R. E. Hanneman

Characterization and mapping of Rpi1, a late-blight resistance locus from diploid (1EBN) Mexican Solanum pinnatisectum

Abstract. Solanum is a diverse genus with over 200 species occupying a range of habitats from the Southwestern United States to Central Chile. Germplasm evaluations have focused on species that can be crossed with S. tuberosum, while Mexican diploid (2n=2x=24) Solanum species with an Endosperm Balance Number (EBN) of 1 have received less attention because of poor crossability due to their ploidy and EBN. Recent changes in Phytophthora infestans populations have increased the need for new sources of genetic resistance to this fungus. We have characterized resistance to P. infestans in the Mexican 2x(1EBN) species S. pinnatisectum. An interspecific hybrid between resistant S. pinnatisectum and susceptible S. cardiophyllum plants was backcrossed to S. cardiophyllum to generate a family segregating for late-blight resistance. The diploid (1EBN) genetic map generated with 99 RFLP markers revealed extensive synteny with previously published potato maps. A single dominant late-blight resistance locus (Rpi1) from S. pinnatisectum was mapped to chromosome 7, a region previously not associated with late-blight resistance. Characterization of the P. infestans isolate used for disease evaluations revealed that it possessed the avirulence gene corresponding to the R9 resistance locus, indicating that Rpi1 could possibly correspond to R9.

Support of the endosperm balance number hypothesis utilizing some tuber-bearingSolanum species

The normal development of the endosperm appears to depend on a ratio of two female genomes to one male genome in most angiosperms. However, successful crosses between some tuber-bearingSolarium species do not conform to this ratio. An endosperm balance number (EBN) hypothesis has been proposed to reconcile the two female genomes: one male genome ratio proposal and the anomalous crossing behavior of some species. Crosses performed to test the EBN hypothesis are reported in this paper. The results support the hypothesis and demonstrate its usefulness in predicting the success or failure of a cross and the ploidy of the offspring. A format for conducting and reporting crossing experiments is proposed that facilitates the comparison of results of different experiments.ResumenEl desarrollo normal del endosperma, parece depender en la proporción de dos genomios provenientes de la planta hembra y uno de la planta macho; sin embargo, cruzamientos entre algunas especies tuberíferas deSolanum se han conseguido y no conforman dicha proporción. Se ha propuesto la hipótesis de factures de balance en el endosperma (EBN) para reconciliar la proporción de genomios 2:1 (hembra: macho) y el comportamiento anormal en cruzamientos de algunas especies. Cruzamientos de prueba de dicha hipótesis se reporta en el presente artículo y los resultados obtenidos concuerdan con ella, demostrando su utilidad en la predictión de éxito o fracaso del cruzamiento, asimismo, el nivel de ploidía de la progene. Un esquema para conducir y reportar experimentos de cruzamientos es propuesto facilitando la comparación de resultados de diferentes experimentos.

Origin of chloroplast DNA diversity in the Andean potatoes.

SummaryWide chloroplast DNA (ctDNA) diversity has been reported in the Andean cultivated tetraploid potato, Solanum tuberosum ssp. andigena. Andean diploid potatoes were analyzed in this study to elucidate the origin of the diverse ctDNA variation of the cultivated tetraploids. The ctDNA types of 58 cultivated diploid potatoes (S. stenotomum, S. goniocalyx and S. phureja), 35 accessions of S. sparsipilum, a diploid weed species, and 40 accessions of the wild or weed species, S. chacoense, were determined based on ctDNA restriction fragment patterns of BamHI, HindIII and PvuII. Several different ctDNA types were found in the cultivated potatoes as well as in weed and wild potato species; thus, intraspecific ctDNA variation may be common in both wild and cultivated potato species and perhaps in the higher plant kingdom as a whole. The ctDNA variation range of cultivated diploid potatoes was similar to that of the tetraploid potatoes, suggesting that the ctDNA diversity of the tetraploid potato could have been introduced from cultivated diploid potatoes. This provided further evidence that the Andean cultivated tetraploid potato, ssp. andigena, could have arisen many times from the cultivated diploid populations. The diverse but conserved ctDNA variation noted in the Andean potatoes may have occurred in the early stage of species differentiation of South American tuber-bearing Solanums.

