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During analysis of kiwifruit derived from hybrids between the high AsA species Actinidia eriantha and A. chinensis var chinensis, we observed bimodal segregation of fruit AsA concentration suggesting major gene segregation. To test this hypothesis we performed whole-genome sequencing on pools of high and low AsA fruit from tetraploid A. chinensis var. deliciosa x A. eriantha backcross families. Pool-GWAS revealed a single QTL spanning more than 5 Mbp on chromosome 26, which we denote as qAsA26.1. A co-dominant PCR marker was used to validate this association in four diploid (A. chinensis x A. eriantha) x A. chinensis backcross families, showing that the eriantha allele at this locus increases fruit AsA levels by 250 mg/100 g fresh weight. Inspection of genome composition and recombination in other A. chinensis genetic maps confirmed that the qAsA26.1 region bears hallmarks of suppressed recombination. The molecular fingerprint of this locus was examined in leaves of backcross validation families by RNASEQ. This confirmed strong allelic expression bias across this region as well as differential expression of transcripts on other chromosomes. This evidence suggests that the region harboring qAsA26.1 constitutes a supergene, which may condition multiple pleiotropic effects on metabolism.

Genomic selection (GS) is a breeding tool, which is rapidly gaining popularity for plant breeding, particularly for traits that are difficult to measure. One such trait is ascochyta blight resistance in pea (Pisum sativum L.), which is difficult to assay because it is strongly influenced by the environment and depends on the natural occurrence of multiple pathogens. Here we report a study of the efficacy of GS for predicting ascochyta blight resistance in pea, as represented by ascochyta blight disease score (ASC), and using nucleotide polymorphism data acquired through genotyping-by-sequencing. The effects on prediction accuracy of different GS models and different thresholds for missing genotypic data (which modified the number of single nucleotide polymorphisms used in the analysis) were compared using cross-validation. Additionally, the inclusion of marker × environment interactions in a genomic best linear unbiased prediction (GBLUP) model was evaluated. Finally, different ways of combining trait data from two field trials using bivariate, spatial, and single-stage analyses were compared to results obtained using a mean value. The best prediction accuracy achieved for ASC was 0.56, obtained using GBLUP analysis with a mean value for ASC and data quality threshold of 70% (i.e., missing SNP data in

Background Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea. Results To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel’s r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways. Conclusions The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1080-9) contains supplementary material, which is available to authorized users.

During analysis of kiwifruit derived from hybrids between the high vitamin C (ascorbic acid, AsA) species Actinidia eriantha and A. chinensis, we observed bimodal segregation of fruit AsA concentration suggesting major gene segregation. To test this hypothesis, we performed whole-genome sequencing on pools of hybrid genotypes with either high or low AsA fruit. Pool-GWAS (genome-wide association study) revealed a single Quantitative Trait Locus (QTL) spanning more than 5 Mbp on chromosome 26, which we denote as qAsA26.1. A co-dominant PCR marker was used to validate this association in four diploid (A. chinensis &times, A. eriantha) &times, A. chinensis backcross families, showing that the A. eriantha allele at this locus increases fruit AsA levels by 250 mg/100 g fresh weight. Inspection of genome composition and recombination in other A. chinensis genetic maps confirmed that the qAsA26.1 region bears hallmarks of suppressed recombination. The molecular fingerprint of this locus was examined in leaves of backcross validation families by RNA sequencing (RNASEQ). This confirmed strong allelic expression bias across this region as well as differential expression of transcripts on other chromosomes. This evidence suggests that the region harbouring qAsA26.1 constitutes a supergene, which may condition multiple pleiotropic effects on metabolism.

