A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Tuber shape and eye depth variation in a diploid family of Andean potatoes.

BACKGROUND: Tuber appearance is highly variable in the Andean cultivated potato germplasm. The diploid backcross mapping population 'DMDD' derived from the recently sequenced genome 'DM' represents a sample of the allelic variation for tuber shape and eye depth present in the Andean landraces. Here we evaluate the utility of morphological descriptors for tuber shape for identification of genetic loci responsible for the shape and eye depth variation. RESULTS: Subjective morphological descriptors and objective tuber length and width measurements were used for assessment of variation in tuber shape and eye depth. Phenotypic data obtained from three trials and male-female based genetic maps were used for quantitative trait locus (QTL) identification. Seven morphological tuber shapes were identified within the population. A continuous distribution of phenotypes was found using the ratio of tuber length to tuber width and a QTL was identified in the paternal map on chromosome 10. Using toPt-437059, the marker at the peak of this QTL, the seven tuber shapes were classified into two groups: cylindrical and non-cylindrical. In the first group, shapes classified as 'compressed', 'round', 'oblong', and 'long-oblong' mainly carried a marker allele originating from the male parent. The tubers in this group had deeper eyes, for which a strong QTL was found at the same location on chromosome 10 of the paternal map. The non-cylindrical tubers classified as 'obovoid', 'elliptic', and 'elongated' were in the second group, mostly lacking the marker allele originating from the male parent. The main QTL for shape and eye depth were located in the same genomic region as the previously mapped dominant genes for round tuber shape and eye depth. A number of candidate genes underlying the significant QTL markers for tuber shape and eye depth were identified. CONCLUSIONS: Utilization of a molecular marker at the shape and eye depth QTL enabled the reclassification of the variation in general tuber shape to two main groups. Quantitative measurement of the length and width at different parts of the tuber is recommended to accompany the morphological descriptor classification to correctly capture the shape variation.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

Applications of biotechnology and genomics in potato improvement.

Potato is the third most important global food crop and the most widely grown noncereal crop. As a species highly amenable to cell culture, it has a long history of biotechnology applications for crop improvement. This review begins with a historical perspective on potato improvement using biotechnology encompassing pathogen elimination, wide hybridization, ploidy manipulation and applications of cell culture. We describe the past developments and new approaches for gene transfer to potato. Transformation is highly effective for adding single genes to existing elite potato clones with no, or minimal, disturbances to their genetic background and represents the only effective way to produce isogenic lines of specific genotypes/cultivars. This is virtually impossible via traditional breeding as, due to the high heterozygosity in the tetraploid potato genome, the genetic integrity of potato clones is lost upon sexual reproduction as a result of allele segregation. These genetic attributes have also provided challenges for the development of genetic maps and applications of molecular markers and genomics in potato breeding. Various molecular approaches used to characterize loci, (candidate) genes and alleles in potato, and associating phenotype with genotype are also described. The recent determination of the potato genome sequence has presented new opportunities for genomewide assays to provide tools for gene discovery and enabling the development of robustly unique marker haplotypes spanning QTL regions. The latter will be useful in introgression breeding and whole-genome approaches such as genomic selection to improve the efficiency of selecting elite clones and enhancing genetic gain over time.

Exogenous abscisic acid and sugar induce a cascade of ripening events associated with anthocyanin accumulation in cultured Pinot Noir grape berries.

