GBStools: A Unified Approach for Reduced Representation Sequencing and Genotyping

Reduced representation sequencing methods such as genotyping-by-sequencing (GBS) enable low-cost measurement of genetic variation without the need for a reference genome assembly. These methods are widely used in genetic mapping and population genetics studies, especially with non-model organisms. Variant calling error rates, however, are higher in GBS than in standard sequencing, in particular due to restriction site polymorphisms, and few computational tools exist that specifically model and correct these errors. We developed a statistical method to remove errors caused by restriction site polymorphisms, implemented in the software package GBStools. We evaluated it in several simulated data sets, varying in number of samples, mean coverage and population mutation rate, and in two empirical human data sets (N = 8 and N = 63 samples). In our simulations, GBStools improved genotype accuracy more than commonly used filters such as Hardy-Weinberg equilibrium p-values. GBStools is most effective at removing genotype errors in data sets over 100 samples when coverage is 40X or higher, and the improvement is most pronounced in species with high genomic diversity. We also demonstrate the utility of GBS and GBStools for human population genetic inference in Argentine populations and reveal widely varying individual ancestry proportions and an excess of singletons, consistent with recent population growth.

Phylogeny of tribe Brassiceae (Brassicaceae) based on chloroplast restriction site polymorphisms and nuclear ribosomal internal transcribed spacer and chloroplast trnL intron sequences

Using sequence data from the ITS region (internal transcribed spacers ITS-1 and ITS-2 of nuclear DNA and the 5.8 rRNA gene), chloroplast DNA (cpDNA) sequence data from the trnL intron, and cpDNA restriction site polymorphism data, we examined the evolutionary relationships of the tribe Brassiceae (Brassicaceae). A group of approximately 50 genera, it is distinguished by the presence of conduplicate cotyledons and (or) heteroarthrocarpic fruit. cpDNA restriction site data and ITS, trnL, and combined ITS/trnL sequence data were obtained for 22, 104, 94, and 87 taxa in the tribe, respectively. Results from maximum parsimony analyses of the cpDNA and ITS and ITS/trnL sequence data showed a monophyletic origin for the tribe, with the inclusion of controversial members Calepina, Conringia, and Orychophragmus. ITS- and ITS/trnL-based clades corresponded to taxonomic subtribes: Vellinae, Zillinae, and Savignyinae; but as with previous cpDNA studies, there was little support for subtribes Brassicinae, Raphaninae, and Moricandiinae. Although there was no support for the Rapa/Oleracea or Nigra cpDNA lineages, many cpDNA subclades within each of them were evident in the ITS- and ITS/trnL-based phylogeny. The trnL sequence data provided little or no resolution of tribal or subtribal limits. The ITS data indicated polyphyletic origins for Brassica, Diplotaxis, and Erucastrum.Key words: Brassiceae, Calepina, Conringia, Orychophragmus, ITS, trnL, cpDNA restriction site polymorphisms.

Variation in TaqI-digested DNA of Sugar and Black Maples Is Independent of Taxon and Plant Origin

Morphological distinctions between sugar maples and black maples are not consistently evident, and molecular assessment of genetic diversity is lacking for these taxa. We examined restriction-site polymorphisms in the ndhA intron of the chloroplast DNA (cpDNA) in populations of sugar maples and black maples representing their zones of allopatry and sympatry in eastern North America. Restriction-site analysis of the ndhA intron after digestion with HinfI and Sau3AI yielded no polymorphisms. Restriction digestion of the ndhA intron with TaqI revealed two cpDNA haplotypes that were neither geographically localized nor taxon specific. Although testing additional accessions of sugar maples and black maples for cpDNA variation will further elucidate patterns of genetic variation, our initial results suggest that the taxa are either exchanging genes or share an ancestral cpDNA polymorphism.

Roles of RuvC and RecG in Phage λ Red-Mediated Recombination

ABSTRACT The recombination properties of Escherichia colistrains expressing the red genes of bacteriophage λ and lacking recBCD function either by mutation or by expression of λ gam were examined. The substrates for recombination were nonreplicating λ chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+recG+ , ruvC recG +,ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome’s restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in arecG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flankinglac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher inrecG than in recG +. These observations favor models in which RecG tends to push invading 3′-ended strands back out of recombination intermediates.