Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.     

A method to generate multilocus barcodes of pinned insect specimens using MiSeq

For molecular insect identification, amplicon sequencing methods are recommended because they offer a cost‐effective approach for targeting small sets of informative genes from multiple samples. In this context, high‐throughput multilocus amplicon sequencing has been achieved using the MiSeq Illumina sequencing platform. However, this approach generates short gene fragments of <500 bp, which then have to be overlapped using bioinformatics to achieve longer sequence lengths. This increases the risk of generating chimeric sequences or leads to the formation of incomplete loci. Here, we propose a modified nested amplicon sequencing method for targeting multiple loci from pinned insect specimens using the MiSeq Illumina platform. The modification exists in using a three‐step nested PCR approach targeting near full‐length loci in the initial PCR and subsequently amplifying short fragments of between 300 and 350 bp for high‐throughput sequencing using Illumina chemistry. Using this method, we generated 407 sequences of three loci from 86% of all the specimens sequenced. Out of 103 pinned bee specimens of replicated species, 71% passed the 95% sequence similarity threshold between species replicates. This method worked best for pinned specimens aged between 0 and 5 years, with a limit of 10 years for pinned and 14 years for ethanol‐preserved specimens. Hence, our method overcomes some of the challenges of amplicon sequencing using short read next generation sequencing and improves the possibility of creating high‐quality multilocus barcodes from insect collections.     

Brenneria alni,causal agent bark canker of Alnus subcordata

In 2014, bark cankers were observed on Caucasian alder (Alnus subcordata) trees in Iran. The disease was characterized by a dark watery liquid often exuding from longitudinal cankers in the bark of the tree trunks which stained the surface. Symptomatic tissue from A. subcordata was sampled from a number of sites in the Mazandaran province. Isolations were performed on nutrient agar supplemented with sucrose (SNA) and yielded bacterial colonies that were uniform, round and whitish. The bacterial strains isolated from alder trees in Iran were similar to Brenneria alni based on phenotypic and genotypic (nucleotide sequences of the 16S rRNA, and housekeeping genes gyrB, and infB) characteristics. The pathogenicity of the representative strains was determined by inoculating stem pieces of A. subcordata. All tested strains caused longitudinal necrotic lesions 30 days after inoculation and were re-isolated from this tissue. To our knowledge, this is the first report of the occurrence of B. alni in Iran, and on A. subcordata globally.     

Geographical pattern of genetic diversity in Capsella bursa‐pastoris (Brassicaceae)—A global perspective

We analyzed the global genetic variation pattern of Capsella bursa‐pastoris (Brassicaceae) as expressed in allozymic (within‐locus) diversity and isozymic (between‐locus) diversity. Results are based on a global sampling of more than 20,000 C. bursa‐pastoris individuals randomly taken from 1,469 natural provenances in the native and introduced range, covering a broad spectrum of the species’ geographic distribution. We evaluated data for population genetic parameters and F‐statistics, and Mantel tests and AMOVA were performed. Geographical distribution patterns of alleles and multilocus genotypes are shown in maps and tables. Genetic diversity of introduced populations is only moderately reduced in comparison with native populations. Global population structure was analyzed with structure, and the obtained cluster affiliation was tested independently with classification approaches and macroclimatic data using species distribution modeling. Analyses revealed two main clusters: one distributed predominantly in warm arid to semiarid climate regions and the other predominantly in more temperate humid to semihumid climate regions. We observed admixture between the two lineages predominantly in regions with intermediate humidity in both the native and non‐native ranges. The genetically derived clusters are strongly supported in macroclimatic data space. The worldwide distribution patterns of genetic variation in the range of C. bursa‐pastoris can be explained by intensive intra‐ and intercontinental migration, but environmental filtering due to climate preadaption seems also involved. Multiple independent introductions of genotypes from different source regions are obvious. “Endemic” genotypes might be the outcome of admixture or of de novo mutation. We conclude that today's successfully established Capsella genotypes were preadapted and found matching niche conditions in the colonized range parts.     

Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human respiratory samples and the rhizosphere

Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883^T (=CCUG 62895^T). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42 °C, acidification of sucrose and adonitol, lysine decarboxylase and β-galactosidase activity, and esculin hydrolysis.     

Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time‐intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185‐bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per‐amplicon‐variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi‐quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus‐specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.