A. H. del Rio

Assessing changes in the genetic diversity of potato gene banks. 1. Effects of seed increase

Abstract Effects of gene bank seed-increases on the genetic integrity of potato germ plasm is a major concern of gene bank managers. Thus the Association of Potato Inter-gene-bank Collaborators (APIC), a consortium of world potato gene bank leaders, initiated this joint research project using RAPD markers to determine genetic relationships between increased generations within accessions. Solanum jamesii (2n=2x=24) and S. fendleri (2n=4x=48), two wild potato species native to North America, were used as plant material. These species represented two major breeding systems found among Solanum species: outcrossing diploids and inbreeding disomic tetraploids, respectively. Comparisons were made between populations one generation apart and between sister populations generated from a common source. Fourteen such comparisons within S. jamesii accessions had an average similarity of 96.3%, and 21 such comparisons within S. fendleri accessions had an average similarity of 96.0%. No pairs of populations were significantly different, despite the fact that RAPD markers easily separated all of these very similar accessions within their respective species. Only one of six S. jamesii accessions analyzed showed a significant change in gene frequencies among generations. These findings indicate that there has been minimal loss or change of genetic diversity in ex situ germplasm using the gene bank techniques standard at NRSP-6 and other world potato gene banks.

Assessing changes in the genetic diversity of potato gene banks. 2. In situ vs ex situ

Abstract An important question in the conservation of potato germ plasm is whether germ plasm in the gene bank, although stable, still represents the in situ populations from which it was collected, sometimes many decades ago. The answer would direct objective decisions regarding the value of re-collections and in situ preservation. The present study was undertaken as a project of the Association of Potato Inter-gene-bank Collaborators (APIC). It measured genetic differentiation between potato germ plasm maintained in the US gene bank for many years and current in situ populations re-collected from the same original sites in the wild. Solanum jamesii and Solanum  fendleri from the United States were used as representatives of potato germ plasm. Re-collections were carried out in 1992 at the same locations at which gene bank-conserved accessions had been collected in 1958 and 1978. RAPD markers revealed significant genetic differences between gene bank-conserved and re-collected in situ populations for all seven comparisons of S. jamesii (diploid outcrosser), and 12 of 16 comparisons within S.  fendleri (tetraploid inbreeder). The average genetic similarities were 65.2% for S.  jamesii and 80.4% for S.  fendleri. Possible explanations and consequences of these unexpectedly large differences are discussed.

Reduction of species in the wild potato Solanum section Petota series Longipedicellata: AFLP, RAPD and chloroplast SSR data

Abstract.Species boundaries were assessed with three molecular markers [AFLPs, RAPDs and chloroplast simple sequence repeats (cpSSRs)] for all six species of wild potatoes (Solanum section Petota) assigned to ser. Longipedicellata: Solanum fendleri, S. hjertingii, S. matehualae, S. papita, S. polytrichon and S. stoloniferum. These tetraploid (2n = 4x = 48) species grow in the southeastern United States (S. fendleri) and Mexico (all six species), and a recent morphological analysis supported only three species: (1) S. polytrichon, (2) S. hjertingii (including S. matehualae) and (3) S. stoloniferum (including S. fendleri and S. papita). We analyzed all six species of ser. Longipedicellata (tetraploid) and also analyzed diploids in ser. Bulbocastana, ser. Pinnatisecta, ser. Polyadenia and ser. Tuberosa; tetraploids in ser. Acaulia and hexaploids in ser. Demissa. Concordant with morphological data, AFLP and RAPD results support the synonymy of S. hjertingii and S. matehualae, and completely intermix S. papita and S. fendleri. However, accessions of S. stoloniferum have a tendency to cluster but with exceptions, and S. polytrichon is completely intermixed with S. fendleri and S. papita. The cpSSRs fail to distinguish any of the species in ser. Longipedicellata. Combined morphological and molecular data support only two species in ser. Longipedicellata: S. hjertingii and S. stoloniferum.

