Search

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.

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.

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

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

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.

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.

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.

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.

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.