Tomato spotted wilt virus (TSWV) is an internationally significant pathogen with a wide host range, vectored by thrips. We have studied the sequence variation and evolutionary mechanisms at play in parts of the L, M and S subgenomes of 23 New Zealand TSWV isolates collected between 1992 and 2009, aiming to identify the possible geographic origins of isolates. Maximum-likelihood-based phylogenetic analyses of New Zealand and overseas TSWV isolates placed the L and M subgenome sequences of two isolates (MAF04 and PFR04) in distinct clades composed primarily of Korean, Japanese and Chinese isolates, in contrast to the remaining 21 isolates, which clustered with a cosmopolitan group of isolates. The nucleocapsid (N) gene sequences of MAF04 and PFR04 plus MAF02 clustered with Japanese isolates. Consequently, we postulate that these isolates may represent a distinct incursion into New Zealand, but we do not have enough evidence to indicate an incursion pathway. Alternately, these isolates may have arrived with an incursion that included a mixture of TSWV isolates of diverse international origins. The sequences of four of the TSWV isolates contained a number of sites with a mixture of nucleotides, suggesting that these isolates either consisted of several sequence variants or were from plants with mixed infections. One isolate (MAF02) was shown to be a either a reassortant or an S subgenome recombinant. Large amounts of low-level polymorphism were detected with low amino acid change fixation rates (purifying selection). Negative selection was indicated at four amino acid sites in the New Zealand TSWV N gene sequences.

Genetic diversity, population structure and genome-wide marker-trait association analysis was conducted for the USDA pea (Pisum sativum L.) core collection. The core collection contained 285 accessions with diverse phenotypes and geographic origins. The 137 DNA markers included 102 polymorphic fragments amplified by 15 microsatellite primer pairs, 36 RAPD loci and one SCAR (sequence characterized amplified region) marker. The 49 phenotypic traits fall into the categories of seed macro- and micro-nutrients, disease resistance, agronomic traits and seed characteristics. Genetic diversity, population structure and marker-trait association were analyzed with the software packages PowerMarker, STUCTURE and TASSEL, respectively. A great amount of variation was revealed by the DNA markers at the molecular level. Identified were three sub-populations that constituted 56.1%, 13.0% and 30.9%, respectively, of the USDA Pisum core collection. The first sub-population is comprised of all cultivated pea varieties and landraces; the second of wild P. sativum ssp. elatius and abyssinicum and the accessions from the Asian highland (Afghanistan, India, Pakistan, China and Nepal); while the third is an admixture containing alleles from the first and second sub-populations. This structure was achieved using a stringent cutoff point of 15% admixture (q-value 85%) of the collection. Significant marker-trait associations were identified among certain markers with eight mineral nutrient concentrations in seed and other important phenotypic traits. Fifteen pairs of associations were at the significant levels of P ≤ 0.01 when tested using the three statistical models. These markers will be useful in marker-assisted selection to breed pea cultivars with desirable agronomic traits and end-user qualities.

Mendel's paper 'Versuche über Pflanzen-Hybriden' is the best known in a series of studies published in the late 18th and 19th centuries that built our understanding of the mechanism of inheritance. Mendel investigated the segregation of seven gene characters of pea (Pisum sativum), of which four have been identified. Here, we review what is known about the molecular nature of these genes, which encode enzymes (R and Le), a biochemical regulator (I) and a transcription factor (A). The mutations are: a transposon insertion (r), an amino acid insertion (i), a splice variant (a) and a missense mutation (le-1). The nature of the three remaining uncharacterized characters (green versus yellow pods, inflated versus constricted pods, and axial versus terminal flowers) is discussed.

Timmerman-Vaughan, G., Larsen, R., Murray, S., McPhee, K., and Coyne, C. 2009. Analysis of the accumulation of Pea enation mosaic virus genomes in seed tissues and lack of evidence for seed transmission in pea (Pisum sativum). Phytopathology 99:1281-1288. Pea enation mosaic virus (PEMV) is an important virus disease of pea. International movement of commercial pea cultivars and germplasm can be problematic due to uncertainty about seed transmission of the viruses responsible for the disease. Whether PEMV is seedborne was assessed by collecting developing seed from infected plants and determining the relative concentrations of the PEMV-1 and PEMV-2 viral genomes using quantitative real-time reverse-transcription polymerase chain reaction. The relative accumulation of PEMV-1 and PEMV-2 was ≈1,240 and 13,000 times higher, respectively, in leaf than in embryo tissues. Accumulation of PEMV-1 and PEMV-2 RNA was also significantly higher in pod walls and seed coats than in cotyledons or embryo axes. No evidence was obtained for seed transmission of PEMV in pea. Although PEMV-1 and PEMV-2 genomic RNAs were found in developing seed, no PEMV symptoms were observed in the field on more than 50,000 plants from seed derived from PEMV-infected source plants. These data demonstrate that PEMV is seedborne in pea but do not support a previous report that PEMV is seed transmitted. Absence of seed transmission may result from the low abundance of PEMV viral genomes in embryo tissue.

