Allelic Diversity of Acetyl Coenzyme A Carboxylase accD/bccp Genes Implicated in Nuclear-Cytoplasmic Conflict in the Wild and Domesticated Pea (Pisum sp.).

Reproductive isolation is an important component of species differentiation. The plastid accD gene coding for the acetyl-CoA carboxylase subunit and the nuclear bccp gene coding for the biotin carboxyl carrier protein were identified as candidate genes governing nuclear-cytoplasmic incompatibility in peas. We examined the allelic diversity in a set of 195 geographically diverse samples of both cultivated (Pisum sativum, P. abyssinicum) and wild (P. fulvum and P. elatius) peas. Based on deduced protein sequences, we identified 34 accD and 31 bccp alleles that are partially geographically and genetically structured. The accD is highly variable due to insertions of tandem repeats. P. fulvum and P. abyssinicum have unique alleles and combinations of both genes. On the other hand, partial overlap was observed between P. sativum and P. elatius. Mapping of protein sequence polymorphisms to 3D structures revealed that most of the repeat and indel polymorphisms map to sequence regions that could not be modeled, consistent with this part of the protein being less constrained by requirements for precise folding than the enzymatically active domains. The results of this study are important not only from an evolutionary point of view but are also relevant for pea breeding when using more distant wild relatives.

Out of southern Africa: Origin, biogeography and age of the Aizooideae (Aizoaceae).

The Aizooideae is an early-diverging lineage within the Aizoaceae. It is most diverse in southern Africa, but also has endemic species in Australasia, Eurasia and South America. We derived a phylogenetic hypothesis from Bayesian and Maximum Likelihood analyses of plastid DNA-sequences. We find that one of the seven genera, the fynbos-endemic Acrosanthes, does not belong to the Aizooideae, but is an ancient sister-lineage to the subfamilies Mesembryanthemoideae & Ruschioideae. Galenia and Plinthus are embedded inside Aizoon and Aizoanthemum is polyphyletic. The Namibian endemic Tetragonia schenckii is sister to Tribulocarpus of the Sesuvioideae. For the Aizooideae, we explored their possible age by means of relaxed Bayesian dating and used Bayesian Binary MCMC reconstruction of ancestral areas to investigate their area of origin. Early diversification occurred in southern Africa in the Eocene-Oligocene, with a split into a mainly African lineage and an Eurasian-Australasian-African-South American lineage. These subsequently radiated in the early Miocene. For Tetragonia, colonisation of Australasia via long-distance dispersal from Eurasia gave rise to the Australasian lineage from which there were subsequent dispersals to South America and Southern Africa. Despite the relatively old age of the Aizooideae, more than half the species have radiated since the Pleiocene, coinciding with the large and rapid diversification of the Ruschioideae. The lineage made up of Tetragonia schenckii &Tribulocarpus split from the remainder of the Sesuvioideae already in the mid Oligocene and its disjunct distribution between Namibia and north-east Africa may be the result of a previously wider distribution within an early Arid African flora. Our reconstruction of ancestral character-states indicates that the expanding keels giving rise to hygrochastic fruits originated only once, i.e. after the split of the Sesuvioideae from the remainder of the Aizoaceae and that they were subsequently lost many times. Variously winged and spiky fruits, adapted to dispersal by wind and animals, have evolved independently in the Aizooideae and the Sesuvioideae. There is then a greater diversity of dispersal systems in the earlier lineages than in the Mesembryanthemoideae and Ruschioideae, where dispersal is mainly achieved by rain.

A phylogenetic hypothesis for the recently diversified Ruschieae (Aizoaceae) in southern Africa.

