represent recent extensions of the range of the species. Group B accessions were distributed from Lake Tiberias south and were generally sympatric with the southern group A accessions. Although group B accessions occupied a smaller area than group A, the SGR sequence diversity in this group (28 alleles in 33 accessions) exceeded that for group A.

Distinguishing between Bread Wheat and Spelt Grains Using Molecular Markers and Spectroscopy.

The increasing demand for spelt products requires the baking industry to develop accurate and efficient tools to differentiate between spelt and bread wheat grains. We subjected a 272-sample spelt-bread wheat set to several potential diagnostic methods. DNA markers for γ-gliadin-D ( GAG56D), γ-gliadin-B ( GAG56B), and the Q-gene were used, alongside phenotypic assessment of ease-of-threshing and near-infrared spectroscopy (NIRS). The GAG56B and GAG56D markers demonstrated low diagnostic power in comparison to the Q-gene genotyping, which showed full accordance with the threshing phenotype, providing a highly accurate distinction between bread wheat and spelt kernels. A highly reliable Q classification was based on a three-waveband NIR model [Kappa (0.97), R-square (0.93)], which suggested that this gene influences grain characteristics. Our data ruled out a protein concentration bias of the NIRS-based diagnosis. These findings highlight the Q gene and NIRS as important, valuable, but simple tools for distinguishing between bread wheat and spelt.

Prevalence, Development, and Significance of Ascochyta Blight Caused by Peyronellaea pinodes in Pisum elatius Populations Growing in Natural Ecosystems.

Wild Pisum populations prevail in Israel in regions with diverse climatic conditions. A comprehensive survey was conducted in the winters of 2007-08 and 2008-09 at two sites in northern Israel, aiming to (i) document the density of Pisum elatius plants in natural ecosystems and elucidate factors related to their initial infection by Ascochyta blight and (ii) determine the factors governing disease development over time on individual plants. The surveyors identified P. elatius plants growing in designated quadrats, inspected each plant visually, and recorded the incidence and severity of its Ascochyta blight symptoms. Ascochyta blight, caused by Peyronellaea pinodes, was ubiquitous in Pisum elatius populations at both survey sites in both seasons. However, the total leaf area exhibiting disease symptoms of individual plants was very low, and stem and pod infections were rarely observed. Based on analyses of the survey data, it was suggested that, in natural ecosystems, the teleomorph stage of Peyronellaea pinodes serves as the main source of the primary and the secondary inoculum of the disease. In addition, it was found that infected leaves dropped off soon after infection, thereby precluding development of stem lesions. The plants continued growing and did not die; thus, they overcame the disease and could be considered "cured". This phenomenon was examined and confirmed in artificially inoculated, potted-plant experiments. It would be worthwhile to exploit the potential of this unique resistance mechanism as a tool for Ascochyta blight management in pea breeding.

The Temperature Response and Aggressiveness of Peyronellaea pinodes Isolates Originating from Wild and Domesticated Pisum sp. in Israel.

Domesticated pea fields are grown in relatively close proximity to wild pea species in Israel. Despite the major role attributed to ascochyta blight in causing yield losses in domesticated pea, very limited information is available on the pathogens prevailing in natural ecosystems. The objectives of this study were (i) to identify the species causing ascochyta blight symptoms on leaves, stems, and petioles of domesticated pea and wild Pisum plants in Israel, and (ii) to quantify the temperature response(s) and aggressiveness of such pathogens originating from Pisum plants growing in sympatric and allopatric contexts. Eighteen fungal isolates were examined and identified; three of them were sampled from Pisum sativum, 11 from Pisum fulvum, and four from Pisum elatius. All isolates were identified as Peyronellaea pinodes. Spore germination and mycelial growth took place over a wide range of temperatures, the lower and upper cardinal temperatures being 2 to 9 and 33 to 38°C, respectively; the optimal temperatures ranged from 22 to 26°C. At an optimal temperature, disease severity was significantly higher for plants maintained under moist conditions for 24 h postinoculation than for those exposed to humidity for 5 or 10 h. Analyses of the data revealed that temperature responses, spore germination rates, and aggressiveness of isolates sampled from domesticated pea plants did not differ from those of isolates sampled from adjacent or distant wild populations. Host specificity was not observed. These observations suggest that Israel may be inhabited by a single metapopulation of P. pinodes.

Experimental growing of wild pea in Israel and its bearing on Near Eastern plant domestication.