Genetic heterogeneity estimated by RAPD polymorphism of four tuber-bearing potato species differing by breeding system

Most wild potato germplasm in genebanks is collected, preserved, and evaluated as botanical seed populations that may be highly heterozygous and heterogeneous. However, some species are selfers so potentially very homozygous, perhaps also homogeneous. Intrapopulation heterogeneity increases sampling error that can undermine consistency in seed regeneration in the genebank, screening results, germplasm collecting, and estimates of taxonomic relationships. Thus, knowledge of genetic heterogeneity (GH) can predict the need to commit more resources for larger sample sizes or replication when populations of a given species are being regenerated, evaluated, collected, and classified. This study investigated withinpopulation GH in 32 potato populations comprising four different breeding systems observed inSolatium species:S. fendleri (2n=4x=48, disomic selfer),S. jamesii (2n=2x=24, outcrosser),S. sucrense (2n=4x=48, tetrasomic outcrosser), andS. verrucosum (2n=2x=24, selfer). RAPD markers were used to estimate heterogeneity among 24 individuals per population. Populations ofS. verrucosum were quite homogeneous with an average GH of 6.0%. Similarly low heterogeneity was detected among the eight populations ofS. fendleri (average GH=7.1%). In contrast,S. jamesii andS. sucrense had a much higher GH of 29.4% and 44.1%, respectively. These results demonstrate and quantify the great difference in intrapopulation heterogeneity among wild potato species. Calculations based on intrapopulation heterogeneity indicate that samples should be composed of 25 to 30 random plants for low sample variation that is uniform for all species.ResumenLa mayor parte de germoplasma de papa silvestre existente en los bancos de genes es recolectada, preservada y evaluada como poblaciones de semilla botánica, las mismas que pueden ser sumamente heterocigotas y heterogéneas. Sin embargo, algunas especies son autógamas, y de este modo potencialmente homocigotas y puede ser que sean también homogéneas. La heterogeneidad intrapoblacional, aumenta el error de muestreo, y puede atentar contra varios aspectos entre ellos: la consistencia de regeneratión de la semilla en el banco de genes, los resultados del tamizado, la recolección de germoplasma y los estimados de la relación taxonémica. De este modo, el conocimiento de la heterogeneidad genética (GH) puede predecir la necesidad de mayores recursos para un tamaño dado de muestra o réplica, cuando la población de una especie determinada está siendo regenerada, evaluada, recolectada y clasificada. Este estudio investigó dentro de la población GH, 32 poblaciones de papa en cuatro sistemas diferentes de mejoramiento, observados en especies deSolanum: S. fendleri (2n =4x =48, disómico autógamo),S. jamesii (2n= 2x=24, de polinización cruzada),S. sucrense (2n= 4x=48, tetrasómico de polinizacién cruzada) yS. verrucosum (2n=2x=24, autógamo). Para estimar la heterogeneidad entre 24 individuos por población se utilizaron marcadores RAPD. Las poblaciones deS. verrucosum fueron totalmente homogéneas con un promedio GH de 6.0%. De la misma manera se détectó una baja heterogeneidad entre ocho poblaciones deS. fendleri (promedio GH = 7.1%). En cambioS. jamesii y S. sucrense tuvieron un GH mucho más alto, 29.4% y 44.1% respectivamente. Estos resultados demuestran y cuantifican la gran diferencia que existe en heterogeneidad intrapoblacional entre las especies silvestres de papa. Cálculos basados en la heterogeneidad de una intrapoblación indican que las muestras deben estar compuestas de 20–25 plantas tomadas al azar para mantener la variación que es uniforme para todas las especies.

