Asymmetric single‐strand conformation polymorphism: An accurate and cost‐effective method to amplify and sequence allelic variants

Rodríguez, Flor; Cai, Danying; Teng, Yuanwen; Spooner, David M.

doi:10.3732/ajb.1000251

Cited by 16 publications

(13 citation statements)

References 27 publications

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“…We used the ten nuclear orthologs that were identified by Arbizu et al (2014b) as a useful subset to recover dominant topologies in Daucus (the subset determined by the result obtained by the concatenated data set of all 94 orthologs). DNA sequences of 53 accessions of the D. guttatus complex for these ten nuclear orthologs were obtained using Asymmetric PCR Single-Strand Conformation Polymorphism, an efficient alternative technique for isolating allelic variants of Daucus (Rodríguez et al 2011). Sanger sequencing (Sanger et al 1977) was conducted at the Biotechnology Center of the University of Wisconsin-Madison.…”

Section: Methodsmentioning

confidence: 99%

Integrated Molecular and Morphological Studies of the <I>Daucus guttatus</I> Complex (Apiaceae)

Arbizu¹,

Simon²,

Flores³

et al. 2016

Systematic Botany

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Abstract-In a previous study using 94 nuclear orthologs, we reported the species status of the Daucus guttatus complex to be unresolved, partitioned into three clades. In the present study, a subset of ten of these 94 orthologs was used to infer the phylogeny of the D. guttatus complex and related species. A near parallel set of accessions, planted in a common garden, was used for morphological analyses. The molecular trees are highly resolved for most of the clades, grouping accessions of the D. guttatus complex into four clades. Bayesian concordance analysis and a coalescent approach gave slightly different topologies. Morphological data likewise support four taxa in the complex. Moreover, herbarium research from a companion study informs nomenclature for taxa of the complex. We identify these four clades as D. bicolor, D. conchitae, D. guttatus, and D. setulosus; internested in or among these segregates are the phenetically distinctive species D. glochidiatus, D. involucratus, D. littoralis, and D. pusillus. Our research redefines species variation in the D. guttatus complex, clarifies species names, interspecific relationships, confirms a useful subset of nuclear orthologs for studies of dominant topologies of Daucus, and discovers morphological characters allowing proper identification of the four species of the D. guttatus complex and related species.

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Section: Methodsmentioning

confidence: 99%

Integrated Molecular and Morphological Studies of the <I>Daucus guttatus</I> Complex (Apiaceae)

Arbizu¹,

Simon²,

Flores³

et al. 2016

Systematic Botany

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“…SSCP protocols followed Rodriguez et al . (). Taxa were cloned if they displayed high numbers (greater than approximately 5/1000 nucleotides) of polymorphisms (but otherwise had clear single bands) after direct sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR product was then gel purified with QIAquick Gel Extraction Kit (Qiagen), ligated into a pGEM T-Vector (Promega, Madison, WI, USA), cloned in Escherichia coli DHB-5a competent cells (Invitrogen, Carlsbad, CA, USA), re-amplified and sequenced. SSCP protocols followed Rodriguez et al (2011). Taxa were cloned if they displayed high numbers (greater than approximately 5/1000 nucleotides) of polymorphisms (but otherwise had clear single bands) after direct sequencing.…”

Section: Dna Extraction Amplification and Sequencingmentioning

confidence: 99%

The South American radiation ofLepechinia(Lamiaceae): phylogenetics, divergence times and evolution of dioecy

Drew

Sytsma

2012

Bot J Linn Soc

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Lepechinia (Lamiaceae) is a diverse and widespread genus that is important in indigenous New World culture. We present a phylogenetic analysis of 72 accessions representing 31 species of Lepechinia using data from three plastid (trnL‐F, ycf1, ycf1‐rps15 spacer) and four nuclear [internal transcribed spacer (ITS), external transcribed spacer (ETS), granule‐bound starch synthase I (GBSS)I, pentatricopeptide repeat region (PPR)‐AT3G09060] DNA regions. Data were analysed using parsimony, maximum likelihood and Bayesian approaches. Divergence time estimation using BEAST shows Lepechinia had a mid/late Miocene origin, perhaps as a response to global cooling patterns. Cladogenesis in most South American Lepechinia is shown to have occurred within the past 5 Myr, presumably as a response to climatic and orogenic events. Dioecy has arisen multiple times in Lepechinia, once in North America and at least twice in South America, and not necessarily involving gynodioecy as an intermediary step. Dioecy and gynodioecy are demonstrated to be associated with several floral characters, including flower size, number and colour. © 2012 The Linnean Society of London