BACKGROUND: Polymerase chain reaction (PCR)-based DNA diagnostics have grown in importance for assessing food quality and safety. PCR diagnostic reliability and sensitivity depend on the quality of the DNA extractions, including the extent of DNA degradation and the presence of PCR inhibitors. RESULTS: An approach has been described that quantifies the extent of DNA degradation in soy food samples using anchored real-time PCR (qPCR) assays that amplify target sequences ranging from 1000 bp, based on two endogenous soy sequences. DNA degradation was quantified for model foods produced in the laboratory (cooked soy meal, tofu) as well as purchased soy-containing food products. Considerably less than 1% of the total DNA extracted from samples was available for amplification of the longest amplicons (830 and 1022 bp) from the most highly processed food products (e.g., soy-based infant formula). CONCLUSION: The utility of anchored qPCR assays was demonstrated for characterizing the amount of DNA that is available for amplifying different-length PCR products from a range of soy-containing processed food products. This approach should be useful for estimating the amount of amplifiable DNA in food ingredients in cases where food processing has caused degradation of DNA. Copyright © 2009 Society of Chemical Industry

A set of 110 diploid putative introgression lines (ILs) containing chromatin introgressed from the undomesticated species Hordeum bulbosum L. (bulbous barley grass) into cultivated barley (Hordeum vulgare L.) has been identified using a high-copy number retrotransposon-like PCR marker, pSc119.1, derived from rye (Secale cereale L.). To evaluate these lines, 92 EST-derived markers were developed by marker sequencing across four barley cultivars and four H. bulbosum genotypes. Single nucleotide polymorphisms and insertions/deletions conserved between the two species were then used to develop a set of fully informative cleaved amplified polymorphic sequence markers or size polymorphic insertion/deletion markers. Introgressed chromatin from H. bulbosum was confirmed and genetically located in 88 of these lines using 46 of the EST-derived PCR markers. A total of 96 individual introgressions were detected with most of them (94.8%) extending to the most distal marker for each respective chromosome arm. Introgressions were detected on all chromosome arms except chromosome 3HL. Interstitial or sub-distal introgressions also occurred, with two located on chromosome 2HL and one each on 3HS, 5HL and 6HS. Twenty-two putative ILs that were positive for H. bulbosum chromatin using pSc119.1 have not had introgressions detected with these single-locus markers. When all introgressions are combined, more than 36% of the barley genetic map has now been covered with introgressed chromatin from H. bulbosum. These ILs represent a significant germplasm resource for barley improvement that can be mined for diverse traits of interest to barley breeders and researchers.

Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from

Advances have been made in our understanding of Ascochyta blight resistance genetics through mapping candidate genes associated with QTL regions and demonstrating the importance of epistatic interactions in determining resistance. Ascochyta blight disease of pea (Pisum sativum L.) is economically significant with worldwide distribution. The causal pathogens are Didymella pinodes, Phoma medicaginis var pinodella and, in South Australia, P. koolunga. This study aimed to identify candidate genes that map to quantitative trait loci (QTL) for Ascochyta blight field disease resistance and to explore the role of epistatic interactions. Candidate genes associated with QTL were identified beginning with 101 defence-related genes from the published literature. Synteny between pea and Medicago truncatula was used to narrow down the candidates for mapping. Fourteen pea candidate sequences were mapped in two QTL mapping populations, A26 × Rovar and A88 × Rovar. QTL peaks, or the intervals containing QTL peaks, for the Asc2.1, Asc4.2, Asc4.3 and Asc7.1 QTL were defined by four of these candidate genes, while another three candidate genes occurred within 1.0 LOD confidence intervals. Epistasis involving QTL × background marker and background marker × background marker interactions contributed to the disease response phenotypes observed in the two mapping populations. For each population, five pairwise interactions exceeded the 5% false discovery rate threshold. Two candidate genes were involved in significant pairwise interactions. Markers in three genomic regions were involved in two or more epistatic interactions. Therefore, this study has identified pea defence-related sequences that are candidates for resistance determination, and that may be useful for marker-assisted selection. The demonstration of epistasis informs breeders that the architecture of this complex quantitative resistance includes epistatic interactions with non-additive effects.

Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations (p < 0.001) were found with SNPs in the glucan water dikinase (GWD), starch branching enzyme I (SBEI) and the starch synthase III (SSIII) genes, and with a SSR allele in the SBEII gene. SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato.

driven by environmental influences (Charles-Edwards, 1982).Agenotype’sultimateperformanceisdetermined Seed yield in pea (Pisum sativum L.) is a physiologically complex by how it integrates genotype and environmental influtrait that is strongly influenced by both genotype and environment. Seed yield can be described in terms of its components, which include ences. The end result is seed yield, which has often been plant number per unit area, seed weight, and seed number. These described as the product of its components: number of yield components show interdependence and plasticity in response to plants per unit area, number of seeds per unit area environment; therefore, selection based on a single component is un- (number of pods per plant, number of seeds per pod), likely to succeed in increasing yield in breeding programs. To improve and mean seed weight (Moot and McNeil, 1995). These our understanding of the genetic basis of seed yield determination in yield components show interdependence or plasticity pea and to identify genetic loci involved, quantitative trait loci (QTL) (Wilson, 1987). For example, compensation is observed for yield per se, yield components (seed weight, seed number, and between the number of pods per plant and number of harvest index [HI]) and developmental traits (node of first flower [NFF], seeds per pod (Moot and McNeil, 1995), or between seed number of flowering nodes [NFN], total node number [TNN]) were number and seed weight (Sarawat et al., 1994). Since mapped. The QTL mapping was conducted using F2–derived families of a cross between Primo, a marrowfat cultivar, and OSU442-15, a selecting for a particular yield component may be inbluepea breedingline. Linkagemaps containing108 locion 11linkage effective as a means of increasing yield per se, alternate groups (LGs) were constructed for 227 families derived from this approaches have been proposed. Hedley and Ambrose cross. Traits were measured in three replicated field trials conducted (1985)suggestedselectingforplantsthatproduceahigh, in New Zealand during the summers of 1997–1998, 1998–1999, and stable plant HI, while Wilson (1987) suggested that se2002–2003.Thetrialsweremanagedtoensuremaximumexpressionof lecting weakly competitive plants may increase yield yield potential. The QTL affecting yield-related traits were associated stability, based on evidence that weak competitors may with 19 genomic regions on LGs I, II, III, IV, VI, and VII. The QTL be more successful within a pea crop than vigorous for different yield-related traits were colocalized, suggesting some genotypes that perform well as single plants. Bourion basis for understanding the reproductive plasticity that is observed et al. (2002) suggested that progress in seed yield and in pea at the genetic level. yield stability may be achieved using lines with different developmental and architectural features. For the genotypes that they examined, this would involve focusing P ea is grownthroughout the world for diverse uses primarily on NFF and on leaf appearance rate. as food and feed. Field pea is the world’s third most With the development of molecular linkage maps and significant grainlegume after soybean andcommonbean. statistical approaches for mapping QTL, it is possible Theaimswhendevelopingpeacultivarsincludeobtaining to identify and tag genetic loci controlling genetically high yields, disease resistance, quality attributes, and complex traits. Quantitative trait loci mapping provides adaptation to a range of environmental conditions. Re- information on the minimum number of genetic loci ducing yield variability is an important goal because pea that may be involved in determining a trait phenotype, exhibits poor stability of yield across years and environ- the genetic map locations of those loci and estimates of ments compared with other crops. the effects of the loci on trait values—information that Yield is a complex trait that, from a crop physiology ispotentially veryuseful tothe plantbreeder. Molecular perspective, is the culmination of a series of processes markers associated with QTL may also prove to be (phenological and canopy development, radiation inter- useful for marker-assisted plant breeding. In addition, ception, biomass production and partitioning) that are QTL mapping offers the opportunity to use map-based cloning approaches to identify the genes underlying the

Resistance to pea seed-borne mosaic virus (PSbMV) pathotype P-1 in peas is conferred by sbm1 with recessive inheritance. PSbMV is an economically important pathogen with world-wide distribution that causes significant losses in pea yield and reduces seed and produce quality. The sbm1 gene was previously mapped to linkage group VI on molecular linkage maps of the pea genome. To improve plant breeders' ability to develop varieties resistant to PSbMV, two random amplified polymorphic DNA markers (G05―2537 and L01―910) and one restriction fragment length polymorphism (P446) linked to sbm1 have been identified. The genomic sequences for these markers have been characterized and the information used to develop three simple polymerase chain reaction-based STS (sequence tagged site) assays. Linkage analysis in two F 2 populations showed that the most tightly linked of these three STS loci (sG05―2537) is approximately 4 cM from sbm1. Characterization of a collection of resistant and susceptible germplasm demonstrated a strong correlation between STS alleles and sbm1 alleles, indicating the utility of these markers for marker-assisted selection in breeding programmes using a range of germplasm sources.