Lack of association between genetic and geographic origin characteristics for the wild potatoSolarium sucrense Hawkes

Collecting germplasm to broaden breeding resources is an essential activity of genebanks. Research to understand how genetic diversity is partitioned in nature might help to identify collections rich in diversity. Previous studies among wild populations ofSolanum fendleri (a disomic polyploid selfer) andS. jamesii (a diploid outcrosser) revealed no significant associations between genetic and ecogeographic variation. Even physical separation did not predict genetic differences. In this study, 28 populations ofS. sucrense Hawkes (2n=4x=48), a Bolivian species with another breeding system (polysomic polyploid oucrosser), were evaluated. The objective was to assess whether genetic differences between populations are predicted by differences in geographic parameters at the natural site of origin. Genetic differentiation was estimated by using 216 RAPD markers. The average genetic distance (GD) found between pairs of populations was 31% (ranging from 8% to 44%). Correlations of GD with latitude, longitude, altitude and distance were not significant. Multiple regression analysis also confirmed that GD was not explained by the geographic parameters used. We conclude that geographic origin data is not very useful in gauging interpopulation genetic diversity in the genebank.ResumenColeccionar germoplasma para ampliar las fuentes de mejoramiento es una actividad fundamental de los bancos genéticos. La investigación para comprender cómo se distribuye la diversidad genética en la naturaleza contribuye a identificar las colecciones ricas en diversidad. Estudios realizados previamente entre poblaciones silvestres deS. fendleri (un poliploide disómico autógamo) yS. jamesii (un diploide alógamo) no revelaron asociaciones significativas entre la variación genética y ecogeográfica. Incluso la separación física no predijo diferencias genéticas. En este estudio se evaluaron 28 poblaciones deS. sucrense Hawkes (2n=4x=48), una especie boliviana con otro sistema de selección (un poliploide polisómico alógamo). El objetivo era evaluar si las diferencias genéticas entre poblaciones son predecibles por las diferencias en los parámetros geográficos de sus lugares naturales de origen. La diferenciación genética se estimó usando 215 marcadores RAPD. El promedio de Distancia Genética (GD) encontrado entre pares de población fue 31% (fluctuando entre 8% a 44%). La correlación de GD con latitud, longitud, altitud y distancia no fue significativa. El análisis de regresión múltiple confirmó igualmente que el GD no se explicó por los parámetros geográficos utilizados. En conclusión, el origen geográfico no es un dato muy util para calibrar la diversidad genética entre poblaciones del banco de germoplasma.

Genetic equivalence of putative duplicate germplasm collections held at CIP and US potato genebanks

A common effort among members of the Association of Potato Inter-Genebank Collaborators (APIC) has yielded a global inventory of wild potato genetic resources that is freely accessible to researchers and breeders. In that database there are a number of accessions that originated from distributed progeny of a single original germplasm collection. The logical assumption has been that although these samples are in different locations, they should be genetically equivalent. This study tests this hypothesis by comparing 17 pairs of accessions of 16 different potato species, which are reputed duplicates preserved in the potato genebanks of The International Potato Center (CIP) in Peru and of the U.S.A. (USPG). The RAPD marker analysis revealed that even though the average genetic similarity of reputed duplicates was quite high, there were a few with significant differences. Similarly, SSR markers identified three reputed duplicates that were genetically different. SSRs revealed a loss of markers for some inter-genebank comparisons, a probable indication of genetic drift. Duplicate potato collections between CIP and USPG are in most cases genetically identical. The few exceptions merit further investigation regarding causes and the impact on useful traits.ResumenUn esfuerzo común entre los miembros de la Asociación de Colaboradores de Bancos de Germoplasma de Papa (APIC) ha dado como resultado un inventario global de recursos genéticos de papa silvestre, de libre acceso a investigadores y mejoradores. En la base de datos existe un número de accesiones que se han generado a partir de la progenie distribuida de la colección original. La suposición lógica ha sido que aunque estas muestras están en diferentes lugares, deberían ser genéticamente equivalentes. Este estudio prueba esta hipótesis al comparar 17 pares de accesiones de 16 especies diferentes de papa, las cuales son consideradas como duplicados mantenidos en el banco de germoplasma del Centro Internacional de la Papa (CIP) en Perú y en los Estados Unidos de América (USPG). El análisis con el marcador RAPD reveló que a pesar de que la similaridad genética de duplicados putativos fue bastante alta, sin embargo, hubo algunos con diferencias significativas. Igualmente los marcadores SSR identificaron tres duplicados putativos genéticamente diferentes. Los SSR revelaron una pérdida de marcadores para algunas comparaciones entre bancos de genes, probable indicación de deriva genética. Los duplicados de las colecciones entre CIP y USPG son, en la mayoría de los casos, genéticamente idénticos. Las pocas excepciones que existen ameritan investigación futura para determinar las causas y su efecto en caracteres de interés.