The use of Endosperm Balance Number and 2n gametes to transfer exotic germplasm in potato.

SummaryA triploid hybrid (2n=3x=36) between a colchicine-induced 4x(2EBN) Solanum brevidens (a non-tuber-bearing species) and 2x(2EBN) S. chacoense (a tuber-bearing species) was used as a vehicle for germplasm transfer to S. tuberosum Group Tuberosum. The use of 2n gametes from the triploid allowed the unique opportunity for transferring exotic germplasm from Series Etuberosa to Gp. Tuberosum material. The triploid hybrid used had a pollen stainability of less than 0.1%. Observations of microsporogenesis revealed that metaphase I pairing configurations were primarily 12 bivalents and 12 univalents with occasional trivalents. Anaphase I separations were irregular, often with lagging univalents. Meiotic observations and pollen morphology suggest that the stainable pollen produced by the hybrid was 2n=3x=36. A single pentaploid hybrid (2n=5x=60) was produced by the fertilization of a rare 2n egg from the triploid with a normal male gamete from the clone ‘Wis AG 231’ (2n=4x=48). Limited crosses to other 1, 2 and 4EBN species and cultivars were unsuccessful. The pentaploid hybrid had a more regular meiosis than the triploid and dramatically improved pollen stainability (37% stainable pollen). Stylar blocks prevented estimates of male fertility in crosses. Female fertility in 47 crosses with nine cultivars averaged 19 seeds per fruit. Although S. brevidens is non-tuber-bearing, and the triploid produced only stolons, the pentaploid hybrid tuberized well under field conditions, despite being very late. Results suggest that the tuberization response is a dosage and/or threshold effect. This approach to the incorporation of 1EBN germplasm indicates the utility of the EBN concept coupled with 2n gametes. Further, it demonstrates a means for the introgression of 1EBN species genes into Gp. Tuberosum material.

Ploidy levels of progeny from diploid-tetraploid crosses in the potato

SummaryThe discovery of an efficient means for obtaining haploids of the potato is slowly revolutionizing, reorienting, and stimulating potato research and breeding. This report is part of a study concerned with their use. as hybrids with species, in gene transfer through diploid-tetraploid hybridization. Such hybridization has been shown to be quite easily accomplished. In this paper, the ploidy level of the progeny of 4x × 2x and 2x × 4x crosses are given. The 4x × 2x cross gave 91.9% (4x), 7.6% (3x). and 0.6% (2x) based on an examination of 344 individuals, while 676 individuals of the 2x × 4x cross were found to be 94.8% (4x), 1.9% (3x), and 3.3 %(2x). The high seed set pollinators gave 98.4% (4x) and 1.6% (3x) progeny based on 551 individuals. The fact that they produced a higher frequency of tetraploids and a lower frequency of triploids than did the other 2x pollinators suggests that the triploid frequency is not dependent on seed set and also that in crosses where seed set is above average the additional seeds are likely to be tetraploid. The high frequency of tetraploids. plus the relative ease of crossing between diploids and tetraploids makes this method of gene transfer both highly effective and quite efficient.ResumenEl descubrimiento de un mé todo eficiente para obtener haploídes de la papa esta lentamente revolucionando, reorientando y estimulan — do las investigaciones y mejoramiento de la papa.Este reporte es parte del estudio concerniente a su uso en la hybridation con especies y la introductión de germoplasma por medio de la cruza de diploide × tetraploide. Este tipo de cruzamiento ha sido efectuado facilmente. Aquí se reporta el nivel de poliploidia de las progenies en las cruzas (4x × 2x) y (2x × 4x). En la cruza 4x × 2x se encontró que 91.9% fueron tetraploides (4x), 7.6% (3x) y 0.6% (2x), estos datos estan basados en el conteo de 344 individuos. De los 676 individuos de la cruza 2x × 4x se encontraron 94.8% tetraploids (4x), 1.9% con (3x) y 3.3% (2x).El conteo de 551 plantulas de las progenies donde se usó polinizadores superiores se encontró 98.4% individuos tetraploides (4x) y 1.6% de plantas triploides (3x).El hecho de que éstos polinizadores superiores produjeron una frecuencia de tetraploides mas alto que la frecuencia de triploides, cosa que no sucedió con los otros polinizadores diploides, hace suponer, que la frecuenca de los triploides, no depende del número de semillas y también que, en cruzas donde el número de semillas es más alto que el promedio, es probable que éste número adicional de semillas sean tetraploides. La alta frecuencia de tetraploides. más la relativa facilidad de la cruza de diploides por tetraploides hace éste método efectivo y eficiente en la transferencia de genes de especis diploides a las variedades cultivadas tetraploides.

