Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y.

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.

Cytoplasmic diversity of potato relatives preserved at Plant Breeding and Acclimatization Institute in Poland.

Among different types of potato cytoplasmic genomes, some are associated with male sterility or affect agronomic traits. The goal of this study was to analyze types of chloroplast and mitochondrial genomes of selected potato relatives originating from collection of the Institute of Plant Industry, Saint Petersburg, Russia, and preserved in Poland. Using chloroplast and mitochondrial markers the cytoplasm types were determined for 401 genotypes belonging to 43 seed accessions of 28 Solanum species. Among characterized genotypes, 201 (50.1%), 156 (38.9%) and 44 (11%) had cytoplasm types W, D, M, respectively. No accessions with the T, P or A cytoplasm were found. Within cytoplasm W, genotypes with the subtypes: W/α and W/ß were identified, but not with W/γ. In S. famatinae, we detected unusual product of the T marker with 65 bp insertion earlier seen exclusively in S. vernei. Among the genotypes of S. leptophyes, two profiles of the ALM_4/ALM_5 marker were observed. S. famatinae and S. vernei come from Argentina, provinces Catamarca and Tucumán. Possibly the insertion in marker T occurred independently in two species, or the accessions were misidentified. Segregation of the ALM_4/ALM_5 marker within S. leptophyes indicates that potato seed accessions are heterogeneous not only due to nuclear DNA polymorphisms but have diversified cytoplasm, too. Our findings are important for exploitation of the tested material in potato breeding. Male-fertile cytoplasm types give a chance of avoiding fertility problems and widening the range of crosses in future generations of breeding materials.

Quantitative trait loci analysis of potato tuber greening.

A conversion of amyloplasts into chloroplasts in the potato tuber after light exposure is known as tuber greening and is one of the major causes of tuber loss. We report here the first mapping of the factors affecting tuber greening in potato. We used an F1 mapping population of diploid potatoes and DArTseq™ markers to construct a genetic map. The individuals of the mapping population, parents and standards were phenotyped for two tuber greening parameters: external tuber greening and internal greening depth on 0-5 scales in three years 2015, 2016 and 2018. The results were used for the analysis of Quantitative Trait Loci (QTLs) by an interval QTL mapping. Two most important QTLs were covering large regions of chromosomes VII and X and had the strongest effect on both greening parameters in data sets obtained in particular years and in the mean data set. Variance observed in the mean tuber greening could be ascribed in 16.9% to the QTL on chromosome VII and in 23.4% to the QTL on chromosome X. The QTL on chromosome VII explained 13.1%, while the QTL on chromosome X explained up to 17.7% of the variance in the mean tuber greening depth. Additional, minor QTLs were year- and/or trait-specific. The QTLs on chromosomes VII and X determine big parts of the observed tuber greening variation and should be investigated further in order to identify the genes underlying their effects but also should be taken into account when selecting non-greening potato lines in the breeding process.

Novel gene Sen2 conferring broad-spectrum resistance to Synchytrium endobioticum mapped to potato chromosome XI.

Key message Sen2 gene for potato wart resistance, located on chromosome XI in a locus distinct from Sen1 , provides resistance against eight wart pathotypes, including the virulent ones important in Europe. Synchytrium endobioticum causes potato wart disease imposing severe losses in potato production, and as a quarantine pathogen in many countries, it results in lost trade markets and land for potato cultivation. The resistance to S. endobioticum pathotype 1(D1) is widespread in potato cultivars but new virulent pathotypes appear and the problem re-emerges. To characterize and map a new gene for resistance to potato wart, we used diploid F1 potato population from a cross of potato clone resistant to S. endobioticum pathotype 1(D1) and virulent pathotypes: 2(G1), 6(O1), 8(F1), 18(T1), 2(Ch1), 3(M1) and 39(P1) with a potato clone resistant to pathotype 1(D1) only. The 176 progeny clones were tested for resistance to eight wart pathotypes with a modified Glynne-Lemmerzahl method. Bimodal distributions and co-segregation of resistance in the population show that a single resistance gene, Sen2, underlies the resistance to eight pathotypes. Resistance to pathotype 1(D1) was additionally conferred by the locus Sen1 inherited from both parents. Sen2 was mapped to chromosome XI using DArTseq markers. The genetic and physical distances between Sen1 and Sen2 loci were indirectly estimated at 63 cM and 32 Mbp, respectively. We developed PCR markers co-segregating with the Sen2 locus that can be applied in marker-assisted selection of potatoes resistant to eight important pathotypes of S. endobioticum. Wide spectrum of the Sen2 resistance may be an indication of durability which can be enhanced by the pyramiding of the Sen2 and Sen1 loci as in 61 clones selected within this study.

Advocating a need for suitable breeding approaches to boost integrated pest management: a European perspective.

