Overcoming hybridization barriers between pea and some of its wild relatives

Pea would benefit from the plasticity and adaptability of its cross-incompatible relatives Pisum fulvum and Lathyrus sativus L., and we have tested reciprocal sexual crossings by manually cross-pollinating plants of genotypes of these three species. Studies of in situ germination of pollen grains on stigmata showed that pollen tubes were generally unable to germinate or could not reach the ovary. A few putative hybrid pods were nevertheless harvested, with one grain per pod germinated in vitro, then micropropagated for flow cytometry, isoenzyme, molecular (ribosomal ITS PCR-RFLP) and genomic in situ hybridization (GISH) studies. One such grain was recovered from an inter-generic cross of P. sativum x L. sativus and four from an inter-specific P. sativum x P. fulvumcross. A strong cross-incompatibility was shown between pea and grass pea, where the putative hybrid turned out to be pea. Conversely, with the interspecific, P. sativum x P. fulvum cross, flow cytometry and isoenzymes with leaf tissues strongly suggested hybridity, while molecular approaches and GISH confirmed the production of inter-specific hybrids, and without the need for a wild type P. sativum accession as a bridging cross.     

Utilization of isozyme polymorphism for cultivar identification of 45 commercial peas (Pisum sativum L.)

Forty five Pisum sativum cultivars were analysed by isozyme electrophoresis with the objective to find protein markers for exact and reproducible discrimination of individual genotypes. The combination of six enzyme systems (acid phosphatase, amylase, esterase, leucine aminopeptidase, shikimate dehydrogenase and phosphoglucomutase) with two electrophoretic techniques (NATIVE-PAGE, isoelectric focusing) and use of seed and leaf tissue enabled to identify all 45 studied cultivars. Critical factors which may affect utilization of isozyme electrophoresis for commercial applications in pea breeding and seed production and testing are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of RAPD markers linked to the rust (<Emphasis Type="Italic">Uromyces fabae</Emphasis>) resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Summary Two RAPD markers linked to gene for resistance (assayed as pustule number cm⁻² leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC₁F₁ [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F₁, F₂, BC₁F₁ and BC₁F₂ generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC₁F₁ individuals. Two RAPD makers, viz., SC10-82₃₆₀ (primer, GCCGTGAAGT), and SCRI-71₁₀₀₀ (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.     

The response of Pisum sativum L. germplasm to high concentrations of soil boron

Summary Tolerance to high levels of boron in the soil is an important aspect of the adaptation of crop varieties to southern Australian conditions. This paper reports investigations aimed at exploring the extent of genetic variation in Pisum sativum and at defining appropriate selection criteria for selection for boron tolerance in breeding programs.A collection of 617 accessions of Pisum was screened in controlled conditions and visually assessed for symptoms of boron toxicity. A high proportion of accessions were sensitive with only 3.5% being more tolerant than any of the Australian varieties. Relatively high proportions of tolerant and moderately tolerant accessions originated from Asia and South America.In a second experiment the responses of selected tolerant accessions were evaluated with respect to different parameters. The objectives were to confirm the performance of the putative boron tolerant accessions and identify appropriate parameters for selecting boron tolerant genotypes. In addition to the visual assessment of boron toxicity, measurements at the time of harvest included dry matter yield and concentration of boron in tissues. Symptom expression was highly correlated with dry matter yield and concentration of boron in tissues under high boron conditions and so could be used as a non-destructive selection criteria. A low degree of symptom expression by tolerant accessions could usually be attributed to low levels of boron in the vegetative tissues. The results of this study indicate that considerable genetic variation exists among exotic accessions of Pisum sativum and tolerance to boron could be transferred to sensitive varieties.     