BACKGROUND AND AIMS: The wild progenitors of the Near Eastern legumes have low germination rates mediated by hardseededness. Hence it was argued that cultivation of these wild legumes would probably result in no yield gain. Based on the meagre natural yield of wild lentil and its poor germination, it was suggested that wild Near Eastern grain legumes were unlikely to have been adopted for cultivation unless freely germinating types were available for the incipient farmers. Unlike wild cereals, data from experimental cultivation of wild legumes are lacking. METHODS: Replicated nurseries of wild pea (Pisum elatius, P. humile and P. fulvum) were sown during 2007-2010 in the Mediterranean district of Israel. To assess the effect of hardseededness on the yield potential, seeds of the wild species were either subjected to scarification (to ensure germination) or left intact, and compared with domesticated controls. KEY RESULTS: Sowing intact wild pea seeds mostly resulted in net yield loss due to poor establishment caused by wild-type low germination rates, while ensuring crop establishment by scarification resulted in net, although modest, yield gain, despite considerable losses due to pod dehiscence. Harvest efficiency of the wild pea plots was significantly higher (2-5 kg seeds h(-1)) compared with foraging efficiency in wild pea populations (ranging from a few grams to 0·6 kg h(-1)). CONCLUSIONS: Germination and yield data from 'cultivation' of wild pea suggest that Near Eastern legumes are unlikely to have been domesticated via a protracted process. Put differently, the agronomic implications of the hardseededness of wild legumes are incompatible with a millennia-long scenario of unconscious selection processes leading to 'full' domestication. This is because net yield loss in cultivation attempts is most likely to have resulted in abandonment of the respective species within a short time frame, rather than perpetual unprofitable cultivation for several centuries or millennia.

Different ecological affinities and aggressiveness patterns among Didymella rabiei isolates from sympatric domesticated chickpea and wild Cicer judaicum.

Domesticated chickpea (Cicer arietinum) and its wild relative C. judaicum grow in sympatric distribution in Israel and both are susceptible to Ascochyta blight caused by Didymella rabiei. C. arietinum was grown for millennia in drier and hotter Levantine spring conditions while C. judaicum grows in the wetter and milder winters. Accordingly, it is possible that D. rabiei isolates originated from C. arietinum are adjusted to the less favorable spring conditions. Here, 60 isolates from both origins were tested in vitro for their hyphal growth at 15 and 25 degrees C. Isolates from C. arietinum had a significantly larger colony area at 25 degrees C than at 15 degrees C (P < 0.001) while no such differences were detected between isolates from C. judaicum. D. rabiei isolates from wild and domesticated origins were used to inoculate nine C. judaicum accessions and two domesticated chickpea cultivars and their aggressiveness patterns were determined using five measures. On domesticated chickpea, isolates from domesticated origin were significantly more aggressive in four out of the five aggressiveness measures than isolates from wild origin. On C. judaicum, isolates from wild origin were generally more aggressive than isolates from domesticated origin. The results suggest that the habitat segregation between wild and domesticated Cicer influences the pathogens ecological affinities and their aggressiveness patterns.

Quantitative trait loci governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.).

Chickpea is a staple protein source in many Asian and Middle Eastern countries. The seeds contain carotenoids such as beta-carotene, cryptoxanthin, lutein and zeaxanthin in amounts above the engineered beta-carotene-containing "golden rice" level. Thus, breeding for high carotenoid concentration in seeds is of nutritional, socio-economic, and economic importance. To study the genetics governing seed carotenoids in chickpea, we studied the relationship between seed weight and concentrations of beta-carotene and lutein by means of high-performance liquid chromatography in segregating progeny from a cross between an Israeli cultivar and wild Cicer reticulatum Ladiz. Seeds of the cross progeny varied with respect to their carotenoid concentration (heritability estimates ranged from 0.5 to 0.9), and a negative genetic correlation was found between mean seed weight and carotenoid concentration in the F(3). To determine the loci responsible for the genetic variation observed, the population was genotyped using 91 sequence tagged microsatellite site markers and two CytP450 markers to generate a genetic map consisting of nine linkage groups and a total length of 344.6 cM. Using quantitative data collected for beta-carotene and lutein concentration and seed weight of the seeds of the F(2) population, we were able to identify quantitative trait loci (QTLs) by interval mapping. At a LOD score of 2, four QTLs for beta-carotene concentration, a single QTL for lutein concentration and three QTLs for seed weight were detected. The results of this investigation may assist in improving the nutritional quality of chickpea.

Construction of BAC and BIBAC libraries and their applications for generation of SSR markers for genome analysis of chickpea, Cicer arietinum L.