Ascochyta blight of pea (Pisum sativum L.) is a fungal disease caused by Mycosphaerella pinodes (Berk. & Bloxham) Verstergren, Phoma medicaginis Malbr. & Roum. var. pinodella (L.K. Jones) Boerema, and Ascochyta pisi Lib. that can result in significant reductions to pea yield and quality. To characterize the genetics of resistance and to identify molecular markers for use in plant breeding, quantitative trait loci (QTLs) affecting Ascochyta blight resistance were mapped in F(2:3) and F(2:4) families produced from a cross between resistant breeding line 3148-A88 and susceptible cultivar Rovar. A linkage map containing 96 loci on 11 linkage groups was constructed for 133 families from this cross. Resistance of progeny lines to natural Ascochyta blight epidemics was examined in field trials at Medina, Western Australia, in 1997, 1998, and 1999. Disease severity was assessed on stems, leaves, and pods by means of separate rating scales. Because pea shows increased susceptibility to Ascochyta blight as it matures, plant reproductive stage was assessed at the time of disease scoring in the 1998 and 1999 trials. Thirteen QTLs were detected for Ascochyta blight resistance on seven linkage groups. Eight of these QTLs were detected in multiple environments or by multiple trait scores. One QTL for plant developmental stage was detected. Linkage of Ascochyta blight resistance QTLs to candidate genes including disease response genes and resistance gene analogs and of the QTL for plant reproductive stage to a pea homolog of the Arabidopsis thaliana CONSTANS gene controlling flowering time in response to photoperiod is discussed.

Background Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. Results A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6–12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. Conclusion This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0143-y) contains supplementary material, which is available to authorized users.

Transformed pea (Pisum sativum L.) lines were produced with two chimeric gene constructs encoding the coat protein (CP) of alfalfa mosaic virus (AMV) strain NZ1. To determine whether transformed lines have improved AMV resistance, progeny of independently derived transgenic lines were tested in the greenhouse and five lines with improved virus resistance were identified. Resistance was observed only in individual plants that accumulated detectable CP product from the transgene, suggesting that resistance is CP mediated. Plants that accumulated detectable amounts of transgene CP product yet were susceptible to AMV were found among the progeny of most lines, indicating that these lines are only partially resistant to the challenging AMV strains, 425 and NZ1(Lincoln). A field test was conducted with the progeny of four independently derived transgenic lines. To test for improved virus resistance under field conditions, plots of transgenic lines and nontransgenic controls were inoculated with two AMV strains, NZ1(Lincoln) and NZ34. Analysis of disease severity in seedlings in inoculated plots confirmed that partial virus resistance can be produced by genetically modifying peas with AMV CP sequences.

Immature cotyledons from six cultivars and breeding lines of Pisum sativum were cocultivated with Agrobacterium tumefaciens and selected on a medium containing kanamycin. Transformed plants were recovered with a success rate of between 0.8 and 3.4% of explants treated. The plasmids used were based on pGA643 and have a nos promoter driving an nptII gene. Progeny from the primary transformants showed stable inheritance of the nos-nptII gene.