Diversity analysis and genotyping in Pisum with sequence tagged microsatellite site (STMS) primers

Pisum sativum specific sequence tagged microsatellite site primers were used to amplify genomic profiles from 15accessions of P. sativum L. that represented the genetic base of the Australian field pea-breeding program and five accessions of the wild related species P. fulvum. The STMS primers were used to assess genetic relationships among the Pisum accessions in two ways. Firstly, to produce RAPD-like multiple banding marker profiles using an adapted RAMS method, for intra- and interspecific diversity analysis. From the 14 flanking primer pairs assessed, 133 markers were obtained. Conservation and reproducibility of markers among individuals within accessions was demonstrated. The largest distance observed among P. sativumaccessions was 22% and among P.fulvum accessions was 40%, similar to that revealed with other PCR-based methods. The maximum distance between P.sativum and P. fulvum accessions was 46%. Phylogenetic clustering of P. sativum accessions, using the neighbour joining method and based on simple matching distances, was distinct and distant to P. fulvum. Secondly, PCR with a higher annealing temperature and fluorescent labeling identified simple and allelic loci markers useful for creating agenotype/fingerprint database for P. sativum cultivars. This is the first report to demonstrate the use of Pisum specific STMS sequences for both diversity analysis and genotype identification. This revised version was published online in August 2006 with corrections to the Cover Date.     

Fingerprinting temperate <Emphasis Type="Italic">japonica</Emphasis> and tropical <Emphasis Type="Italic">indica</Emphasis> rice genotypes by comparative analysis of DNA markers

This paper describes the relative efficiency of three marker systems, RAPD, ISSR, and AFLP, in terms of fingerprinting 14 rice genotypes consisting of seven temperatejaponica rice cultivars, three indica near-isogenic lines, three indica introgression lines, and one breeding line of japonica type adapted to high-altitude areas of the tropics with cold tolerance genes. Fourteen RAPD, 21 ISSR, and 8 AFLP primers could produce 970 loci, with the highest average number of loci (92.5) generated by AFLP. Although polymorphic bands in the genotypes were detected by all marker assays, the AFLP assay discriminated the genotypes effectively with a robust discriminating power (0.99), followed by ISSR (0.76) and RAPD (0.61). While significant polymorphism was detected among the genotypes of japonica and indica through analysis of molecular variance (AMOVA), relatively low polymorphism was detected within the genotypes of japonica rice cultivars. The correlation coefficients of similarity were significant for the three marker systems used, but only the AFLP assay effectively differentiated all tested rice lines. Fingerprinting of backcross-derived resistant progenies using ISSR and AFLP markers easily detected progenies having a maximum rate of recovery for the recurrent parent genome and suggested that our fingerprinting approach adopting the ‘undefined-element-amplifying’ DNA marker system is suitable for incorporating useful alleles from the indica donor genome into the genome of temperate japonica rice cultivars with the least impact of deleterious linkage drag.     

Study of the variation in the somatic embryogenesis ability of some pea lines (Pisum sativum L. and Pisum arvense L.)

Summary Thirty lines of pea were tested in vitro to evaluate their ability to produce somatic embryos. Three distinct genotypic classes were detected (strong, medium and weak). The best responses were obtained in Pisum sativum. Abnormal somatic embryos and secondary embryogenesis seem to constitute the principal obstacle to the development of these structures.     

Maintenance breeding and multiplication of pea and faba bean cultivars. Maintenance of protein peas (Pisum sativum) and field beans (Vicia faba)

Summary The maintenance of protein pea (Pisum sativum)and field bean (Vicia faba)cultivars is a part of the breedingscheme of these pulses at CEBECO-ZADEN. As soon as the cultivar is pure breeding a large quantity ofseed is stored under conditioned circumstances.     