Hybridization between GP. tuberosum haploids and 1EBN wild potato species

Diploid wild potato species, classified as 1EBN (Endosperm Balance Number), do not cross with tetraploid or diploid forms ofS. tuberosum Gp. Tuberosum. The crossing of 2n pollen producing 1EBN clones as well as chromosome-doubled 1EBN clones with Gp. Tuberosum haploids was initiated to overcome this hybridization barrier. The screening of eleven 1EBN species,S. brachistotrichum, S. bulbocastanum, S. cardiophyllum, S. chancayense, S. commersonii, S. etuberosum, S. fernandezianum, S. jamesii, S. mochicense, S. pinnatisectum andS. trifidum, resulted in the identification of 2n pollen producers in eight of the species examined. Direct hybridization with Gp. Tuberosum haploids utilizing the above-mentioned crossing scheme was successful withS. chancayense andS. commersonii. The hybrids obtained, however, were male and female sterile. Abnormal microsporogenesis in the hybrids was postulated to be the result of an interaction between Gp. Tuberosum cytoplasm and nuclear genes contributed by the 1EBN male parent. Analyses of the growth of 1EBN species’ pollen tubes in the stigma/style of Gp. Tuberosum haploids were also conducted. Interspecific incompatibilities were observed with the most severe forms found with the use of species in the Series Etuberosa. The use of the Endosperm Balance Number theory can aid in designing crosses which overcome barriers to successful endosperm development. Sterilities in the hybrids obtained as well as interspecific incompatibilities will also need to be addressed, however, before 1EBN species germplasm can be successfully utilized for the improvement of the cultivated potato.CompendioEspecies silvestres diploides de papa, clasificadas como 1EBN (Número de Balance del Endosperma), no se cruzan con las formas tetraploides o diploides deS. tuberosum Gp. Tuberosum. Se inició el cruzamiento de clones 1EBN productores de polen 2n, al iguao que el de clones 1EBN con cromosomas doblados, con haploides del Gp. Tuberosum, para eliminar esta bar rera a la hibridación. La evaluatión y selectión de once especies 1EBN,S. brachistotrichum, S. bulbocastanum, S. cardiophyllum, S. chancayense, S. commersonni, S. etuberosum, S. fernandezianum, S. jamesii, S. mochicense, S. pinnatisectum yS. trifidum, dió por resultado la identificatión de productores de polen 2n en ocho de las especies examinadas. La hibridación directa con haploides del Gp. Tuberosum utilizando los sistemas de cruzamientos mencionados anteriormente fue exitosa conS. chancayense yS. commersonii. Sin embargo, los híbridos obtenidos fueron totalmente estériles. Se supone que la microesporogenesis anormal en los híbridos es el resultado de una interaction entre el citoplasma del Gp. Tuberosum y los genes nucleares pertenecientes al progenitor masculino 1EBN. Se condujeron también análisis del crecimiento de los tubos polínicos de las especies 1EBN en el estigma/estilo de los haploides del Gp. Tuberosum. Se observaron incompatibilidades interespecíficas con las formas más severas encontradas con el uso de especies en la Serie Etuberosa. El uso de la teoría del Número de Balance del Endosperma puede servir para diseñar cruzamientos que superen las barreras para el desarrollo exitoso del endosperma. Sin embargo, la esterilidad de los híbridos obtenidos, al igual que las incompatibilidades interespecíficas, necesitarán también ser corregidas antes que el germoplasma de especies 1EBN pueda ser exitosamente utilizado para el mejoramiento de la papa cultivada.

