Summary
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.
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
can prove problematic due to the physiological and environmental (abiotic and biotic) factors at play in vineyards. Here we report on the use of
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
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.
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.).
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.
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.
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.
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•We assess N leaching reduction by cover crops in response to climate change scenarios.•Climate change impacted cover crop performance and N leaching events concomitantly.•Risks of N leaching can rise despite a simultaneous increase in cover crop performance.•Fine spatio-temporal resolutions are recommended to analyze cover crop effectiveness.
There is a growing niche market for coloured-skin potatoes, and breeders are developing new cultivars that not only exhibit coloured tuber skin but are also improved for agronomic and nutritional ...traits. Molecular markers provide a mechanism to increase the efficiency of such breeding programmes through marker-assisted selection (MAS) of both parents and elite seedlings in breeding populations. In our study, we used next-generation sequencing and high-resolution DNA melting (HRM) to develop single nucleotide polymorphism (SNP)-based markers from candidate genes associated with tuber skin colour in tetraploid potato, including small amplicon assays and dosage-sensitive unlabelled probes. We present SNP-based HRM markers for
Stan2
and
Stan3
(
D
locus),
dfr
(
R
locus) and
f3′5′h
(
P
locus) as predictors of tuber skin colour in potato, employing a genetic resource of 19 parental accessions for marker development and then demonstrating their application in three segregating populations. To our knowledge, this is not only the first report of HRM unlabelled probe and short amplicon assays at the
D
locus, but also the first report of the implementation of MAS for tuber skin colour, using a combination of markers at the
D
,
R
and
P
loci. Our estimation of allele dosage for these genes highlights the usefulness of this methodology for parental selection targeting oligogenic traits.
Although Sequence-Characterized Amplified Region (SCAR) markers linked to the potato
H1
locus, which confers resistance to pathotypes Ro1 and Ro4 of the potato cyst nematode (PCN)
Globodera ...rostochiensis
, have been reported, robust markers that enable estimation of allele dosage would improve the quality of information obtained from genotyping parental accessions (cultivars/breeding lines) and progeny populations within breeding programmes. With this in mind, we have developed single nucleotide polymorphism (SNP)-based molecular markers flanking the
H1
resistance gene, using genomic re-sequence data from five elite tetraploid accessions. The published TG689 and 57R primer sequences were used in a Basic Local Alignment Search Tool (BLAST) examination of the reference potato genome, and SNPs within the vicinity of these primer regions were identified and targeted for designing probe-based High Resolution Melting (HRM) SNP assays. Evaluation of the subsequently developed HRM markers, TG689_1P and 57R_1P, against the publicly available SCAR markers, TG689 and 57R, indicated that the HRM markers enabled more reliable marker-trait association than the SCARs. Additionally, allelic dosage estimates for the
H1
locus were also derived using the TG689_1P marker, providing a tool to optimise parental and progeny selections in PCN resistance breeding.
A modified cry9Aa2 gene under the transcriptional control of the CaMV 35S promoter was transferred to four potato cultivars ('Iwa', 'Karaka', 'Pacific' and 'Red Rascal') using Agrobacterium-mediated ...gene transfer. Field plots were established for 170 independently regenerated transgenic lines derived from 113 transformation events. Phenotypic changes such as stunted plants, reduced vigour and/or leaf puckering, were observed in 29% of these lines. Foliage from the remaining 117 lines inhibited larval growth of potato tuber moth (Phthorimaea operculella Zeller) compared with non-transgenic controls. A high correlation was observed between greenhouse- and field-grown foliage for insect resistance using a detached leaf bioassay. Independently regenerated lines recovered from the same transformation event exhibited similar insect resistance. However, these lines derived from the same transformed cell differed markedly in phenotypic appearance and tuber yield. The first shoot regenerated from each transformation event was equivalent to the non-transgenic control for yield traits in only eight transformation events. A further 19 transformation events exhibited agronomic performance equivalent to the non-transgenic control when the second or subsequent regenerated shoots were evaluated. In clonal crops, multiple shoots from each transformation event should be independently assessed to ensure recovery of high-performing transgenic lines.
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.
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.
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.
The feasibility of two strategies for transgene pyramiding using
Agrobacterium
-mediated transformation was investigated to develop a transgenic potato (
Solanum tuberosum
L. cv. Iwa) with resistance ...to potato tuber moth (PTM) (
Phthorimaea operculella
(Zeller)). In the first approach,
cry
1Ac9 and
cry
9Aa2 genes were introduced simultaneously using a kanamycin (
npt
II) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a
cry
1Ac9 gene, using a hygromycin resistance (
hpt
) selectable marker gene, into an existing line transgenic for a
cry
9Aa2 gene and a kanamycin resistance (
npt
II) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both
cry
genes in all regenerated lines. The relative steady-state level of the
cry
gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both
cry
genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual
cry
genes.
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