Cytological and Morphological Characterization of Pisum sativum and Pisum fulvum Tetraploids

Two colchicine-induced tetraploid lines, one belonging to Pisum sativum and the other to the wild species Pisum fulvum, were compared with their corresponding diploids and among themselves for chromosome number, meiotic behaviour and for pollen and pod fertility. For both lines, five generations were analyzed; the tetraploid level was maintained throughout the generations. In P. fulvum, aneuploids with 27, 29 and 30 chromosomes were observed, while in P. sativum aneuploids with 29 chromomes were found. Meiotic analysis evidenced a higher number of quadrivalents and a lower number of univalents in P. fulvum. This particular chromosomal behaviour may also have been responsible for determining the higher pollen and pod fertility in P. fulvum. Wild species of this genus seem to have better tolerance for ploidy variations.     

Microsatellite polymorphism in Pisum sativum

Pisum sativum sequences were retrieved from Genbank/EMBL databases and searched for all possible dinucleotide and trinucleotide tandem repeats. One‐hundred and seventy‐one simple sequence repeats (SSRs) were found among 663 sequences. The different dinucleotide or trinucleotide motifs occurred at varying frequencies. CT/AG was the most frequent dinucleotide, and TCT/AGA the most frequent trinucleotide. Forty‐three microsatellite markers were generated from these sequences and used to assess the genetic variability among 12 pea genotypes. Thirty‐one were polymorphic among the genotypes and the average number of variants per marker was 3.6 when considering only polymorphic markers. Overall, the number of variants for a given SSR marker was correlated with the length of the SSR but some 12‐bp long SSRs showed the same degree of polymorphism as longer ones. The groupings resulting from the SSR genotyping among the 12 genotypes gave an interesting insight into the possible origin of one recent cultivar. Database‐derived SSR markers are highly variable. They can provide useful information on the genetic diversity among P. sativum cultivated types.     

Genetics of tolerance to high concentrations of soil boron in peas (Pisum sativum L.)

Summary The inheritance of tolerance to high concentrations of soil boron in pea (Pisum sativum L.) was studied in five cross combinations and their reciprocals. Segregation patterns for boron response in F₂ populations and F₃ derived families were established by visual assessment of leaf damage. The segregation ratios were explained in terms of two major gene loci interacting in an additive manner with incomplete dominance at each locus. Evaluation of selected tolerant and susceptible families indicated that tolerant families contained a significantly lower concentration of boron in shoots than susceptible families.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.     

Molecular tagging of a gene for resistance to brown planthopper in rice (Oryza sativa L.)

An introgression line derived from an interspecific cross between Oryzasativa and Oryza officinalis, IR54741-3-21-22 was found to beresistant to an Indian biotype of brown planthopper (BPH). Genetic analysisof 95 F₃ progeny rows of a cross between the resistant lineIR54741-3-21-22 and a BPH susceptible line revealed that resistance wascontrolled by a single dominant gene. A comprehensive RAPD analysisusing 275 decamer primers revealed a low level of (7.1%) polymorphismbetween the parents.RAPD polymorphisms were either co-dominant (6.9%), dominant forresistant parental fragments (9.1%) or dominant for susceptible parentalfragments (11.6%). Of the 19 co-dominant markers, one primer,OPA16, amplified a resistant parental band in the resistant bulk and asusceptible parental band in the susceptible bulk by bulked segregantanalysis. RAPD analysis of individual F₂ plants with the primerOPA16 showed marker-phenotype co-segregation for all, with only onerecombinant being identified. The linkage between the RAPD markerOPA16₉₃₈ and the BPH resistance gene was 0.52 cM in couplingphase. The 938 bp RAPD amplicon was cloned and used as a probe on122 Cla I digested doubled haploid (DH) plants from aIR64xAzucena mapping population for RFLP inheritance analysis and wasmapped onto rice chromosome 11. The OPA16₉₃₈ RAPD markercould be used in a cost effective way for marker-assisted selection of BPHresistant rice genotypes in rice breeding programs.     

