Marc Ghislain

Marker-assisted sampling of the cultivated Andean potato Solanum phureja collection using RAPD markers

The potato crop originated in the Andean highlands where numerous farmers varieties and non-cultivated wild species exist. An Andean potato collection is held in trust at the International Potato Center (CIP) to preserve the biodiversity of this crop and ensure the supply of germplasm for potato improvement worldwide. A core collection representing the biodiversity of the Andean potato germplasm is under construction using morphological, molecular, and geographic data. One of the eight cultivated potato species, Solanum phureja, has been genotyped using the RAPD technique. A protocol suitable for large germplasm collection genotyping has been developed to process numerous samples at reasonable costs. From 106 RAPD primers evaluated, we have selected 12 primers yielding 102 polymorphic markers, which unambiguously discriminated all 128 accessions but 2 that are possible duplicates. The S. phureja germplasm collected throughout the Andean countries appears to have a homogeneous genetic constitution. There was no clear geographic pattern as indicated by cluster analysis of the RAPD data. A sub-group of 20 accessions has been identified on the basis of the marker data and selected to maximize molecular (RAPD) variance and polymorphism. The probability of capturing equal amounts of marker polymorphism in this sub-group of 20 accessions by random sampling is less than 40%. This set accessions represents our first group of accessions that may constitute a core of the S. phureja collection. This tentative core will be challenged for diversity content by alternate markers and agronomic traits. Hence, the methodology for sampling less than 10% of the base collection, proposed for core collections by Brown (1989), can be based on molecular marker data provided cost-efficient fingerprints are developed.

Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato

Characterization of nearly 1,000 cultivated potato accessions with simple sequence repeats (SSRs; also referred to as microsatellites) has allowed the identification of a reference set of SSR markers for accurate and efficient genotyping. In addition, 31 SSRs are reported here for a potato genetic map, including new map locations for 24 of them. A first criterion for this proposed reference set was ubiquity of the SSRs in the eight landrace cultivar groups of the potato, Solanum tuberosum. All SSRs tested in the present study displayed the same allele phenotypes and allele size range in the diverse germplasm set as in the advanced potato cultivar germplasm in which they were originally discovered. Thirteen of 13 SSR products from all cultivar groups are shown to cross-hybridize with the corresponding SSR product of the source cultivar to ascertain sequence homology. Other important SSR selection criteria are quality of amplification products, locus complexity, polymorphic index content, and well-dispersed location on a potato genetic map. Screening of 156 SSRs allowed the identification of a highly informative and user-friendly set comprising 18 SSR markers for use in characterization of potato genetic resources. In addition, we have identified true- and pseudo-multiplexing SSRs for even greater efficiency.

Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification

Contrasting taxonomic treatments of potato landraces have continued over the last century, with the recognition of anywhere from 1 to 21 distinct Linnean species, or of Cultivar Groups within the single species Solanum tuberosum. We provide one of the largest molecular marker studies of any crop landraces to date, to include an extensive study of 742 landraces of all cultivated species (or Cultivar Groups) and 8 closely related wild species progenitors, with 50 nuclear simple sequence repeat (SSR) (also known as microsatellite) primer pairs and a plastid DNA deletion marker that distinguishes most lowland Chilean from upland Andean landraces. Neighbor-joining results highlight a tendency to separate three groups: (i) putative diploids, (ii) putative tetraploids, and (iii) the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii (triploid), and S. curtilobum (pentaploid). However, there are many exceptions to grouping by ploidy. Strong statistical support occurs only for S. ajanhuiri, S. juzepczukii, and S. curtilobum. In combination with recent morphological analyses and an examination of the identification history of these collections, we support the reclassification of the cultivated potatoes into four species: (i) S. tuberosum, with two Cultivar Groups (Andigenum Group of upland Andean genotypes containing diploids, triploids, and tetraploids, and the Chilotanum Group of lowland tetraploid Chilean landraces); (ii) S. ajanhuiri (diploid); (iii) S. juzepczukii (triploid); and (iv) S. curtilobum (pentaploid). For other classifications, consistent and stable identifications are impossible, and their classification as species is artificial and only maintains the confusion of users of the gene banks and literature.

The genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food crop

Significance We communicate the rather remarkable observation that among 291 tested accessions of cultivated sweet potato, all contain one or more transfer DNA (T-DNA) sequences. These sequences, which are shown to be expressed in a cultivated sweet potato clone (“Huachano”) that was analyzed in detail, suggest that an Agrobacterium infection occurred in evolutionary times. One of the T-DNAs is apparently present in all cultivated sweet potato clones, but not in the crop’s closely related wild relatives, suggesting the T-DNA provided a trait or traits that were selected for during domestication. This finding draws attention to the importance of plant–microbe interactions, and given that this crop has been eaten for millennia, it may change the paradigm governing the “unnatural” status of transgenic crops. Agrobacterium rhizogenes and Agrobacterium tumefaciens are plant pathogenic bacteria capable of transferring DNA fragments [transfer DNA (T-DNA)] bearing functional genes into the host plant genome. This naturally occurring mechanism has been adapted by plant biotechnologists to develop genetically modified crops that today are grown on more than 10% of the world’s arable land, although their use can result in considerable controversy. While assembling small interfering RNAs, or siRNAs, of sweet potato plants for metagenomic analysis, sequences homologous to T-DNA sequences from Agrobacterium spp. were discovered. Simple and quantitative PCR, Southern blotting, genome walking, and bacterial artificial chromosome library screening and sequencing unambiguously demonstrated that two different T-DNA regions (IbT-DNA1 and IbT-DNA2) are present in the cultivated sweet potato (Ipomoea batatas [L.] Lam.) genome and that these foreign genes are expressed at detectable levels in different tissues of the sweet potato plant. IbT-DNA1 was found to contain four open reading frames (ORFs) homologous to the tryptophan-2-monooxygenase (iaaM), indole-3-acetamide hydrolase (iaaH), C-protein (C-prot), and agrocinopine synthase (Acs) genes of Agrobacterium spp. IbT-DNA1 was detected in all 291 cultigens examined, but not in close wild relatives. IbT-DNA2 contained at least five ORFs with significant homology to the ORF14, ORF17n, rooting locus (Rol)B/RolC, ORF13, and ORF18/ORF17n genes of A. rhizogenes. IbT-DNA2 was detected in 45 of 217 genotypes that included both cultivated and wild species. Our finding, that sweet potato is naturally transgenic while being a widely and traditionally consumed food crop, could affect the current consumer distrust of the safety of transgenic food crops.

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophilamelanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

Plant defense genes associated with quantitative resistance to potato late blight in Solanum phureja x dihaploid S. tuberosum hybrids.

Markers corresponding to 27 plant defense genes were tested for linkage disequilibrium with quantitative resistance to late blight in a diploid potato population that had been used for mapping quantitative trait loci (QTLs) for late blight resistance. Markers were detected by using (i) hybridization probes for plant defense genes, (ii) primer pairs amplifying conserved domains of resistance (R) genes, (iii) primers for defense genes and genes encoding transcriptional regulatory factors, and (iv) primers allowing amplification of sequences flanking plant defense genes by the ligation-mediated polymerase chain reaction. Markers were initially screened by using the most resistant and susceptible individuals of the population, and those markers showing different allele frequencies between the two groups were mapped. Among the 308 segregating bands detected, 24 loci (8%) corresponding to six defense gene families were associated with resistance at chi2 > or = 13, the threshold established using the permutation test at P = 0.05. Loci corresponding to genes related to the phenylpropanoid pathway (phenylalanine ammonium lyase [PAL], chalcone isomerase [CHI], and chalcone synthase [CHS]), loci related to WRKY regulatory genes, and other -defense genes (osmotin and a Phytophthora infestans-induced cytochrome P450) were significantly associated with quantitative disease resistance. A subset of markers was tested on the mapping population of 94 individuals. Ten defense-related markers were clustered at a QTL on chromosome III, and three defense-related markers were located at a broad QTL on chromosome XII. The association of candidate genes with QTLs is a step toward understanding the molecular basis of quantitative resistance to an important plant disease.

Assessing genetic diversity of sweet potato (Ipomoea batatas (L.) Lam.) cultivars from tropical America using AFLP.

