Rena Sanetomo

Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.).

Key messageSNPs in candidate genesPain-1,InvCD141(invertases),SSIV(starch synthase),StCDF1(transcription factor),LapN(leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield.AbstractTuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of genes that control the natural variation of TY, TSC, and TSY could increase precision and speed of breeding new cultivars optimized for potato starch production. Diagnostic DNA markers are identified by association mapping in populations of tetraploid potato varieties and advanced breeding clones. A novel association mapping population of 282 genotypes including varieties, breeding clones and Andean landraces was assembled and field evaluated in Northern Spain for TY, TSC, TSY, tuber number (TN) and tuber weight (TW). The landraces had lower mean values of TY, TW, TN, and TSY. The population was genotyped for 183 microsatellite alleles, 221 single nucleotide polymorphisms (SNPs) in fourteen candidate genes and eight known diagnostic markers for TSC and TSY. Association test statistics including kinship and population structure reproduced five known marker–trait associations of candidate genes and discovered new ones, particularly for tuber yield and starch yield. The inclusion of landraces increased the number of detected marker–trait associations. Integration of the present association mapping results with previous QTL linkage mapping studies for TY, TSC, TSY, TW, TN, and tuberization revealed some hot spots of QTL for these traits in the potato genome. The genomic positions of markers linked or associated with QTL for complex tuber traits suggest high multiplicity and genome wide distribution of the underlying genes.

Reciprocal differences in DNA sequence and methylation status of the pollen DNA between F1 hybrids of Solanum tuberosum × S. demissum

Reciprocal differences, mostly caused by cytoplasmic effects, are frequently observed in interspecific hybrids. Previously, we found that crosses onto Solanum demissum were much successful with the pollen of interspecific hybrids between S. tuberosum as female and S. demissum as male (TD hybrids) than the reciprocal ones (DT hybrids). To elucidate this reciprocally different crossability, we analyzed the pollen DNA of TD and DT using methylation-sensitive amplified polymorphism (MSAP) analysis. Using 126 primer combinations, MSAP analysis revealed 57 different bands between bulked pollen DNA samples of TD and DT. Individual examination of 16 TD and 9 DT plants disclosed eight bands uniformly different between TD and DT. Their sequencing results revealed two pairs of bands to be identical to each other, resulting in six distinct sequences. As expected, one band shared high homology with chloroplast DNA, and another one with mitochondrial DNA. However, one band that was apparently different at DNA sequence level and maternally transmitted from S. demissum, showed no homology with any known sequence. The remaining three bands were of DNA methylation level differences with no or uncertain homology to known sequences. To our knowledge, this is the first report detecting reciprocal differences in DNA sequence or DNA methylation other than those in cytoplasmic DNA.

A maternally inherited DNA marker, descended from Solanum demissum (2n = 6x = 72) to S. tuberosum (2n = 4x = 48)

A Mexican hexaploid wild potato species, Solanum demissum (dms), was only used as a female in previous breeding programs. The resulting clones with dms cytoplasm produced abundant, but non-functional pollen. A 170 bp DNA fragment, named Band 1, was originally detected in the F1 hybrid between dms and S. tuberosum. In this study, the sequenced region was extended to 1,032 bp; nevertheless, it did not show any homology to known sequences. This extended region harboring Band 1 was, without introns, all transcribed to mRNA and was maternally inherited from dms to S. tuberosum through backcrosses. Three dms accessions, 168 accessions of 38 cultivated and closely related wild species, and 158 varieties and breeding lines were surveyed, which demonstrated that Band 1 was specific to dms and varieties and breeding lines with dms cytoplasm. Thus, Band 1 is a useful marker to distinguish dms cytoplasm, which enables us to design efficient mating combinations in breeding programs.

Application of a PCR-Based Cytoplasm Genotyping Method for Phylogenetic Analysis in Potato

We previously developed five polymerase chain reaction-based markers (T, S, SAC, D, and A) to distinguish potato cytoplasms into six types (M, P, A, W, T, and D). As the applicability of this genotyping method for phylogenetic studies had been questioned, we applied this method to species distantly related to cultivars (four accessions of two tomato species, and 176 accessions of 29 Solanum species). The T marker uncovered two insertions, which, along with a unique S marker band, supported independency of series Piurana. The A and D markers generated unique bands to A- and D-type cytoplasms, respectively, but the SAC marker generated a similar banding pattern to the cytoplasms of both cultivated and their distantly related species. Consequently, while the cytoplasm type definition is validated only among cultivated potatoes and their closely related wild species, the developed markers, with the exception of the SAC marker, could provide useful phylogenetic information.ResumenNosotros desarrollamos previamente cinco marcadores con base a la reacción en cadena de la polimerasa (T, S, SAC, D, y A) para distinguir los citoplasmas de la papa en seis tipos (M, P, A, W, T, y D). Considerando que la aplicabilidad de este método de caracterizar genotipos para estudios filogenéticos había sido cuestionada, aplicamos este método para especies poco relacionadas a variedades (cuatro introducciones de dos especies de tomate y 176 de 29 especies de Solanum). El marcador T descubrió dos inserciones, las cuales, junto con una banda única del marcador S, respaldaron independencia de la serie Piurana. Los marcadores A y D generaron bandas únicas a citoplasmas de los tipos A y D, respectivamente, pero el marcador SAC generó un patrón de bandas similar a los citoplasmas tanto de las cultivadas como de las especies con relaciones distantes. Consecuentemente, mientras que la definición del tipo de citoplasma se valida solo entre papas cultivadas y sus especies silvestres con relación cercana, los marcadores desarrollados, con la excepción del SAC, pudieron proporcionar información filogenética útil.

