Genomic Selection for Ascochyta Blight Resistance in Pea.

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 <30% of lines). GBLUP and Bayesian Reproducing kernel Hilbert spaces regression (RKHS) performed slightly better than the other models trialed, whereas different missing data thresholds made minimal differences to prediction accuracy. The prediction accuracies of individual, randomly selected, testing/training partitions were highly variable, highlighting the effect that the choice of training population has on prediction accuracy. The inclusion of marker × environment interactions did not increase the prediction accuracy for lines which had not been phenotyped, but did improve the results of prediction across environments. GS is potentially useful for pea breeding programs pursuing ascochyta blight resistance, both for predicting breeding values for lines that have not been phenotyped, and for providing enhanced estimated breeding values for lines for which trait data is available.

Association mapping of starch chain length distribution and amylose content in pea (Pisum sativum L.) using carbohydrate metabolism candidate genes.

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.

Ascochyta blight disease of pea (Pisum sativum L.): defence-related candidate genes associated with QTL regions and identification of epistatic QTL.

KEY MESSAGE: 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.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

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.

Starch phosphorylation in potato tubers is influenced by allelic variation in the genes encoding glucan water dikinase, starch branching enzymes I and II, and starch synthase III.

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.

Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution.

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.

Mendel, 150 years on.

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.

Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer.

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.

Identification of Mendel's white flower character.

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.

Marker development and characterisation of Hordeum bulbosum introgression lines: a resource for barley improvement.

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.

Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products.

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<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.

Validation of quantitative trait loci for Ascochyta blight resistance in pea ( Pisum sativum L.), using populations from two crosses.

Resistance to Ascochyta blight of pea was genetically characterized by mapping quantitative trait loci (QTLs) using two crosses, 3147-A26 (A26, partially resistant) x cultivar Rovar (susceptible) and 3148-A88 (A88, partially resistant) x 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 x Rovar population and 14 putative QTLs from the A88 x 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 x Rovar population, while five plant maturity QTLs were mapped in the A88 x 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.