Didymella pinodes is the major pathogen of the ascochyta blight disease complex of field pea. The disease is endemic in all major field pea producing countries, frequently causing heavy losses in yield and quality. It is the most challenging of field pea diseases to manage, with most fungicides only partially effective and/or not cost-effective. In the absence of effective levels of host resistance, historically, the best management option has been delayed sowing and/or crop rotation to avoid major ascospore showers, but delayed sowing generally incurs a concurrent heavy yield penalty. This review evaluates world-wide progress in understanding critical components of black spot in terms of its management and evaluates opportunities both for new research and for development of more effective and sustainable management of this disease, using improved host resistance as a foundation to build and deploy more effective integrated disease management strategies. In the past decade, research has provided considerable new insights into potential ascochyta blight management strategies, including new insights into inheritance of host resistance, response to selection, and use of molecular technology, that together have demonstrated the potential to improve the level of host resistance. Significant improvements have been reported in the level of partial resistance of field pea with improved agronomic traits. Consequently, while the effect of this level of resistance in reducing D. pinodes infection remains to be quantified, for the first time such novel germplasm offers the prospect of revising and reintegrating the different disease management options at the farm level, based on deployment of such resistance. Combining this improved host resistance with both cultural management options and fungicidal application offers new opportunities. It is likely that previously restrictive cultural management and ineffective fungicidal control measures will re-emerge as effective and profitable practices when used in conjunction with partially-resistant germplasm. While this approach is an effective first stage to better manage ascochyta blight, future 'stacking' of broad antifungal genes on current moderately resistant varieties using genomic tools and/or GM technologies offers an avenue for even more effective control of D. pinodes. © 2013 Elsevier B.V.

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.

The glucosyl residues comprising starch can be phosphorylated at either the C3 or the C6 position of the molecule because of the activities of two distinct dikinase enzymes. After hydrolysis of the starch, the C6 phosphorylation is easy to measure using a routine enzyme assay for glucose 6-phosphate, but the C3 phosphorylation is more difficult to assay. A mass spectrometric (MS) method has been developed that, in a single run, can distinguish and quantify the glucose 3-phosphate and glucose 6-phosphate produced by hydrolysis of starch and can also measure the glucose content to give an accurate estimate of the starting material. The MS method involves quantification by LC/MS with external standards, using normal-phase hydrophilic interaction liquid chromatography and selective reaction monitoring. The MS method has been used to determine degrees of starch phosphorylation in a diverse group of potato lines, revealing threefold differences in phosphorylation between high- and low-phosphate lines. The method was also used to show that cold storage of potato tubers for up to 24weeks had little substantive effect on the levels of starch phosphorylation. MS provided an effective and efficient means of determining both the C6 and the C3 phosphorylation of starch.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is an analytical method for characterizing carbohydrate chain length that has been applied to neutral, charged, and N-linked oligosaccharides and that has been implemented using diverse separation platforms, including polyacrylamide gel electrophoresis and capillary electrophoresis. In this article, we describe three substantial improvements to FACE: (i) reducing the amount of starch and APTS required in labeling reactions and systematically analyzing the effect of altering the starch and 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) concentrations on the reproducibility of the FACE peak area distributions; (ii) implementing FACE on a multiple capillary DNA sequencer (an ABI 3130xl), enabling higher throughput than is possible on other separation platforms; and (iii) developing a protocol for producing quantitative output of peak heights and areas using genetic marker analysis software. The results of a designed experiment to determine the effect of decreasing both the starch and fluorophore concentrations on the sensitivity and reproducibility of FACE electrophoregrams are presented. Analysis of the peak area distributions of the FACE electrophoregrams identified the labeling reaction conditions that resulted in the smallest variances in the peak area distributions while retaining strong fluorescence signals from the capillary-based DNA sequencer.

Background: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. Methodology/Principal Findings: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. Conclusions/Significance: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

Pea ( Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2–0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Resistance to Ascochyta blight of pea was genetically characterized by mapping quantitative trait loci (QTLs) using two crosses, 3147-A26 (A26, partially resistant) × cultivar Rovar (susceptible) and 3148-A88 (A88, partially resistant) × Rovar, with the aim of developing an increased understanding of the genetics of resistance and of identifying linked molecular markers that may be used to develop resistant germplasm. Molecular linkage maps for both crosses were aligned so that the results of QTL mapping could be compared. Ascochyta blight disease severity in response to natural epidemics was measured in field trials conducted in Western Australia and New Zealand. Eleven putative QTLs for Ascochyta blight resistance were identified from the A26 × Rovar population and 14 putative QTLs from the A88 × Rovar population. Six QTLs were associated with the same genomic regions in both populations. These QTLs reside on linkage groups II, III, IV, V, and VII (two QTLs). The severity of Ascochyta blight disease symptoms on pea increases during field epidemics as plants mature; therefore, QTLs for plant reproductive maturity were mapped. Six QTLs were detected for plant maturity in the A26 × Rovar population, while five plant maturity QTLs were mapped in the A88 × Rovar population. QTLs for plant maturity coincide with Ascochyta blight resistance QTLs in four genomic regions, on linkage groups II (two regions), III, and V. The plant maturity and Ascochyta blight resistance QTLs on III were linked in repulsion phase. Therefore, the coincidence of these QTLs may be explained by linkage of distinct loci for the two traits. The QTLs on linkage groups II and V were linked in coupling phase; therefore, linked QTLs for resistance and maturity may be present in these regions, or the Ascochyta blight resistance QTLs detected in these regions are the result of pleiotropic effects of plant-maturity genetic loci.

