Introgression of Two Broad-Spectrum Late Blight Resistance Genes, Rpi-Blb1 and Rpi-Blb3, From Solanum bulbocastanum Dun Plus Race-Specific R Genes Into Potato Pre-breeding Lines.

There is a wealth of resistance genes in the Mexican wild relative of cultivated Solanum, but very few of these species are sexually compatible with cultivated Solanum tuberosum. The most devastating disease of potato is late blight caused by the oomycete Phytophthora infestans (Pi). The wild hexaploid species S. demissum, which it is able to cross with potato, was used to transfer eleven race-specific genes by introgressive hybridization that were subsequently widely used in potato breeding. However, there are now more virulent races of Pi that can overcome all of these genes. The most sustainable strategy for protecting potatoes from late blight is to pyramid or stack broad-spectrum resistance genes into the cultivars. Recently four broad-spectrum genes (Rpi) conferring resistance to Pi were identified and cloned from the sexually incompatible species S. bulbocastanum: Rpi-blb1 (RB), Rpi-blb2, Rpi-blb3, and Rpi-bt1. For this research, a resistant S. bulbocastanum accession was selected carrying the genes Rpi-blb1 and Rpi-blb3 together with race-specific R3a and R3b genes. This accession was previously used to produce a large number of somatic hybrids (SHs) with five commercial potato cultivars using protoplast electrofusion. In this study, three SHs with cv. 'Delikat' were selected and backcross generations (i.e., BC1 and BC2) were obtained using cvs. 'Baltica', 'Quarta', 'Romanze', and 'Sarpo Mira'. Their assessment using gene-specific markers demonstrates that these genes are present in the SHs and their BC progenies. We identified plants carrying all four genes that were resistant to foliage blight in greenhouse and field trials. Functionality of the genes was shown by using agro-infiltration with the effectors of corresponding Avr genes. For a number of hybrids and BC clones yield and tuber number were not significantly different from that of the parent cultivar 'Delikat' in field trials. The evaluation of agronomic traits of selected BC2 clones and of their processing qualities revealed valuable material for breeding late blight durable resistant potato. We show that the combination of somatic hybridization with the additional use of gene specific markers and corresponding Avr effectors is an efficient approach for the successful identification and introgression of late blight resistance genes into the potato gene pool.

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization.

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a 'mutator' phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for 'mutator' phenotypes in somatic hybrids between potato cvs. 'Delikat' and 'Désirée' and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with 'mutator' phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.

Mismatch repair deficiency increases the transfer of antibiosis and antixenosis properties against Colorado potato beetle in somatic hybrids of Solanum tuberosum + S. chacoense.

BACKGROUND: Colorado potato beetle (CPB) has become the biggest enemy of cultivated potato worldwide. One of the most effective sources of resistance to CPB is Solanum chacoense, an accession with a high leptine glycoalkaloid content. The aim of our study was to assay the repellence and toxicity of S. chacoense, its somatic hybrids (SHs) and their backcross progenies (BC1 ) with potato for CPB adults and larvae. Transgenic S. chacoense, deficient in DNA mismatch repair (MMR), was also used to produce SHs, in order to increase homeologous recombination and hence introgression of wild-species DNA into the potato gene pool. RESULTS: Wild-type SH was highly resistant to CPB. Resistance to CPB of BC1 progenies showed a 1:3 inheritance pattern. MMR-deficient SHs performed better in the resistance analysis. Most MMR-deficient SHs had a similar toxicity as S. chacoense and an intensely repellent effect on CPB adults. Resistance of SHs and BC1 clones may be attributed to leptine biosynthesis, which was confirmed using a RAPD marker. CONCLUSION: This is the first report of SHs and their progenies exhibiting both antibiosis and antixenosis against CPB. Resistant SHs are an important step forward in combating this voracious pest of potato. © 2016 Society of Chemical Industry.

Characterization of the multiple resistance traits of somatic hybrids between Solanum cardiophyllum Lindl. and two commercial potato cultivars.

Interspecific somatic hybrids between commercial cultivars of potato Solanum tuberosum L. Agave and Delikat and the wild diploid species Solanum cardiophyllum Lindl. (cph) were produced by protoplast electrofusion. The hybrid nature of the regenerated plants was confirmed by flow cytometry, simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphism (MFLP) markers and morphological analysis. Somatic hybrids were assessed for their resistance to Colorado potato beetle (CPB) using a laboratory bioassay, to Potato virus Y (PVY) by mechanical inoculation and field trials, and foliage blight in a greenhouse and by field trials. Twenty-four and 26 somatic hybrids of cph + cv. Agave or cph + cv. Delikat, respectively, showed no symptoms of infection with PVY, of which 3 and 12, respectively, were also resistant to foliage blight. One hybrid of cph + Agave performed best in CPB and PVY resistance tests. Of the somatic hybrids that were evaluated for their morphology and tuber yield in the field for 3 years, four did not differ significantly in tuber yield from the parental and standard cultivars. Progeny of hybrids was obtained by pollinating them with pollen from a cultivar, selfing or cross-pollination. The results confirm that protoplast electrofusion can be used to transfer the CPB, PVY and late blight resistance of cph into somatic hybrids. These resistant somatic hybrids can be used in pre-breeding studies, molecular characterization and for increasing the genetic diversity available for potato breeding by marker-assisted combinatorial introgression into the potato gene pool.

Novel somatic hybrids (Solanum tuberosum L.+Solanum tarnii) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight.

Solanum tarnii, a wild diploid, tuber-bearing Mexican species belonging to the series Pinnatisecta is highly resistant to Potato virus Y (PVY) and Colorado potato beetle and shows a strong hypersensitive reaction to Phytophthora infestans. Therefore, it could be a potential source of resistance to pathogens for potato breeders. S. tarnii (2n=2x=24) is reproductively isolated from tetraploid Solanum tuberosum and hence difficult to include in potato breeding programmes. In this study, interspecific somatic hybrids were produced for the first time by protoplast electrofusion of the cells of potato cv. Delikat (Solanum tuberosum L.) and Solanum tarnii. The hybrid nature of the regenerants was confirmed by simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers and by morphological analysis and flow cytometry. Selected somatic hybrids were successfully backcrossed with cv. Delikat. Parental lines, primary somatic hybrids and BC1 progeny were assessed for resistance to PVY by mechanical inoculation, grafting and exposure to viruliferous aphid vectors in the field, and resistance to late blight (P. infestans) by detached leaflet and whole tuber tests. The somatic hybrids showed no symptoms of viral infection and most of them displayed high levels of resistance to foliage blight. The BC1 progenies were highly resistant to PVY and a few were resistant to foliage blight. Selected hybrids and BC1 clones were evaluated in the field for tuber quality and tuber yield. Some BC1 clones produced yields of good quality tubers. The results confirm that both the resistance to PVY and to late blight of S. tarnii is expressed in somatic hybrids, and PVY resistance is transferred to BC1 progeny, whereas blight resistance is harder to transfer. Somatic hybridization again proved to be a valuable tool for producing pre-breeding material with increased genetic diversity.

The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.