Cultivated potato chloroplast DNA differs from the wild type by one deletion- Evidence and implications*

SummaryThe chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.

Endosperm Balance Number and the polar-nuclei activation hypotheses for endosperm development in interspecific crosses of Solanaceae and Gramineae, respectively.

The Endosperm Balance Number (EBN) and the polar-nuclei activation (PNA) hypotheses have been developed to interpret, explain and predict interspecific and interploidy crossabilities in the Solanums and the Gramineae, respectively. Although these two hypotheses evolved independently, they share a number of common features. Assignment of EBNs and ‘activation/response values’ (AVs/RVs) depend on plumpness, size, and germinability of hybrid seeds. Also, both hypotheses emphasize the importance of a balanced parental genic contribution for the normal development of endosperm. However, in the EBN hypothesis a 2 maternal∶1 paternal EBN ratio is a prerequisite for successful interspecific crossability, while the PNA hypothesis is based on the stimulative strength of the male nuclei to initiate mitotic divisions in the primary endosperm nucleus and is idependent on a 2∶1 ratio between the RV of the polar nuclei and the AV of the male gamete. Differences and similarities betweeen the EBN and PNA hypotheses are summarized and contrasted. It is proposed that EBN and PNA be considered as the same concept.

A possible heterotic threshold in the potato and its implications for breeding

SummaryComplex hybrids containing genomes from three different Solanum tuberosum Groups were synthesized (3-way hybrids), utilizing 2n gametes in 4x−2x crosses. Ten such families were compared to nine analogous two-Group (2-way) hybrid families and nine (1-way) families representing conventional Gp. Tuberosum breeding materials. The three types of crosses, representing three descending levels of heterozygosity, were placed in four field trials.The 3-way hybrids were never significantly superior to the 2-way hybrids for vigor, yield, or tuber type. When yields were adjusted for maturity differences, the 3-way hybrids tended to be inferior to the 2-way hybrids for yield. This suggests that there may be a heterotic threshold in the cultivated potato, beyond which point more heterozygosity does not result in greater vigor or more yield.While the 2-way and 3-way hybrids did not significantly differ from each other, they both dramatically surpassed the conventional 1-way crosses for vigor and yield (42%). The evidence of a possible heterotic threshold indicates that more sophisticated methods such as cell fusion and bilateral sexual polyploidization may not be necessary to exploit the full potential of the hybrid approach in the potato. A simple and direct “2-way” hybridization approach may be optimal, or at least would seem comparable with other hybrid approaches, and is a technology ready for immediate and widespread implementation.

Rapid ploidy screening of tuber-bearingSolanum (potato) species through pollen diameter measurement

Determination of the sporophytic ploidy of numerous individuals by chromosome counting is a difficult and time consuming necessity for many genetic and taxonomic research problems. The potential for microscopic pollen diameter measurement as a fast, easy and reliable alternative method for estimating sporophytic ploidy was investigated. Contrary to popular assumptions, die diameters of large, well-stained pollen grains from plants of the same ploidy were shown to be quite consistent among a wide variety of tuber-bearingSolanum (potato) species, fitting remarkably well with expectations based on a linear relationship between ploidy and pollen grain volume (r = 0.97). Seventy-six ploidy predictions on a total of 83 coded samples were correct (92%). Although it is not effective in distinguishing 4x and 6x sporophytes, microscopic pollen diameter measurement is a very rapid and reliable method by which to distinguish 2x from 4x or 6x sporophytes.