Diagnostic capacity is a key component of phytosanitary systems designed to exclude or control unwanted pests and diseases. Because of their sensitivity, speed, and application to previously intractable problems, molecular methods are increasingly being used for diagnosis of pests or pathogens with either DNA or RNA genomes. This article describes methods for extracting nucleic acids suitable for PCR-or reverse transcription-polymerase chain reaction (RT-PCR)-based sequence detection from plant species in diverse botanical taxa. A modified hexadecyltrimethylammonium bromide (CTAB) extraction procedure produced total nucleic acid (T-NA) extracts suitable for PCR or RT-PCR from 62 of the 68 species or cultivars tested. For the remaining 8 taxonomically diverse plant species, commercially available DNA extraction kits yielded nucleic acids that were suitable for PCR. Goodquality RNA extractions were obtained from leaf tissue in most cases, by use of either a modified phenol-guanidine isothiocyanate method or proprietary reagents. When examining the suitability of the extraction methods for tissues other than young leaves such as roots, bark, flowers, and rhizomes/bulbs/tubers, a wider range of modifications to the methods was required to obtain suitable T-NA or RNA extractions. The value of PCR and RT-PCR assays based on chloroplastndhB gene sequences as endogenous positive controls was demonstrated by using both RNA and T-NA extracted from a wide range of botanical taxa, including both dicotyledonous and monocotyledonous plants.

Quantitative trait loci (QTLs) affecting seed weight in pea (Pisum sativum L.) were mapped using two populations, a field-grown F2 progeny of a cross between two cultivated types (‘Primo’ and ‘OSU442-15’) and glasshouse-grown single-seed-descent recombinant inbred lines (RILs) from a wide cross between a P. sativum ssp. sativum line (‘Slow’) and a P. sativum ssp. humile accession (‘JI1794’). Linkage maps for these crosses consisted of 199 and 235 markers, respectively. QTLs for seed weight in the ‘Primo’ x ‘OSU442-15’ cross were identified by interval mapping, bulked segregant analysis, and selective genotyping. Four QTLs were identified in this cross, demonstrating linkage to four intervals on three linkage groups. QTLs for seed weight in the ‘JI1794’ x ‘Slow’ cross were identified by single-marker analyses. Linkage were demonstrated to four intervals on three linkage groups plus three unlinked loci. In the two crosses, only one common genomic region was identified as containing seed-weight QTLs. Seed-weight QTLs mapped to the same region of linkage group III in both crosses. Conserved linkage relationships were demonstrated for pea, mungbean (Vigna radiata L.), and cowpea (V. unguiculata L.) genomic regions containing seed-weight QTLs by mapping RFLP loci from the Vigna maps in the ‘Primo’ x ‘OSU442-15’ and ‘JI1794’ x ‘Slow’ crosses.