The Ruschieae is a large tribe of about 1600 species of succulent perennials. They form a major component of the arid parts of the Greater Cape Floristic Region, both in numbers of species and in their density of coverage. So far phylogenetic relationships within the tribe have been unresolved, largely through the paucity of variable molecular characters and this is ascribed to the tribe's recent and rapid radiation. Our phylogeny is based on 10 chloroplast gene regions and represents a nearly complete sampling of the 100 currently recognised genera of the Ruschieae. These chloroplast regions yielded relatively few phylogenetically informative characters, consequently providing only limited resolution in and poor support for many parts of the phylogeny. Nevertheless, for the first time, we provide well-supported evidence that taxa with mostly mesomorphic, often ephemeral leaves and weakly persistent fruits form a basal grade of lineages in the Ruschieae. These lineages subtend a large polytomy of taxa with almost exclusively xeromorphic, persistent leaves and strongly persisting fruits. Among the basal grade of lineages, those occurring within the winter-rainfall region typically shed their leaves or form (at least partly) a protective, dry sheath around the apical bud during the dry summer months, as a means of escaping the summer drought. This contrasts with taxa of the basal grade from outside the winter-rainfall region, in which the leaves persist. Our results show that, in both strongly and weakly persistent fruits, specialised characteristics of the fruit evolved repeatedly and so these structures are highly homoplasious. Perhaps as a consequence of repeated changes towards increased persistence and specialisation of leaves and fruits, several clades show little morphological cohesion. However, as in other groups in the Cape Flora, most clades in the Ruschieae represent regional groupings. Our analysis of sequences of the nuclear gene 'chloroplast-expressed glutamine synthetase' (ncpGS) revealed extensive paralogy within the Ruschieae, but found an intact reading frame in all its members. More data on the cytology of the Ruschieae is needed to evaluate whether the paralogy observed is due to gene duplication or polyploidy.

Phenylpropanoid Content of Chickpea Seed Coats in Relation to Seed Dormancy.

The physical dormancy of seeds is likely to be mediated by the chemical composition and the thickness of the seed coat. Here, we investigate the link between the content of phenylpropanoids (i.e., phenolics and flavonoids) present in the chickpea seed coat and dormancy. The relationship between selected phenolic and flavonoid metabolites of chickpea seed coats and dormancy level was assessed using wild and cultivated chickpea parental genotypes and a derived population of recombinant inbred lines (RILs). The selected phenolic and flavonoid metabolites were analyzed via the LC-MS/MS method. Significant differences in the concentration of certain phenolic acids were found among cultivated (Cicer arietinum, ICC4958) and wild chickpea (Cicer reticulatum, PI489777) parental genotypes. These differences were observed in the contents of gallic, caffeic, vanillic, syringic, p-coumaric, salicylic, and sinapic acids, as well as salicylic acid-2-O-ß-d-glucoside and coniferaldehyde. Additionally, significant differences were observed in the flavonoids myricetin, quercetin, luteolin, naringenin, kaempferol, isoorientin, orientin, and isovitexin. When comparing non-dormant and dormant RILs, significant differences were observed in gallic, 3-hydroxybenzoic, syringic, and sinapic acids, as well as the flavonoids quercitrin, quercetin, naringenin, kaempferol, and morin. Phenolic acids were generally more highly concentrated in the wild parental genotype and dormant RILs. We compared the phenylpropanoid content of chickpea seed coats with related legumes, such as pea, lentil, and faba bean. This information could be useful in chickpea breeding programs to reduce dormancy.

Evolutionary relationships and taxonomy of Microtea (Microteaceae), a basal lineage in the core Caryophyllales.

