Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

Influence of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> strain on the production of transgenic peas (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants. KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean. The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids. The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons. Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants. We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures.Communicated by P. Debergh     

Evaluating cadmium toxicity in the root meristem of <Emphasis Type="Italic">Pisum</Emphasis><Emphasis Type="Italic">sativum</Emphasis> L.

The effect of cadmium (Cd) was studied on root tips of Pisum sativum L. Seeds of P. sativum were treated with a series of concentrations ranging from 0.125, 0.250, 0.500 and 1.000 mM for 6 h. The effect of Cd was analyzed by studying the percentage seed germination, radicle length (RL), mitotic index (MI) and chromosomal aberrations (CAs) in root tip. The results revealed that Cd had significant impeding effect on the root meristem activity of P. sativum at 0.500 and 1.000 mM as noticed by reduction in seed germination percentage and RL compared to control. Furthermore, it also reduced MI in dose-related manner compared to control. Additionally, the variation in the percentage of mitotic abnormalities was observed. The overall percentage of aberrations generally increased with increasing concentrations of Cd. Among these abnormalities laggards, bridges, stickiness, precocious separation and fragments were most common. The obtained results demonstrated that the Cd treatment leads to a significant reduction in MI and increase in CAs. Overall results allow us to suggest that the Cd has clastogenic effect on the crop.     

Spectral properties and the number of photosynthetic reaction centers in chlorophyll-deficient mutants of <Emphasis Type="Italic">Pisum sativum</Emphasis>

Spectral and photochemical properties were analyzed on intact chloroplasts and pigment-protein complexes isolated with gel electrophoresis from pea (Pisum sativum L.) leaves of parental variety Torsdag and of chlorophyll-deficient mutants chlorotica 2004 and 2014. Measurements of chlorophyll absorption and fluorescence spectra and of second derivative low-temperature (–196°C) spectra clarified exact positions of fluorescence maxima and revealed the chlorophyll forms of individual complexes in samples investigated. The chlorotica 2004 mutant, whose hybrids yield the heterosis effect, was characterized by the decreased accumulation of chlorophyll forms absorbing at 690, 697, and 708 nm, known to constitute the core antenna in the vicinity of photosystem I (PSI) reaction center. In the chlorotica 2014 mutant, whose hybrids are low productive, the interaction between PSI and PSII complexes was weakened, but no other difference from the parental variety was observed. The analysis of PSI and PSII photochemical activities, as well as estimates of light-harvesting antenna size and the number of reaction centers revealed that the chlorotica 2004 mutant is deficient in the number of PSI reaction centers by a factor of 1.7. This deficiency resulted from the mutation-induced disorder in biosynthesis of chlorophyll a-protein complex of PSI. It appears that gene interactions between the 2004 mutant and the parental variety Torsdag enhance the functional and metabolic activity of leaves in their hybrids, thereby yielding the heterosis effect.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 172–183.Original Russian Text Copyright © 2005 by Ladygin, Vaishlya.This revised version was published online in April 2005 with a corrected cover date.     

Sorbitol accumulation during natural and accelerated ageing of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) seeds

The aim of the present study was to determine the effect of accelerated ageing on the composition and content of the soluble carbohydrates in pea seeds of six genotypes differing in the composition of raffinose family oligosaccharides. A gradual decrease in the concentration of higher homologues of raffinose was observed along with seed ageing. At the same time the seeds lost vigor, viability and germination capacity. No increase in the concentration of reducing sugars was recorded, but sorbitol accumulated in pea embryos. Sorbitol accumulation may indicate seed quality deterioration during storage.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Duration of irradiation rather than quantity and frequency of high irradiance inhibits photosynthetic processes in the lichen <Emphasis Type="Italic">Lasallia pustulata</Emphasis>

Lichen thalli were exposed to 4 regimes differing in irradiance and duration of irradiation. Photosynthetic efficiency of thalli was monitored by chlorophyll fluorescence parameters and xanthophyll cycle analysis. Maximal quantum yield of photosystem 2 (F_V/F_M) decreased gradually with time in long-term treatment. The effect of additional short-term high irradiance (HI) treatment applied each 24 h was not significant. Nevertheless, short-term HI applied repeatedly on thalli kept in the dark led to a significant decrease of F_V/F_M. Non-photochemical quenching recorded during the long-term treatment corresponded to the content of zeaxanthin (Z). In short-term treatment, however, proportion of Z (and antheraxanthin) to total amount of xanthophyll cycle pigments recovered to the initial values every 24 h after each repeated short-term HI event in thalli kept in dark. Thus duration of irradiation rather than irradiance and frequency of HI events is important for a decrease in primary photosynthetic processes in wet thalli of Lasallia pustulata. Rapidly responding photoprotective mechanisms, such as conversion of xanthophyll cycle pigments, are involved mainly in short-term irradiation events, even at HI.     