Large-insert bacterial artificial chromosome (BAC) libraries, plant-transformation-competent binary BAC (BIBAC) libraries, and simple sequence repeat (SSR) markers are essential for many aspects of genomics research. We constructed a BAC library and a BIBAC library from the nuclear DNA of chickpea, Cicer arietinum L., cv. Hadas, partially digested with HindIII and BamHI, respectively. The BAC library has 14,976 clones, with an average insert size of 121 kb, and the BIBAC library consists of 23,040 clones, with an average insert size of 145 kb. The combined libraries collectively cover ca. 7.0 x genomes of chickpea. We screened the BAC library with eight synthetic SSR oligos, (GA)10, (GAA)7, (AT)10, (TAA)7, (TGA)7, (CA)10, (CAA)7, and (CCA)7. Positive BACs were selected, subcloned, and sequenced for SSR marker development. Two hundred and thirty-three new chickpea SSR markers were developed and characterized by PCR, using chickpea DNA as template. These results have demonstrated that BACs are an excellent source for SSR marker development in chickpea. We also estimated the distribution of the SSR loci in the chickpea genome. The SSR motifs (TAA)n and (GA)n were much more abundant than the others, and the distribution of the SSR loci appeared non-random. The BAC and BIBAC libraries and new SSR markers will provide valuable resources for chickpea genomics research and breeding (the libraries and their filters are available to the public at http://hbz.tamu.edu).

Cytogenetics of semi-fertile triploid and aneuploid intergeneric vine cacti hybrids.

Crosses between the diploid Hylocereus polyrhizus, as the female parent, and the tetraploid Selenicereus megalanthus, as the male parent, yielded triploid and aneuploid hybrids. The fruits of these hybrids combined the attractive appearance of Hylocereus fruits with the delicious taste of S. megalanthus fruits. The aim of this work was to assess the fertility and breeding potential of the triploid and aneuploid hybrids with a view to developing an improved vine cactus crop. Pollen mother cells at metaphase I revealed univalents, bivalents, trivalents, and occasionally quadrivalents. Chromosome distribution at anaphase I revealed different classes of chromosome segregation as well as lagging chromosomes. At metaphase II, parallel and tripolar spindles were observed. The occurrence of triads was frequent, whereas dyads were rarely observed. Pollen stainability varied among the clones studied ranging from 9.8% to 18.6%. The diameters of the stained pollen grains varied widely, probably as a result of the number of chromosomes. Despite the allotriploid origin of our hybrids, functional female and male gametes were produced in considerable proportions, most likely as a result of balanced chromosome segregation. The triploid and aneuploid clones studied yielded viable seeds whose number per fruit was strongly dependent on the pollen donor.

Chromosome doubling in vine cacti hybrids.

We performed reciprocal crosses between the tetraploid Selenicereus megalanthus and the diploid Hylocereus species, H. undatus and H. polyrhizus. S. megalanthus x H. undatus gave rise to viable hexaploids and 6x-aneuploid hybrids rather than to the expected triploids. No genuine hybrids were obtained in the reciprocal cross. The pollen diameter of the tetraploid S. megalanthus varied widely, indicating the occurrence of unreduced gametes, while that of H. undatus pollen was very uniform, indicating an extremely low frequency of unreduced gametes. This finding suggests that the hexaploids were formed by chromosome doubling after the formation of the hybrid triploid zygote rather than by fusion of unreduced gametes of the two species.

Biometric Analyses of the Inheritance of Resistance to Didymella rabiei in Chickpea.

ABSTRACT Historically, the response of chickpea (Cicer arietinum L.) to Didymella rabiei (causal agent of Ascochyta blight) has been mainly related to as complete resistance and it was commonly assayed with qualitative (nonparametric) scales. Two reciprocal populations, derived from intra-specific crosses between a moderately resistant late flowering Israeli cultivar and a highly susceptible early flowering Indian accession, were tested at F(3) and F(4) generations in 1998 and 1999, respectively. A quantitative (parametric) assessment (percent disease severity) was used to evaluate the chickpea field response to Ascochyta blight. The transformed relative area under the disease progress curve (tRAUDPC) was calculated for each experimental unit for further analyses. Heritability estimates of the tRAUDPC were relatively high (0.67 to 0.85) in both generations for both reciprocal populations. The frequency distributions of tRAUDPC of the populations were continuous and significantly departed from normality (Shapiro-Wilk W test; P of W < 0.0001), being all platykurtic and skewed toward either the resistant or the susceptible parental lines. The presence of major genes was examined by testing the relationship between the F(3) and F(4) family means and the within-family variances (Fain's test). Analyses of these relationships suggested that segregation of a single (or few) quantitative trait locus with major effect and possibly other minor loci was the predominant mode of inheritance. The correlation estimates between the resistance and days to flower (r = -0.19 to -0.44) were negative and significantly (P = 0.054 to 0.001) different from zero, which represents a breeding constraint in the development of early flowering cultivars with Ascochyta blight resistance.