Currently, European farmers do not have access to sufficient numbers and diversity of crop species/varieties. This prevents them from designing cropping systems more resilient to abiotic and biotic stresses. Crop diversification is a key lever to reduce pest (pathogens, animal pests and weeds) pressures at all spatial levels from fields to landscapes. In this context, plant breeding should consist of: (1) increased efforts in the development of new or minor crop varieties to foster diversity in cropping systems, and (2) focus on more resilient varieties showing local adaptation. This new breeding paradigm, called here 'breeding for integrated pest management (IPM)', may boost IPM through the development of cultivars with tolerance or resistance to key pests, with the goal of reducing reliance on conventional pesticides. At the same time, this paradigm has legal and practical implications for future breeding programs, including those targeting sustainable agricultural systems. By putting these issues into the context, this article presents the key outcomes of a questionnaire survey and experts' views expressed during an EU workshop entitled 'Breeding for IPM in sustainable agricultural systems'. © 2017 Society of Chemical Industry.

Genetic composition of interspecific potato somatic hybrids and autofused 4x plants evaluated by DArT and cytoplasmic DNA markers.

KEY MESSAGE: Using DArT analysis, we demonstrated that all Solanum × michoacanum (+) S. tuberosum somatic hybrids contained all parental chromosomes. However, from 13.9 to 29.6 % of the markers from both parents were lost in the hybrids. Somatic hybrids are an interesting material for research of nucleus-cytoplasm interaction and sources of new nuclear and cytoplasmic combinations. Analyses of genomes of somatic hybrids are essential for studies on genome compatibility between species, its evolution and are important for their efficient exploitation. Diversity array technology (DArT) permits analysis of the composition of nuclear DNA of somatic hybrids. The nuclear genome compositions of 97 Solanum × michoacanum (+) S. tuberosum [mch (+) tbr] somatic hybrids from five fusion combinations and 11 autofused 4x mch were analyzed for the first time based on DArT markers. Out of 5358 DArT markers generated in a single assay, greater than 2000 markers were polymorphic between parents, of which more than 1500 have a known chromosomal location on potato genetic or physical map. DArT markers were distributed along the entire length of 12 chromosomes. We noticed elimination of markers of wild and tbr fusion components. The nuclear genome of individual somatic hybrids was diversified. Mch is a source of resistance to Phytophthora infestans. From 97 mch (+) tbr somatic hybrids, two hybrids and all 11 autofused 4x mch were resistant to P. infestans. The analysis of the structure of particular hybrids' chromosomes indicated the presence of markers from both parental genomes as well as missing markers spread along the full length of the chromosome. Markers specific to chloroplast DNA and mitochondrial DNA were used for analysis of changes within the organellar genomes of somatic hybrids. Random and non-random segregations of organellar DNA were noted.

Mapping of quantitative trait loci for tuber starch and leaf sucrose contents in diploid potato.

KEY MESSAGE: Most QTL for leaf sucrose content map to positions that are similar to positions of QTL for tuber starch content in diploid potato. In the present study, using a diploid potato mapping population and Diversity Array Technology (DArT) markers, we identified twelve quantitative trait loci (QTL) for tuber starch content on seven potato chromosomes: I, II, III, VIII, X, XI, and XII. The most important QTL spanned a wide region of chromosome I (42.0­104.6 cM) with peaks at 63 and 84 cM which explained 17.6 and 19.2% of the phenotypic variation, respectively. ADP-glucose pyrophosphorylase (AGPase) is the key enzyme for starch biosynthesis. The gene encoding the large subunit of this enzyme, AGPaseS-a, was localized to chromosome I at 102.3 cM and accounted for 15.2% of the variance in tuber starch content. A more than 100-fold higher expression of this gene was observed in RT-qPCR assay in plants with the marker allele AGPaseS-a1334. This study is the first to report QTL for sucrose content in potato leaves. QTL for sucrose content in leaves were located on eight potato chromosomes: I, II, III, V, VIII, IX, X and XII. In 5-week-old plants, only one QTL for leaf sucrose content was detected after 8 h of darkness; four QTL were detected after 8 h of illumination. In 11-week-old plants, 6 and 3 QTL were identified after dark and light phases, respectively. Of fourteen QTL for leaf sucrose content, eleven mapped to positions that were similar to QTL for tuber starch content. These results provide genetic information for further research examining the relationships between metabolic carbon molecule sources and sinks in potato plants.

Single-nucleotide polymorphisms and reading frame shifts in RNA2 recombinant regions of tobacco rattle virus isolates Slu24 and Deb57.