The basal position of the small American genus Microtea within the core Caryophyllales was suggested only recently in accordance with molecular phylogeny. However, the specific relationships within the genus were not traced. The results of our phylogenetic analysis based on the matK chloroplast gene suggest the monophyly of Microtea, and Ancistrocarpus and other related genera should be included in it. Microtea is divided into two major sister clades: clade A consisting of M.glochidiata, M.maypurensis and M.tenuifolia, and clade B comprising M.debilis, M.sulcicaulis, M.scabrida, M.celosioides, and M.papillosa. The nrDNA dataset (ITS), although containing only a limited number of accessions, shows the same species number in clade A, and the remaining species studied (M.debilis, M.scabrida and M.celosioides) form clade B. Subgeneric status is assigned to clades A and B corresponding with the names Microteasubgen.Ancistrocarpus subgen. nov. and Microteasubgen.Microtea, respectively. The diagnostic characters at the subgeneric level are as follows: length of pedicels, number of flowers at each node, number of stamens and styles. A multivariate analysis of 13 distinguishing morphological characters supports the results of phylogenetic analysis. All species have similar pericarp and seed ultrasculpture and anatomy, and they share the reticulate pericarp surface (independent of presence or absence of finger-shaped outgrowths on its surface) and rugose or slightly alveolate seed ultrasculpture. On the basis of morphological characters, we accept 10 Microtea species. A checklist includes a new diagnostic key, morphological descriptions and distribution patterns of each species. Galeniacelosioides is the oldest legitimate name available for the plants previously known as Microteapaniculata, for which the combination Microteacelosioides is validated here. The neotypes of Galeniacelosioides and Microteasprengelii were designated from the collections of Prinz Wied at BR. The name M.foliosa is discussed and finally synonymized with M.scabrida. The lectotypes of Ancistrocarpusmaypurensis (≡Microteamaypurensis), Microteadebilisvar.ovata (=M.debilis), M.glochidiata, M.maypurensisvar.angustifolia (=M.tenuifolia), M.glochidiataf.lanceolata (=M.maypurensis), M.longebracteata (=M.celosioides), M.paniculatavar.latifolia (=M.scabrida), M.portoricensis, M.scabrida, M.sulcicaulis, and Potamophilaparviflora (=M.maypurensis) are designated. Microteasulcicaulis is reported for the first time as native to Bolivia, and M.maypurensis is reported from Indonesia (Java), where it is found as an alien plant with an unclear invasion status.

Genetic structure of wild pea (Pisum sativum subsp. elatius) populations in the northern part of the Fertile Crescent reflects moderate cross-pollination and strong effect of geographic but not environmental distance.

Knowledge of current genetic diversity and mating systems of crop wild relatives (CWR) in the Fertile Crescent is important in crop genetic improvement, because western agriculture began in the area after the cold-dry period known as Younger Dryas about 12,000 years ago and these species are also wild genepools of the world's most important food crops. Wild pea (Pisum sativum subsp. elatius) is an important source of genetic diversity for further pea crop improvement harbouring traits useful in climate change context. The genetic structure was assessed on 187 individuals of Pisum sativum subsp. elatius from fourteen populations collected in the northern part of the Fertile Crescent using 18,397 genome wide single nucleotide polymorphism DARTseq markers. AMOVA showed that 63% of the allelic variation was distributed between populations and 19% between individuals within populations. Four populations were found to contain admixed individuals. The observed heterozygosity ranged between 0.99 to 6.26% with estimated self-pollination rate between 47 to 90%. Genetic distances of wild pea populations were correlated with geographic but not environmental (climatic) distances and support a mixed mating system with predominant self-pollination. Niche modelling with future climatic projections showed a local decline in habitats suitable for wild pea, making a strong case for further collection and ex situ conservation.

Correction: Genetic structure of wild pea (Pisum sativum subsp. elatius) populations in the northern part of the Fertile Crescent reflects moderate cross-pollination and strong effect of geographic but not environmental distance.

[This corrects the article DOI: 10.1371/journal.pone.0194056.].

A Combined Comparative Transcriptomic, Metabolomic, and Anatomical Analyses of Two Key Domestication Traits: Pod Dehiscence and Seed Dormancy in Pea (Pisum sp.).

The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography-electrospray ionization/mass spectrometry and Laser desorption/ionization-mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence.

Geographical gradient of the eIF4E alleles conferring resistance to potyviruses in pea (Pisum) germplasm.

BACKGROUND: The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. METHODOLOGY/PRINCIPAL FINDINGS: Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. CONCLUSIONS/SIGNIFICANCE: This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.

Estimation of pea (Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses.

Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5'untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10(-3) per locus per generation, with a 95% confidence interval of 1.2 × 10(-4) to 2.7 × 10(-2). The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.