Fruit quality is dependent on various factors including flavour, texture and colour. These factors are determined by the ripening process, either climacteric or non-climacteric. In grape berry, which is non-climacteric, the process is signalled by a complex set of hormone changes. Abscisic acid (ABA) is one of the key hormones involved in ripening, while sugar availability also plays a significant role in certain ripening aspects such as anthocyanin production. To understand the relative influence of hormone and sugar signalling in situ can prove problematic due to the physiological and environmental (abiotic and biotic) factors at play in vineyards. Here we report on the use of in vitro detached berry culture to investigate the comparative significance of ABA and sugar in the regulation of Pinot noir berry anthocyanin production under controlled conditions. Using a factorial experimental design, pre-véraison berries were cultured on media with various concentrations of sucrose and ABA. After 15 days of in vitro culture, the berries were analysed for changes in metabolites, hormones and gene expression. Results illustrated a stimulatory effect of sucrose and ABA on enhancing berry colour and a corresponding increase in anthocyanins. Increased ABA concentration was able to boost anthocyanin production in berries when sucrose supply was low. The sucrose and ABA effects on berry anthocyanins were primarily manifested through the up-regulation of transcription factors and other genes in the phenylpropanoid pathway, while in other parts of the pathway a down-regulation of key proanthocyanindin transcription factors and genes corresponded to sharp reduction in berry proanthocyanidins, irrespective of sucrose supply. Similarly, increased ABA was correlated with a significant reduction in berry malic acid and associated regulatory genes. These findings suggest a predominance of berry ABA over berry sugar in coordinating the physiological and genetic regulation of anthocyanins and proanthocyanins in Pinot noir grape berries.

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance.

BACKGROUND: The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). RESULTS: A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. CONCLUSIONS: A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event.

Understanding spatial and temporal variability of N leaching reduction by winter cover crops under climate change.

Winter cover crops are sown in between main spring crops (e.g. cash and forage crops) to provide a range of benefits, including the reduction of nitrogen (N) leaching losses to groundwater. However, the extent by which winter cover crops will remain effective under future climate change is unclear. We assess variability and uncertainty of climate change effects on the reduction of N leaching by winter oat cover crops. Field data were collected to quantify ranges of cover crop above-ground biomass (7 to 10 t DM/ha) and N uptake (70 to 180 kg N/ha) under contrasting initial soil conditions. The data were also used to evaluate the APSIM-NextGen model (R2 from 62 to 96% and RMSEr from 7 to 50%), which was then applied to simulate cover crop and fallow conditions across four key agricultural locations in New Zealand, under baseline and future climate scenarios. Cover crops reduced N leaching risks for all location/scenario combinations but with large variability in space and time (e.g. 21 to 47% of fallow) depending on the climate change scenario. For instance, end-of-century estimates for northern (warmer) locations mostly showed non-significant effects of climate change on cover crop effectiveness and N leaching. In contrast for southern (colder) locations, there was a systematic increase in N leaching risks with climate change intensity despite a concomitant, but less than proportional, increase in cover crop effectiveness (up to ~5% of baseline) due to higher winter yields and N uptake. This implies that climate change may not only modify the geography of N leaching hotspots, but also the extent by which cover crops can locally reduce pollution risks, in some cases requiring complementary adaptive measures. The patchy- and threshold-nature of leaching events indicates that fine spatio-temporal resolutions are better suited to evaluate cover crop effectiveness under climate change.

Towards disease resistance in potatoes using intragenic approaches.

Disease resistance is an important objective of global potato breeding programmes. The use of resistant cultivars is a significant tool for disease management. Recent advances in plant molecular genetics have identified several genes for resistance to potato diseases from within the germplasm pool available to potato breeders. Antimicrobial peptides, such as Snakin-1 (StSN1) and Snakin-2 (StSN2), have been isolated recently from potato tubers. Overexpression of the StSNI and StSN2 genes in potato is known to provide broad spectrum activity against a wide range of bacterial and fungal pathogens. We describe the use of intragenic gene transfer technology towards disease resistance in potatoes. An expression cassette was constructed with the 5' promoter and 3' terminator regions of a potato gene encoding a chlorophyll a/b binding protein (StLhca3). The coding regions of the StSN1 and StSN2 genes of potato were cloned individually between these regulatory regions. The resulting Lhca3-StSNi-Lhca3 and Lhca3-StSN2-Lhco3 chimeric genes were individually cloned into a potato-derived T-DNA-like region for potato transformation. Potato cultivar Iwa was co-cultivated with Agrobocterium harbouring intragenic binary vectors with the StSN1 and StSN2 genes. Regenerated potato plants were screened using PCR to identify lines transformed with the disease resistance genes without the presence of foreign DNA.

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

BACKGROUND: The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. RESULTS: To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. CONCLUSION: We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.