The sweet potato genebank at the International Potato Center (CIP) maintains 5,526 cultivated I. batatas accessions from 57 countries. Knowledge of the genetic structure in this collection is essential for rational germplasm conservation and utilization. Sixty-nine sweet potato cultivars from 4 geographical regions (including 13 countries) of Latin America were randomly sampled and fingerprinted using AFLP markers. A total of 210 polymorphic and clearly scorable fragments were generated. A geographic pattern of diversity distribution was revealed by mean similarity, multidimensional scaling (MDS), and analysis of molecular variance (AMOVA). The highest genetic diversity was found in Central America, whereas the lowest was in Peru-Ecuador. The within-region variation was the major source of molecular variance. The between-regions variation, although it only explains 10.0% of the total diversity, is statistically significant. Cultivars from Peru-Ecuador, with the lowest level of within region diversity, made the most significant contribution to the between region differentiation. These results support the hypothesis that Central America is the primary center of diversity and most likely the center of origin of sweet potato. Peru-Ecuador should be considered as a secondary center of sweet potato diversity.

Systematics, Diversity, Genetics, and Evolution of Wild and Cultivated Potatoes

The common potato, Solanum tuberosum L., is the third most important food crop and is grown and consumed worldwide. Indigenous cultivated (landrace) potatoes and wild potato species, all classified as Solanum section Petota, are widely used for potato improvement. Members of section Petota are broadly distributed in the Americas from the southwestern United States to the Southern Cone of South America. The latest comprehensive taxonomic treatment of section Petota was published by John (Jack) Hawkes in 1990; it recognized seven cultivated species and 228 wild species, divided into 21 taxonomic series. Since 1990, intensive field collections from throughout the range of the group, coupled with morphological and molecular studies, have halved the number of species and elucidated new ingroup and outgroup relationships. The recent sequencing of the potato genome has greatly accelerated investigation of all aspects of potato biology and allows us to address new questions not conceivable before. The purpose of this review is to provide a historical overview and update since 1990 of the systematics, diversity, genetics, domestication, evolution, and breeding of Solanum section Petota that will serve as a reference for the next generation of studies in the potato.

RAPD variation in sweetpotato (Ipomoea batatas (L.) Lam) cultivars from South America and Papua New Guinea

The island of New Guinea is considered a secondary center on diversity for sweetpotato, because of its range of isolated ecological niches and large number of cultivars found within a small area. Information of genetic diversity in Papua New Guinea (PNG) sweetpotato is essential for rationalizing the global sweetpotato germplasm collection. Using random amplified polymorphic DNA (RAPD), we compared the genetic variation and genetic diversity in 18 PNG cultivars versus 18 cultivars from South America. The analysis of molecular variance revealed large genetic diversity in both groups of cultivars. The within-group (among individuals) variation accounted for 90.6% of the total molecular variance. However, the difference between PNG and South American groups is statistically significant, although it explained only 9.4% of the total molecular variance. The PNG cultivars are also less divergent than their South American ancestors as the mean genetic distance in PNG group is significantly smaller than that of South American group. The lower level of genetic diversity in PNG cultivars was also reflected by multidimensional scaling. This study shows that PNG cultivars, after many years of isolated evolution in an unique agro-ecological environment are substantially divergent from their ancestors in South America. The genetic diversity level in PNG cultivars is significantly lower than that in South American cultivars. It thus provides a baseline for continuing studies of genetic diversity in different sweetpotato gene pools.

Multilevel Agrobiodiversity and Conservation of Andean Potatoes in Central Peru Species, Morphological, Genetic, and Spatial Diversity

Abstract Botanical species and morphological and genetic diversity represent different yet linked units of conservation. These features, and their spatial distribution in the central Peruvian Andean highlands of Huancavelica, were used as a basis for characterizing and quantifying potato agrobiodiversity at different scales. Results show that individual farm households maintain high levels of cultivar, morphological, and genetic diversity. At the regional level, all cultivated species, with the exception of Solanum ajanhuiri, were found to be present. Tetraploid native potatoes were most abundant, followed by diploids, triploids, and pentaploids. Morphological characterization of 2481 samples belonging to 38 in situ collections resulted in the identification of 557 unique cultivars. Genetic fingerprinting of 989 samples belonging to 8 in situ collections resulted in the identification of 406 unique cultivars. The principal source of genetic variation is found within rather than between geographically distanced subpopulations. High levels of cultivar diversity are found, particularly at elevations between 3850 and 4150 m.