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“…DNA obtained from leaves of young plants grown from seeds in a greenhouse was extracted by the CTAB method [66] and qualified and quantified in 1% agarose gels with marker CsCl-purified λ DNA digested with Pst I. All DNA amplification, and SSCP sequencing followed [49]. In brief, SSCP involved running SSCP, extracting the bands of interest, and sequencing them.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, our study stimulated us to develop single strand conformation polymorphism (SSCP) that separates alleles by their different physical conformations, not by size, alleviating all three of these problems [49]. Asymmetric PCR single-strand conformation polymorphism is an efficient alternative technique for isolating allelic variants of highly heterozygous individuals that eliminates two common problems encountered in cloning: PCR recombination and heteroduplex fixation.…”

Section: Introductionmentioning

confidence: 99%

Single copy nuclear gene analysis of polyploidy in wild potatoes (Solanum section Petota)

et al. 2012

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BackgroundRecent genomic studies have drastically altered our knowledge of polyploid evolution. Wild potatoes (Solanum section Petota) are a highly diverse and economically important group of about 100 species widely distributed throughout the Americas. Thirty-six percent of the species in section Petota are polyploid or with diploid and polyploid cytotypes. However, the group is poorly understood at the genomic level and the series is ideal to study polyploid evolution. Two separate studies using the nuclear orthologs GBSSI and nitrate reductase confirmed prior hypotheses of polyploid origins in potato and have shown new origins not proposed before. These studies have been limited, however, by the use of few accessions per polyploid species and by low taxonomic resolution, providing clade-specific, but not species-specific origins within clades. The purpose of the present study is to use six nuclear orthologs, within 54 accessions of 11 polyploid species, 34 accessions of 29 diploid species of section Petota representing their putative progenitors, and two outgroups, to see if phenomena typical of other polyploid groups occur within wild potatoes, to include multiple origins, loss of alleles, or gain of new alleles.ResultsOur results increase resolution within clades, giving better ideas of diploid progenitors, and show unexpected complexity of allele sharing within clades. While some species have little diversity among accessions and concur with the GBSSI and nitrate reductase results, such as S. agrimonifolium, S. colombianum, S. hjertingii, and S. moscopanum, the results give much better resolution of species-specific progenitors. Seven other species, however, show variant patterns of allele distributions suggesting multiple origins and allele loss. Complex three-genome origins are supported for S. hougasii, and S. schenckii, and one of the ten accessions of S. stoloniferum. A very unexpected shared presence of alleles occurs within one clade of S. verrucosum from Central America, and S. berthaultii from South America in six polyploid species S. demissum, S. hjertingii, S. hougasii, S. iopetalum, S. schenckii, and S. stoloniferum.ConclusionsOur results document considerable genomic complexity of some wild potato polyploids. These can be explained by multiple hybrid origins and allele losses that provide a clear biological explanation for the taxonomic complexity in wild potato polyploids. These results are of theoretical and practical benefit to potato breeders, and add to a growing body of evidence showing considerable complexity in polyploid plants in general.

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Asymmetric single‐strand conformation polymorphism: An accurate and cost‐effective method to amplify and sequence allelic variants

Cited by 16 publications

References 27 publications

Integrated Molecular and Morphological Studies of the <I>Daucus guttatus</I> Complex (Apiaceae)

Integrated Molecular and Morphological Studies of the <I>Daucus guttatus</I> Complex (Apiaceae)

The South American radiation ofLepechinia(Lamiaceae): phylogenetics, divergence times and evolution of dioecy

Single copy nuclear gene analysis of polyploidy in wild potatoes (Solanum section Petota)

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