Two previously sequenced tobacco rattle virus (TRV) isolates, Slu24 and Deb57, from Polish potato fields have recombinant RNA2 species. The 3'-proximal region of the Slu24 RNA2 is derived from the 3' terminus of its supporting RNA1, while that of the Deb57 RNA2 is derived from the 3' terminus of the unrelated RNA1 from the isolate SYM or PpK20. Gene structure annotation revealed open reading frames encoding truncated 16-kDa proteins in the recombinant regions of the RNA2 of Deb57 and Slu24. Reading frame shifts, single nucleotide substitutions and deletions occurred during recombination, including shifts from a stop codon or replacements of an internal stop codon. In the recombinant region of the Deb57 RNA2, the first reading frameshift event starts from the AUG start codon of the truncated 16-kDa protein. The second frameshift event, caused by a single nucleotide deletion upstream of the stop codon, leads to the splitting of the stop codon into two amino acid codons and the continuation of translation. In addition, a U-to-A substitution changes a potential internal stop codon UAA, which is caused by recombination-related frame shifts, into the codon AAA, encoding lysine. The replacement of this internal stop codon with an amino acid codon prevented the premature translation termination of the truncated 16-kDa protein. These recombination-related reading frame shifts are the driving force underlying the major differences in the translated amino acids, consequently leading to their translation into distinct polypeptides. Conversely, single nucleotide substitutions in the recombinant regions of the RNA2 of Deb57 or Slu24 resulted in only minor changes in the translated amino acids.

The genomic RNA1 and RNA2 sequences of the tobacco rattle virus isolates found in Polish potato fields.

Four tobacco rattle virus (TRV) isolates were identified from tobacco bait seedlings planted in soil samples from Polish potato fields. Sequence analysis of the genomic RNA1 of the isolates revealed significant similarity to the isolates Ho and AL recently found in Germany. Multiple sequence alignments of the genomic RNA2 indicated that the two isolates from northern Poland (Deb57 and Slu24) are in a cluster with the isolates PSG and PLB found in the Netherlands. The remaining two isolates, from central Poland (11r21 and Mlo7), are in a distinct group with the unique isolate SYM found in England. The RNA2 sequences of the studied isolates range from 1998 nt to 2739 nt in length, and all carry deletions of the 2b and/or 2c genes. The isolate Mlo7 has an atypical RNA2 structure, having its cp gene located in its central region.

Late blight resistance gene from Solanum ruiz-ceballosii is located on potato chromosome X and linked to violet flower colour.

BACKGROUND: Phytophthora infestans (Mont.) de Bary, the causal organism of late blight, is economically the most important pathogen of potato and resistance against it has been one of the primary goals of potato breeding. Some potentially durable, broad-spectrum resistance genes against this disease have been described recently. However, to obtain durable resistance in potato cultivars more genes are needed to be identified to realize strategies such as gene pyramiding or use of genotype mixtures based on diverse genes. RESULTS: A major resistance gene, Rpi-rzc1, against P. infestans originating from Solanum ruiz-ceballosii was mapped to potato chromosome X using Diversity Array Technology (DArT) and sequence-specific PCR markers. The gene provided high level of resistance in both detached leaflet and tuber slice tests. It was linked, at a distance of 3.4 cM, to violet flower colour most likely controlled by the previously described F locus. The marker-trait association with the closest marker, violet flower colour, explained 87.1% and 85.7% of variance, respectively, for mean detached leaflet and tuber slice resistance. A genetic linkage map that consisted of 1,603 DArT markers and 48 reference sequence-specific PCR markers of known chromosomal localization with a total map length of 1204.8 cM was constructed. CONCLUSIONS: The Rpi-rzc1 gene described here can be used for breeding potatoes resistant to P. infestans and the breeding process can be expedited using the molecular markers and the phenotypic marker, violet flower colour, identified in this study. Knowledge of the chromosomal localization of Rpi-rzc1 can be useful for design of gene pyramids. The genetic linkage map constructed in this study contained 1,149 newly mapped DArT markers and will be a valuable resource for future mapping projects using this technology in the Solanum genus.

A resistance gene against potato late blight originating from Solanum × michoacanum maps to potato chromosome VII.

Solanum ×  michoacanum (Bitter.) Rydb. is a diploid, 1 EBN (Endosperm Balance Number) nothospecies, a relative of potato originating from the area of Morelia in Michoacán State of Mexico that is believed to be a natural hybrid of S. bulbocastanum × S. pinnatisectum. Both parental species and S. michoacanum have been described as sources of resistance to Phytophthora infestans (Mont.) de Bary. The gene for resistance to potato late blight, Rpi-mch1, originating from S. michoacanum was mapped to the chromosome VII of the potato genome. It confers high level of resistance since the plants possessing it showed only small necrotic lesions or no symptoms of the P. infestans infection and we could ascribe over 80% of variance observed in the late blight resistance test of the mapping population to the effect of the closest marker. Its localization on chromosome VII may correspond to the localization of the Rpi1 gene from S. pinnatisectum. When mapping Rpi-mch1, one of the first genetic maps made of 798 Diversity Array Technology (DArT) markers of a plant species from the Solanum genus and the first map of S. michoacanum, a 1EBN potato species was constructed. Particular chromosomes were identified using 48 sequence-specific PCR markers, originating mostly from the Tomato-EXPEN 2000 linkage map (SGN), but also from other sources. Recently, the first DArT linkage map of 2 EBN species Solanum phureja has been published and it shares 197 DArT markers with map obtained in this study, 88% of which are in the concordant positions.