Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples

Tracking disease resistance deployment in potato breeding by enrichment sequencing

Following the molecular characterisation of functional disease resistance genes in recent years, methods to track and verify the integrity of multiple genes in varieties are needed for crop improvement through resistance stacking. Diagnostic resistance gene enrichment sequencing (dRenSeq) enables the highconfidence identification and complete sequence validation of known functional resistance genes in crops. As demonstrated for tetraploid potato varieties, the methodology is more robust and cost-effective in monitoring resistances than whole-genome sequencing and can be used to appraise (trans)gene integrity efficiently. All currently known NB-LRRs effective against viruses, nematodes and the late blight pathogen Phytophthora infestans can be tracked with dRenSeq in potato and hitherto unknown polymorphisms have been identified. The methodology provides a means to improve the speed and efficiency of future disease resistance breeding in crops by directing parental and progeny selection towards effective combinations of resistance genes

Detection of Novel QTLs for Late Blight Resistance Derived from the Wild Potato Species Solanum microdontum and Solanum pampasense

peer-reviewedWild potato species continue to be a rich source of genes for resistance to late blight in potato breeding. Whilst many dominant resistance genes from such sources have been characterised and used in breeding, quantitative resistance also offers potential for breeding when the loci underlying the resistance can be identified and tagged using molecular markers. In this study, F1 populations were created from crosses between blight susceptible parents and lines exhibiting strong partial resistance to late blight derived from the South American wild species Solanum microdontum and Solanum pampasense. Both populations exhibited continuous variation for resistance to late blight over multiple field-testing seasons. High density genetic maps were created using single nucleotide polymorphism (SNP) markers, enabling mapping of quantitative trait loci (QTLs) for late blight resistance that were consistently expressed over multiple years in both populations. In the population created with the S. microdontum source, QTLs for resistance consistently expressed over three years and explaining a large portion (21–47%) of the phenotypic variation were found on chromosomes 5 and 6, and a further resistance QTL on chromosome 10, apparently related to foliar development, was discovered in 2016 only. In the population created with the S. pampasense source, QTLs for resistance were found in over two years on chromosomes 11 and 12. For all loci detected consistently across years, the QTLs span known R gene clusters and so they likely represent novel late blight resistance genes. Simple genetic models following the effect of the presence or absence of SNPs associated with consistently effective loci in both populations demonstrated that marker assisted selection (MAS) strategies to introgress and pyramid these loci have potential in resistance breeding strategies.Department of Agriculture, Food and the Marine, Irelan

The effect of pyramiding Phytophthora infestans resistance genes RPi-mcd1 and RPi-ber in potato

Despite efforts to control late blight in potatoes by introducing Rpi-genes from wild species into cultivated potato, there are still concerns regarding the durability and level of resistance. Pyramiding Rpi-genes can be a solution to increase both durability and level of resistance. In this study, two resistance genes, RPi-mcd1 and RPi-ber, introgressed from the wild tuber-bearing potato species Solanum microdontum and S. berthaultii were combined in a diploid S. tuberosum population. Individual genotypes from this population were classified after four groups, carrying no Rpi-gene, with only RPi-mcd1, with only RPi-ber, and a group with the pyramided RPi-mcd1 and RPi-ber by means of tightly linked molecular markers. The levels of resistance between the groups were compared in a field experiment in 2007. The group with RPi-mcd1 showed a significant delay to reach 50% infection of the leaf area of 3 days. The group with RPi-ber showed a delay of 3 weeks. The resistance level in the pyramid group suggested an additive effect of RPi-mcd1 with RPi-ber. This suggests that potato breeding can benefit from combining individual Rpi-genes, irrespective of the weak effect of RPi-mcd1 or the strong effect of RPi-ber

GpaXItarl originating from Solanum tarijense is a major resistance locus to Globodera pallida and is localised on chromosome 11 of potato

Resistance to Globodera pallida Rookmaker (Pa3), originating from wild species Solanum tarijense was identified by QTL analysis and can be largely ascribed to one major QTL. GpaXItarl explained 81.3% of the phenotypic variance in the disease test. GpaXItarl is mapped to the long arm of chromosome 11. Another minor QTL explained 5.3% of the phenotypic variance and mapped to the long arm of chromosome 9. Clones containing both QTL showed no lower cyst counts than clones with only GpaXItarl. After Mendelising the phenotypic data, GpaXItarl could be more precisely mapped near markers GP163 and FEN427, thus anchoring GpaXItarl to a region with a known R-gene cluster containing virus and nematode resistance genes

Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin

The origin and widespread occurrence of Sli based self-compatibility in potato

Self-compatible (SC) diploid potatoes allow innovative potato breeding. Therefore, the Sli gene, originally described in S. chacoense, has received much attention. In elite S. tuberosum diploids, spontaneous berry set is occasionally observed. We aimed to map SC from S. tuberosum origin. Two full-sib mapping populations from non-inbred diploids were used. Bulks were composed based on both pollen tube growth and berry set upon selfing. After DNA sequencing of the parents and bulks we generated k-mer tables. Set algebra and depth filtering was used to identify bulk-specific k-mers. Coupling and repulsion phase k-mers, transmitted from the SC parent, mapped in both populations to the distal end of chromosome 12. Intersection between the k-mers from both populations, in coupling phase with SC, exposed a shared haplotype of approximately 1.5 Mb. Subsequently we screened read archives of potatoes and wild relatives for k-mers specific to this haplotype. The well-known SC clones US-W4 and RH89-039-16, but surprisingly, also S. chacoense clone M6 were positives. Hence, the S. tuberosum source of SC seems identical to Sli. Furthermore, the candidate region drastically reduced to 333 kb. Haplotype specific KASP markers were designed and validated on a panel of diploid clones including another renown SC dihaploid G254. Interestingly, k-mers specific to the SC haplotype were common in tetraploid varieties. Pedigree information suggests that the SC haplotype was introduced into tetraploid varieties via the founder ‘Rough Purple Chili’. We show that Sli is surprisingly widespread and indigenous to the cultivated gene pool of potato

The origin and widespread occurrence of Sli based self-compatibility in potato

Self-compatible (SC) diploid potatoes allow innovative potato breeding. Therefore, the Sli gene, originally described in S. chacoense, has received much attention. In elite S. tuberosum diploids, spontaneous berry set is occasionally observed. We aimed to map SC from S. tuberosum origin. Two full-sib mapping populations from non-inbred diploids were used. Bulks were composed based on both pollen tube growth and berry set upon selfing. After DNA sequencing of the parents and bulks we generated k-mer tables. Set algebra and depth filtering was used to identify bulk-specific k-mers. Coupling and repulsion phase k-mers, transmitted from the SC parent, mapped in both populations to the distal end of chromosome 12. Intersection between the k-mers from both populations, in coupling phase with SC, exposed a shared haplotype of approximately 1.5 Mb. Subsequently we screened read archives of potatoes and wild relatives for k-mers specific to this haplotype. The well-known SC clones US-W4 and RH89-039-16, but surprisingly, also S. chacoense clone M6 were positives. Hence, the S. tuberosum source of SC seems identical to Sli. Furthermore, the candidate region drastically reduced to 333 kb. Haplotype specific KASP markers were designed and validated on a panel of diploid clones including another renown SC dihaploid G254. Interestingly, k-mers specific to the SC haplotype were common in tetraploid varieties. Pedigree information suggests that the SC haplotype was introduced into tetraploid varieties via the founder ‘Rough Purple Chili’. We show that Sli is surprisingly widespread and indigenous to the cultivated gene pool of potato