Microtubule Orientation in the Brassinosteroid Mutants <Emphasis Type="Italic">lk,lka</Emphasis> and <Emphasis Type="Italic">lkb</Emphasis> of Pea

Short brassinosteroid (BR) mutants lk, lka and lkb of pea (Pisum sativum L.) were investigated by immunofluorescence microscopy to elucidate the role of brassinosteroids in cell elongation via an effect on the microtubules (MTs). This study adds to our knowledge the fact that brassinolide (BL) can cause MT realignment in azuki bean and rescue the MT organization of BR mutants in Arabidopsis. It provides novel information on both cortical and epidermal cells and presents detailed information about the ratios of all MT orientations present, ranging from transverse (perpendicular to the elongating axis) to longitudinal (parallel to the elongating axis). Experiments were conducted in vivo using intact plants with direct application of a small amount of brassinolide (BL) to the internode. Employing a BR-receptor mutant, lka, and the BR-synthesis mutants, lk and lkb, allowed the identification and isolation of any BR-induced responses in the MT cytoskeleton following BL application. Increases in growth rate were noted in all pea lines including WT following BL application. These increases were strong in the BR-synthesis mutants, but weak in the BR-receptor mutant. Immunofluorescence revealed significant differences in the average MT orientation of cortical cells of mutants versus WTs. Importantly, these mutants possessed abundant MTs, unlike the BR-deficient bul1-1 mutant in Arabidopsis. Following BL application, the epidermal and cortical cells of lk and lkb plants showed a large and significant shift in MT orientation towards more transverse, whereas lka plants showed a small and nonsignificant response in these cells. These results suggest that the BR response pathway is linked to the regulation of MT orientation.     

Mutation mechanism of chlorophyll-less barley mutant <Emphasis Type="Italic">NYB</Emphasis>

NYB is chlorophyll-less barley mutant, which is controlled by a recessive nuclear gene. The mutation mechanism is revealed. The activities of enzymes transforming 5-aminolevulinic acid into protochlorophyllide were the same in both NYB and the wild type (WT), but the activity of the protochlorophyllide oxidoreductase (POR) in WT was much higher than that of NYB. Most of the photosystem 2 apoproteins were present in both WT and NYB, suggesting that the capability of protein synthesis was probably fully preserved in the mutant. Thus chlorophyll (Chl) biosynthesis in NYB was hampered at conversion form protochlorophyllide (Pchlide) into chlorophyllide. The open reading frame of porB gene in NYB was inserted with a 95 bp fragment, which included a stop codon. The NYB mutant is a very useful material for studies of Chl biosynthesis, chloroplast signalling, and structure of light-harvesting POR-Pchlide complex (LHPP).     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Identification and detection of genetic relatedness among important varieties of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) grown in India

Among the cool season legume crops grown in India and the Indian sub-continent, peas are very popular and preferred by the growers as well as consumers for various uses. The third largest area in pea cultivation is occupied by India after Canada and Russia. Among the important and popular varieties of peas that are grown in India, several are from exotic background. But very little work has been done to carry out the genetic diversity present in the widely adapted Indian pea varieties using DNA markers. Twenty-four most popular and widely adapted varieties were subjected to RAPD analysis to find out the genetic relatedness among them using 60 decamer primers. All the primers used in our study were found to be polymorphic and seven of them showed 100% polymorphism. Out of 579 amplified products, 433 showed polymorphism (74.8%). On an average, 9.65 bands were amplified per primer. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA grouped all the tall type varieties together, whereas, dwarf types formed two different clusters based upon their pedigree. The arithmetic mean heterozygosity (H _av) value and marker index (MI) was found to be 0.496 and 4.787, respectively, thus this indicated the efficiency of RAPD as a marker system. Moreover, the calculated value of probability of identical match by chance suggested that about 10⁵³ genotypes can be unambiguously distinguish by employing 60 RAPD primers.     