Vernalization response of wild chickpea.

• Response to low temperature during early growth in cultivated chickpea (Cicer arietinum) and its wild progenitor C. reticulatum was investigated to clarify the evolutionary processes under domestication in this crop. • Parental lines and their F2 and F3 progeny were exposed to cold treatment (4°C) for 30°d after seed imbibition and compared with controls. • Cold treatment caused a 19-d advance in flowering time in wild chickpea, but only a 3-d advance in cultivated chickpea. It also promoted apical dominance of the main stem of the wild chickpea, whereas apical dominance was constitutive in the cultivated type. F3 progeny showed significant genetic variation affecting the response of flowering time to low temperature. We suggest that selection against alleles conferring vernalization requirements was a major step in the evolution of cultivated chickpea. The reduced low-temperature response was fundamental both for the ancient conversion of chickpea from an autumn- to a spring-sown crop ('summer crop') in west Asia, and for its spread into the lower-latitude regions of India and east Africa. • Attempts to improve yield and/or resistance to biotic and abiotic stresses through introgression with wild chickpea species carry the risk of reintroducing vernalization sensitive alleles into the cultigen.

A new hyperrecombinogenic mutant of Nicotiana tabacum.

We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six- to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma-irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.

Cytology and mating systems in the climbing cacti Hylocereus and Selenicereus.

Chromosome numbers and meiotic behavior are reported for the climbing cacti species Hylocereus undatus, Hylocereus polyrhizus, and Selenicereus megalanthus. The Hylocereus spp. are diploid (2n = 22), while S. megalanthus is a tetraploid (2n = 44). Irregular chromosome disjunction at anaphase I in pollen mother cells of S. megalanthus is probably the major cause of its reduced pollen viability and may contribute to low seed set, low number of viable seeds and, consequently, low fruit mass. A pollination study confirmed self-incompatibility in H. polyrhizus and a weakened incompatibility reaction in H. undatus and S. megalanthus. Major crossability barriers do not exist between the Hylocereus spp. investigated. Reciprocal intergeneric crosses were successful between Hylocereus spp. and S. megalanthus, suggesting that an Hylocereus sp. might be one of the diploid progenitors of the tetraploid S. megalanthus. The implications of the results on cacti nomenclature and systematics are briefly discussed.

Rapid elimination of low-copy DNA sequences in polyploid wheat: a possible mechanism for differentiation of homoeologous chromosomes.

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects.

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed that Aegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation of Triticum and Aegilops diploid species.

Isolation of two differentially expressed wheat ACC synthase cDNAs and the characterization of one of their genes with root-predominant expression.

Two partial 1-aminocyclopropane-1-carboxylic acid (ACC) synthase cDNA clones (pWAS1, 1089 bp; and pWAS3, 779 bp) were isolated by polymerase chain reaction (PCR) using cDNA to total mRNA purified from etiolated wheat seedlings as template and degenerate oligonucleotides synthesized based on the regions of the ACC synthase amino acid sequence that are highly conserved among different plants. Northern analysis showed that the expression of the corresponding genes are differentially regulated. While the transcripts of pWAS1 were found in all the tissues of wheat that were tested with a maximum level at the early stages of spike development, pWAS3 mRNA was present almost exclusively in the root. A 5590 bp genomic clone, TA-ACS2, corresponding to pWAS3 cDNA has been isolated. The TA-ACS2 sequence consists of a 589-bp 5'-upstream region, 2743 bp of transcribed region with four exons and three introns and a 3'-downstream region of 2257 bp. Expression in Escherichia coli confirmed the ACC synthase activity of TA-ACS2 polypeptide. Sequence comparisons show that the two wheat ACC synthases are more similar to each other and to the rice ACC synthase, OS-ACS1, at the nucleotide level than at the amino acid level. The amino acid sequence of TA-ACS2 is most similar (66.1% identity) to that of broccoli. The chromosomal location of both wheat ACC synthase genes have been determined by aneuploid analysis. TA-ACS1 is located on the short arm of chromosomes 7A and 7D and on the long arm of chromosome 4A. TA-ACS2 is located on the long arm of homoeologous group 2 chromosomes.