Introducing resistance genes through plant breeding remains an important and effective means of protecting plants from diseases. Plant species commonly carry genes for disease resistance within their collective germplasm base. Disease resistance can either be monogenic (ie. encoded by a single gene) or quantitative (ie. encoded by a number of genes). The first step in disease resistance breeding is to identify accessions carrying the resistance phenotype. The conventional process of breeding for resistance involves making controlled crosses and selecting sexual progeny for improved disease resistance. While relatively straight-forward for dominantly-inherited single gene resistance, this process becomes progressively more difficult and time-comsuming for resistance encoded by single genes with recessive inheritance and for quantitatively inherited resistance. Through the process of genetic linkage mapping, molecular markers which are linked to disease resistance genes can be identified, and these can then be applied in plant breeding programmes to assist in resistance gene introgression. Our research group has identified DNA tags for a number of genes for resistance to diseases affecting peas (Pisum sativum L.). For example, DNA markers linked to recessive genes for resistance to pea seed-borne mosaic virus (PSbMV) pathotype P-1 (Timmerman et al. 1993) and to powdery mildew fungus (Timmerman et al. 1994) have been identified. These genes are termed sbm-1 and er-1, respectively. In more recent research, we have identified markers linked to the dominantly inherited gene for resistance to pea enation mosaic virus (PEMV). This gene is termed En. The molecular markers linked to these three monogenic disease resistance genes have been applied in a field pea breeding programme to develop germplasm containing multiple disease resistance phenotypes. DNA tags linked to sbm-1 and er-1 have been used in conjunction with limited direct testing for disease resistance. Although widely distributed throughout the world, PEMV does not occur in New Zealand; therefore breeding for resistance to PEMV requires the use of overseas disease testing or DNA tags. The DNA tags linked to En have been used in the early stages of cultivar development without direct testing for disease resistance. The genes contributing to quantitatively inherited disease resistance can also be characterised using linkage maps and DNA markers and DNA tags can be developed (Michelmore 1995). Experiments to map the genes for resistance to Ascochyta blight of peas are currently underway, using a QTL mapping experimental design. Similar experiments have been carried out by our research group to map genes for two other quantitatively inherited traits, seed weight (Timmerman-Vaughan et al. 1996) and green seed colour (McCallum et al. 1997). Ascochyta blight is a serious disease of peas which is caused by a trio of fungal pathogens: Mycosphaerella pinodes, Ascochyta pisi and Phoma medicaginis. Accessions with improved resistance to Ascochyta blight have been identified among peas bred by the Crop & Food Research field pea breeding programme. Using these accessions as the resistant parents, large populations of F 2 progeny and their descendants have been developed. The genotypes of these segregating

A linkage map for a set of 51 F2-derived recombinant inbred lines has been constructed from the segregation data of ≈850 morphological, isozyme, RFLP, STS, RAPD, and AFLP markers. The final map consists of seven clear linkage groups with a total length of nearly 900 cM. The wide variety of loci placed on this map permits its comparison with partial maps that have been developed in other programs. For the most part, the arrangement of loci agrees with that in previous maps, and no evidence for translocation heterozygosity in this cross is apparent. Although some clustering of markers is observed, for the most part the markers are well-distributed, and few gaps greater than 5 cM are found in the coverage. The availability of this first “complete” and highly saturated map for pea should permit more efficient comparison of the partial maps that have been generated in a number of different crosses, as well as provide a firm basis for future mapping and molecular studies in this species.

Developmental, environmental, and genetic factors affecting seed color were studied in the progeny of a cross between two white-flowered (aa) green cotyledon (ii) field peas (Pisum sativum L.): the pale large-seeded Marrowfat cultivar Primo and the greener small-seeded Prussian Blue OSU442-15. Changes in chlorophyll and carotenoid content during seed development of the parental genotypes were determined by high performance liquid chromatography analysis. Both cultivars accumulated similar pigment quantities per seed, but pigment loss was greater during maturation of `Primo'. Bleached and unbleached mature seed tissues also were compared for pigment composition. Lutein was the predominant pigment in bleached cotyledons of both cultivars. Only trace amounts of pheophytins were detected in unbleached seed. In both genotypes, chlorophyll A : B ratios were ≈1:1 in seed coats compared to 3:1 in cotyledons. Objective measurements of seed color in terms of luminance (lightness) and chrominance (hue and saturation) were made in YUV color space by video image analysis. Inheritance of seed color was studied in an F2 population derived from the `Primo' × `OSU442-15' cross and inbred descendants. Quantitative trait loci (QTL) for seed color were localized by interval mapping using a linkage map of 199 molecular markers spanning most of the genome and by bulked segregant analysis and selective genotyping. Four genomic regions affecting seed color were detected. A major gene accounting for 61% of the phenotypic variance in seed lightness (Y luminance component) was identified on linkage group V linked to r locus. Another major gene, which accounted for 56% of the phenotypic variance in seed hue (U chrominance component), was mapped to a linkage group containing group III and IV markers. A QTL with smaller effect on seed hue (U and V chrominance components) was detected on linkage group VII. Support for overdominant allelic interaction for a QTL on linkage group I, adjacent to the legumin locus Lg-J, was obtained by selective genotyping of the seed lightness distributional extremes.