Robert L. Jarret

Sequence characterization of microsatellites in diploid and polyploid Ipomoea

Abstract The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids.

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t1 seed by T0 plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 3∶1 Mendelian ratio in most T1 generation plants.

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.

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa.

SummaryRandom genomic probes were used to detect RFLPs in 19 Musa species and subspecies. A total of 89 phylogenetically informative alleles were scored and analyzed cladistically and phenetically. Results were in general agreement with morphology-based phylogenetic analyses, with the following exceptions: our data unambiguously places M. boman in section Australimusa, and indicates M. beccarii is very closely related to M. acuminata. Additionally, no support was found for the separation of section Rhodochlamys from section Musa. A comparison of morphology-based and RFLP-based phylogenetic analyses is presented.

Genetic diversity and systematic relationships in sweetpotato (Ipomoea batatas (L.) Lam.) and related species as revealed by RAPD analysis

SummaryFifteen 10mer primers, in combination with the Stoffel fragment, were used to detect random amplified polymorphic DNA (RAPDs) among 26 accessions of sweetpotato (I. batatas (L.) Lam.) from Oceania, Peru, the Philippines, and the United States and between 8 Ipomoea species from section Batatas. Phenetic and principal coordinate analysis of the 56 polymorphisms detected within the hexaploid I. batatas clearly delineated the South Pacific and the Peruvian sweetpotato lines. The two U.S. cultivars clustered with the Oceanic materials. Cladistic and phenetic analysis of 8 Ipomoea species supports previously published phylogenies based on morphological and RFLP data. Among the species examined, I. tabascana, I. trifida and the tetraploid forms of I. batatas from Mexico and Ecuador, including I. batatas var. apiculata, are the taxa most closely related to the cultivated hexaploid I. batatas. These findings support the utility of RAPD markers for evaluating genetic diversity in sweetpotato and for establishing taxonomic and evolutionary relationships in Ipomoea.

Random amplified polymorphic DNA and genetic diversity in IndianMusa germplasm

SummaryFifty-seven accessions ofMusa including cultivated clones of 6 genomic groups (AA, AB, AAA, AAB, ABB, ABBB),M. balbisiana Colla (BB),M. acuminata Colla ssp.banksii F. Muell. (AA),M. acuminata Colla ssp.malaccensis Ridl. (AA) andM. velutina Wendl. & Drude were examined for random amplified polymorphic DNA (RAPD) genetic markers using PCR with sixty 10-mer random primers. Forty-nine of 60 tested primers gave reproducible DNA amplification patterns. The number of bands resolved per amplification was primer dependent and varied from 1 to a maximum of 24. The size range of the amplification products also differed with the selected primer sequence/genotype and ranged from 0.29 to 3.0 kb. RAPD data were used to generate Jaccards similarity coefficients which were analyzed phenetically. Phenetic analysis separated clones into distinct groupings that were in agreement with clusterings revealed when data were subsequently analyzed by principal coordinate analysis (PCO). In both the phenetic and the PCO analyses, previously unclassified cultivars grouped with cultivars previously classified for their genomic group based on morphological keys. The implications of RAPD analysis forMusa germplasm classification, clonal identification, and management are discussed.

CHLOROPLAST DNA RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPS) IN MUSA SPECIES

SummaryTaxonomic and phylogenetic determinations within the genus Musa are established using a numerical, morphology-based scoring system. However, within this system, the classification and relationships of some types are disputed. The application of chloroplast DNA (cpDNA) restriction fragment length polymorphism (RFLP) analysis to Musa taxonomy provided valuable, supplemental information about the classification of, and relationships between, Musa species and subspecies. Whole-cell DNA was extracted from lyophilized Musa leaf-blade tissue and digested with various restriction enzymes, Southern blotted onto nylon membranes, and probed using radioactively labeled heterologous orchid cpDNA fragments. Phylogenies were inferred from cpDNA RFLP patterns using PAUP software. The relationships between most species examined were as expected; however, some species (M. beccarii and M. basjoo) did not conform to the conventional morphology-based phylogeny.

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene

Determination of capsinoids by HPLC-DAD in Capsicum species.

Capsicum fruits contain a newly discovered phytochemical called capsinoids. Because little is known about the quantities of these compounds in both sweet and pungent pepper fruits, a high-performance liquid chromatography (HPLC) method was developed to identify and quantify the capsinoids (naturally present E-capsiate and dihydrocapsiate) utilizing fruit obtained from a variety of Capsicum spp. in the U.S. Department of Agricultures Capsicum germplasm collection. Capsinoids were extracted with acetonitrile, filtered, and analyzed using an HPLC system equipped with a C(18) monolithic column, gradient pump, and diode array detector. The elution solvents were acetonitrile and water (60:40) with an isocratic flow rate of 1.0 mL/min. Forty-nine samples representing distinct morphotypes of four cultivated species ( C. annuum var. annuum, C. annuum var. glabriusculum, C. baccatum , C. chinense , and C. frutescens ) contained detectable levels (11-369 microg/g) of E-capsiate quantified at a wavelength of 280 nm. Nine of the E-capsiate-containing samples also had dihydrocapsiate (18-86 micro/g). Gas chromatography with a mass spectrometry detector (GC-MS) confirmed the presence of these compounds in the Capsicum spp.