Comparison of reputed duplicate populations in the russian and us Potato genebanks using RAPD markers

The Association of Potato Intergenebank Collaborators (APIC) produced a global inventory of wild potato genetic resources that is available on the Internet (www.potgenebank.org/ipd). This database shows that, in many cases, several genebanks have samples of the progeny from a single original germplasm collection. The assumption has been that these samples are genetically equivalent, so all the characterization and evaluation data gathered on a seedlot from one genebank can be applied to all the other “duplicate” seedlots in other genebanks. This assumption was tested by comparing 25 pairs of reputed duplicates in the VIE (St. Petersburg, Russia) and US (Sturgeon Bay, USA) potato genebanks using RAPDs. In 23 of 25 populations, reputed duplicates among genebanks had significantly less similarity than replicate samples taken from a single population. The average genetic similarity of reputed duplicates was 93%, and the lowest was 81%. Thus, users of germplasm should be aware that reputed duplicate populations from these genebanks may not be genetically identical.

The effect of genebank seed increase on the genetics of recently collected Potato (Solanum) germplasm

In previous work at the US Potato Genebank, RAPDs were used to detect large differences between genebank-conserved samples and their correspondingin situ populations re-collected from the wild. This work investigates one possible explanation for these differences: a large genetic change in the sample when it undergoes “domestication” by a forced sexual seed increase and subsequent adaptation to cultivation in the genebank. However, when 11Solanum fendleri and 17S. jamesii populations were collected from the wild and compared to their sexual progeny generated at the genebank, no significant differences in RAPD finger-prints were detected. These results show that theory of “genebank domestication” is not supported. However, when plants of one pair of populations were tested individually, the seed increase population was significantly more heterogeneous than its clonally collectedin situ parents. Thus, while genebank populations have the same genes as their wild counterparts, they may contain genotypes not present in the wild.ResumenEn trabajos previos llevados a cabo en el Banco de Germoplasma de Papa de los Estados Unidos, el análisis de RAPDs encontró que existian diferencias genéticas extremas entre el material preservado en el Banco y sus poblaciones correspondientesin situ; las cuales fueron recolectadas de sus habitats naturales. El presente estudio investigó una posible explicación a estas diferencias: la posibilidad que haya ocurrido un cambio genético drástico debido a la “domesticacion” de estas poblaciones cuando fueron forzadas a reproducirse sexualmente y su posterior adaptación para cultivación en el Banco. Sin embargo, cuando 11 poblaciones deSolanum fendleri y 17 deS. jamesii fueron recolectadas de sus sitios originales y comparadas con sus progenies generadas sexualmente en el Banco, el analisis de RAPDs demostró que no habia ninguna diferencia significativa. Estos resultados no respaldan la teoria de “domesticación en el Banco”. Sin embargo, el análisis de plantas individuales en un par de poblaciones demostró que la población generada para multiplición de semillas fue mas heterogénea que su población parental recolectadain situ. Por lo tanto, a pesar que las poblaciones preservadas en el Banco poseen los mismos genes que sus correspondientes poblaciones en habitats naturales, estas pueden expresar genotipos que no se observan en sus habitats originales.