Effects of Solanum demissum chromosomes on crossability in the backcross progeny to Solanum tuberosum

A balance of maternal and paternal genetic factors, conceptually named the endosperm balance number (EBN), is required for normal endosperm development in interspecific crosses in potato. We previously found that Solanum demissum (D), a hexaploid wild species widely used in potato breeding, has a slightly lower EBN than S. tuberosum (T). To explore the genetic nature of the EBN, the berry-setting rate, seed number/berry, and seed weight were evaluated in BC1 [(D × T) × T] plants, each possessing different portions of the S. demissum chromosomes, by reciprocal crosses with D and T, and a quantitative trait locus (QTL) analysis was performed. At least 99 S. demissum-derived QTLs were detected, of which 29 were associated with differential responses to D and T. Three QTLs were possibly co-localized on chromosomes 7A and 10D1, while the remaining 23 QTLs were independently located. The QTLs in the three S. demissum homoeologous chromosomes exhibited three types of interaction: (1) positive, (2) negative, and (3) one positive and one negative effect on the same trait. We found that several major genes, one of which was localized in the S. demissum chromosome 9A, and many minor genes controlled the crossability of BC1 plants. The QTLs responsible for the differential responses to D and T were different between the BC1 plants used as male and female parents, indicating that different genes control the male and female EBNs. Consequently, we conclude that the EBN is represented by the sum of various genetic effects controlled by a large number of genes.

Harimaru: a new potato variety for a local specialty

‘Harimaru’ is a new potato variety bred from a cross between ‘Saikai 35’ as a female parent and ‘Pike’ as a male parent. Marker selection was performed for 1,647 seedlings to combine resistance genes to late blight (R1), Potato virus Y (Rychc), Potato virus X (Rx1), and golden cyst nematode (H1). In total, 194 selected clones were evaluated in the field, among which the best clone was officially released as ‘Harimaru’. Its yield was slightly lower than the local standard variety, ‘May Queen’. However, it produces tasty potatoes, that do not become mushy with long boiling times despite its high starch content. ‘Harimaru’ may become a local specialty potato and its multiple resistance to potato viruses may allow cultivation using homemade seed tubers from the previous season’s crop.

A System for Identification of Potato Varieties Using SNP Dosage

As in autoteraploids, five genotypes are possible in a biallelic locus, allele dosage could be useful for variety identification in potato. DNA sequences of 13 genes associated with tuber yield, tuber starch content or late blight resistance were surveyed for single nucleotide polymorphism (SNP) in eight tetraploid varieties. A total of 193 potential SNPs was found by Sanger-sequencing, of which 58 were measured for nucleotide frequencies using a pyrosequencer. Of the 58 SNPs, 35 yielded distinct clusters, corresponding to allele dosages. Among these, six were highly correlated with another, leaving 29 independent SNP loci for use in variety discrimination. By using dosage scores at the 29 SNP loci, it was possible to differentiate 115 varieties, except for a sport with red tuber skin color, with at least three different SNP dosages. Therefore, using SNP dosages is a simple, fast and reliable tool for variety identification.ResumenComo en los autotetraploides, cinco genotipos son posibles en un locus bialelico, la dosis de alelo podría ser útil para la identificación de variedades en papa. Se estudiaron las secuencias de ADN de 13 genes asociados con el rendimiento de tubérculo, su contenido de almidón, o resistencia al tizón tardío, para un polimorfismo de nucleótido simple (SNP en inglés) en ocho variedades tetraploides. Se encontró un total de 193 SNPs mediante secuenciación Sanger, de los cuales se midieron 58 para frecuencias de nucleótidos utilizando un polisecuenciador. De los 58 SNPs, 35 arrojaron diferentes agrupamientos, correspondientes a dosis de alelo. Entre estos, seis estuvieron altamente correlacionados con otro, dejando 29 loci SNP independientes para utilizarse en la discriminación varietal. Mediante el uso de mediciones de dosis en los 29 loci SNP fue posible diferenciar 115 variedades, excepto una con tubérculo de piel roja, con por lo menos tres diferentes dosis de SNP. De aquí que, el uso de dosis de SNP es una herramienta simple, rápida y confiable, para la identificación varietal.