Random amplified polymorphic DNA (RAPD) markers variability among cultivars and landraces of common beans (Phaseolus vulgaris L.) of south-Brazil

To evaluate the variability among cultivars and landraces of common bean(Phaseolus vulgaris L.), 15 cultivars and 18 landraces of common bean (Phaseolus vulgarisL.), a undefined species of Phaseolus,two landraces of Vigna angularis L., and a landrace of soybean (Glycine maxL.), were screened with fifteen oligonucleotide primers in PCR reactions. An average of 20.3 RAPD bands were scored per primer. A total of 304 amplification products were scored of which 88.8% were polymorphic among Phaseolus genotypes. Based on the RAPD markers, four major clusters were formed. Three clusters corresponded to the soybean, to the two Vigna angularis landraces, and to the Phaseolus sp. landrace, respectively. The fourth cluster include all the landraces and cultivars of Phaseolus vulgaris. This large group could be separated into three subgroups that were correlated with the phaseolin patterns and the average seed weight of the genotypes. The analysis shows that most of the landraces collected in South Brazil (17 out of 18) belong to the Andean gene pool, and most of the cultivars (13 out of 15) belong to the Middle American gene pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity in cultivated Osteospermum as revealed by random amplified polymorphic DNA analysis

A collection of 28 Osteospermum clones and cultivated varieties of different origin were evaluated by random amplified polymorphic DNA (RAPD) analysis. All the clones were identified by 12 decamers selected from a set of 30. This is the first characterization by molecular markers of the genetic material of Osteospermum. The level of genetic diversity among genotypes was assayed and all the accessions tested were then classified into six groups by UPGMA cluster analysis; the clustering of genotypes using the RAPD data proved to be in accordance with their breeding group origin. RAPD analysis can therefore be a useful tool for evaluating genetic variability in other Osteospermum germplasm collections for breeding purposes and for protecting intellectual property rights of improved varieties.     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

Criteria for selecting productive and stable pea cultivars

The beginning and duration of the seed set in the growth cycle determine the level and stability of the yield of a pea (Pisum sativum L.) genotype. The objective of the present study was to identify criteria for selecting genotypes, both in terms of timing of seed set and productivity. Genotypes were initially compared in field experiments for two different levels of inter-plant competition, but using the same photo-thermal conditions. These experiments showed that the initiation and, particularly, the duration of seed set were affected by plant growth rates, indicating that selection on these variables must be done by comparing genotypes under regular cropping conditions. When measuring seed production of the whole plant, we found that mean dry seed weight per podded node all over the plant and the number of podded nodes on any fertile stem were similar to those on the main stem. These results confirmed that branches and main stems have a similar reproductive pattern, and thus that any podded stem of the canopy is representative of every stem of the plant. Lastly, we showed that, when associated, the number of podded nodes, the mean dry seed weight per podded node on the main stem (or on any reproductive stem of the canopy), and the number of basal branches per plant are suitable criteria for selecting for productivity among genotypes with a similar duration of seed set. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular diversity and plant disease resistance: An electrophoretic comparison of near-isogenic lines of wilt-resistant or -susceptible Pisum sativum L. cv. William Massey

Summary The soluble proteins from two near-isogenic lines of Pisum sativum. cv. William Massey have been compared electrophoretically. These lines differ in their physiological response to wilt (Fusarium oxysporum f. sp. pisi), one being susceptible and the other resistant. The total protein profiles derived from the two lines appear to be identical. The resistant line differs electrophoretically from the susceptible line in carrying an esterase component which has been derived from its resistant parent, cultivar Delwiche Commando.     

Somaclonal variation in winter wheat: frequency,occurrence and inheritance

Summary Seventy two landraces of green and sugar pea (Pisum sativum), plus four elite commercial cultivars as controls, were evaluated in 1989–1990 for 19 quantitative traits. The landraces show a broad diversity in most of the traits studied. There are significant positive correlations of flowering date with node of first flower, shoot height and number of days to maturity, which could be relevant parameters for evaluating plant precocity. The analysis of means over each population in some important morpho-biological and quality traits in pod and seed show that there are seven landraces which deserve special attention for having promising breeding value.