Vulnerability of alleles in the US potato genebank extrapolated from RAPDs

Crop genebank managers must be concerned with preserving low-frequency alleles that are vulnerable to being lost from the genebank because they are not sampled as seed increase parents. Traits conferred by such alleles are also vulnerable to being overlooked because they are not sampled in plants evaluated for breeding traits. Mathematical models and recommendations that follow from hypothetical vulnerable alleles within single populations have been made by various researchers. This work was undertaken to empirically detect the prevalence of such alleles in the US Potato Genebank by extrapolation from RAPD markers. Two outcrossing species known to be very heterogeneous were selected,Solanum jamesii Torr. (2n=2x=24) andSolanum sucrense Hawkes (2n=4x=48). RAPD band frequencies at a total of 83 marker loci within each of 15 populations were assessed using 24 plants per population. For both species, about 10% of within-population loci had vulnerable alleles (banded plant frequency < 40%), considering just the particular population in question. However, about half of these were fixed in at least one other population (making that marker invulnerable to loss). The highest within-population banded plant frequency for loci not fixed in any population was, with one exception, always > 40%. So, while several alleles may be vulnerable within a given population, these were nearly always fixed or nearly fixed (invulnerable) in another population. This suggests that the safest strategy for maximizing conservation of alleles is to apportion available resources over many separate populations, rather than adopt a preservation protocol so rigorous that it can only be practically applied to a limited number of populations.ResumenLos administradores del Banco de genes deben estar preocupados en la preservación de alelos de baja frecuencia que son vulnerables a perderse porque no están catalogados como progenitores para el incremento de semilla. Los rasgos conferidos por tales alelos son también vulnerables a ser descuidados por que no están catalogados en plantas evaluadas para características de mejoramiento. Los modelos matemáticos y las recomendaciones que siguen han sido hechas por varios investigadores a partir de alelos hipotéticos vulnerables dentro de una sola población. Este trabajo se hizo para detectar empíricamente la prevalencia de tales alelos en el Banco de genes de papa de EUA por extrapolación de marcadores RAPD. Se seleccionaron dos especies autocruzantes conocidas como muy heterogéneas,Solanum jamesii Torr (2n=2x=24) ySolanum sucrense Hawkes (2n=4x=48). La frecuencia de bandas RAPD de un total de 83 loci dentro de cada una de las 15 poblaciones se evaluó utilizando 24 plantas por población. Para ambas especies, alrededor del 10% dentro de la población loci tenía alelos vulnerables (frecuencia de plantas bandeadas < 40%) considerando exactamente la población particular en cuestión. Sin embargo, cerca de la mitad de estas bandas estaban fijadas por lo menos en otra población (haciendo al marcador vulnerable a perderse). Lo más alto entre la población con frecuencia de plantas para loci no fijados en ninguna población fue con una sola excepción, siempre > 40%. Así, mientras que varios alelos pueden ser vulnerables dentro de una población dada, estos fueron casi siempre fijados o casi fijados (invulnerables) en otra población. Esto sugiere que la estrategia más segura para maximizar la conservación de alelos es prorratear los recursos disponibles sobre muchas poblaciones separadas, en lugar de adoptar un protocolo tan riguroso que sólo puede aplicarse a un número limitado de poblaciones.

RAPD analysis of genetic diversity in Solanum populations to predict need for fine screening.

Populations of two wild potato species known to have high levels of green peach aphid, PLRV, and PVY resistance (Solanum polytrichon PI 184170 andS. stoloniferum PI 160226) were assessed for heterogeneity of RAPD markers among plants within these populations and among other populations of the same species. These species are reputed selfers, so were suspected of being relatively inbred. About 75% of the bands segregating within their species were fixed within these populations, and more than 90% of the total bands observed within these populations were fixed. These levels of homogeneity were not significantly different from those of an inbred population ofS. albicans known to be extremely homogeneous. It can be expected that populations such as these, that are relatively homogeneous, are also more likely to be homogeneous for specific traits of interest. Thus, there may be less need for rigorous fine screening of these materials at the genotype level before making breeding crosses.