Phenols,proline and low-molecular thiol levels in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) plants respond differently toward prolonged exposure to ultraviolet-B and ultraviolet-C radiations

Pea (Pisum sativum L.) seedlings were exposed to low, moderate, and high regimes of ultraviolet-B (UV-B) (ld-B 4.4, md-B 13.3, and hd-B 26.5 kJ m⁻² day⁻¹), or ultraviolet-C (UV-C) (ld-C 0.1, md-C 0.3, and hd-C 0.6 kJ m⁻² day⁻¹) radiations. Concentrations of total phenols, free proline, and low-molecular thiol groups were determined in the last formed (young) and older leaves after irradiation for 7, 10 or 14 consecutive days. Shoot length and weight did not change markedly after 14 days of ld-B and ld-C, but reduced substantially after moderate and high regimes of both UV-B and UV-C. Proline decreased upon high doses of irradiation, while in ld-B treated plants, by contrast, an increase was observed. The reduction in total phenols and thiols was stronger after hd-B than after hd-C irradiations, although an induction was found in ld-B treated plants. In contrast to ld-B, ld-C regime led mainly to reductions or insignificant changes in proline, phenols, and thiols. Therefore, the stress-protection mechanisms are different between low UV-B and UV-C irradiation regimes in regard to proline, phenols, and thiols.     

The ontogenetic basis for floral diversity in <Emphasis Type="Italic">Agonis</Emphasis>, <Emphasis Type="Italic">Leptospermum</Emphasis> and <Emphasis Type="Italic">Kunzea</Emphasis> (Myrtaceae)

Floral development in three species each of Leptospermum and Kunzea, and one species of Agonis, is described and compared. Differences in the number of stamens and their arrangement in the flower at anthesis are determined by the growth dynamics of the bud. In Leptospermum, early expansion of the bud is predominantly in the axial direction and causes the stamen primordia to be initiated in antepetalous chevrons. In Kunzea, early expansion occurs predominantly in the lateral direction and successive iterations of stamen primordia are inserted alternately at more or less the same level. In both genera, further expansion in the lateral plane spreads the stamens into a ring around the hypanthium. Agonis flexuosa is similar to Leptospermum. Other variable factors include the timing at which stamen initiation commences (earlier in Leptospermum than Kunzea), the duration of stamen initiation (hence the total number of stamens produced – varies within genera), and very late differential expansion that forces stamens into secondary antesepalous groups in A. flexuosa and L. myrsinoides.The authors thank Dr H. Toelken for kindly providing some material and the impetus for this project. This research was supported by Australian Research Council grant AS19131815.     

Screening of <Emphasis Type="Italic">cry2</Emphasis> genes in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolates from Argentina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identification of cry2 genes from Bacillus thuringiensis (Bt) was established. Strains from different sources of Argentina were analyzed to study the distribution of cry2 genes. The results showed that cry2Aa/cry2Ab profile was the most abundant irrespective of source and represented 56 of 59 Bt isolates (94.9%). Three different cry2 profiles were found in this collection, one of which was novel.     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

Parasitism <Emphasis Type="Italic">versus</Emphasis> mutualism in the ant-garden parabiosis between <Emphasis Type="Italic">Camponotus femoratus</Emphasis> and <Emphasis Type="Italic">Crematogaster levior</Emphasis>

Ant-gardens represent a special type of association between ants and epiphytes. Frequently, two ant species can share the same nest in a phenomenon known as ‘parabiosis’, but the exact nature (i.e., mutualistic or parasitic) of this interaction is the subject of debate. We thus attempted to clarify the mutual costs and benefits for each partner (ants and plants) in the Crematogaster levior/Camponotus femoratus ant-garden parabiosis. The ants’ response to experimental foliar damage to the epiphytes and to the host tree as well as their behavior and interactions during prey capture were investigated to see if the purported parasitic status of Cr. levior could be demonstrated in either the ant-ant or in the ant-plant interactions. The results show that both species take part in protecting the epiphytes, refuting the role of Cr. levior as a parasite of the ant-garden mutualism. During capture of large prey Ca. femoratus took advantage from the ability of Cr. levior to discover prey; by following Cr. levior trails Ca. femoratus workers discover the prey in turn and usurp them during agonistic interactions. Nevertheless, the trade-off between the costs and benefits of this association seems then to be favorable to both species because it is known that Cr. levior benefits from Ca. femoratus building the common carton nests and furnishing protection from vertebrates. Consequently, parabiosis can then be defined as the only mutualistic association existing between ant species, at least in ant-gardens. Received 31 August 2006 ; revised 8 December 2006 ; accepted 12 December 2006