<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Exome sequencing identifies nonsegregating nonsense <Emphasis Type="Italic">ATM</Emphasis> and <Emphasis Type="Italic">PALB2</Emphasis>variants in familial pancreatic cancer

We sequenced 11 germline exomes from five families with familial pancreatic cancer (FPC). One proband had a germline nonsense variant in ATM with somatic loss of the variant allele. Another proband had a nonsense variant in PALB2 with somatic loss of the variant allele. Both variants were absent in a relative with FPC. These findings question the causal mechanisms of ATM and PALB2 in these families and highlight challenges in identifying the causes of familial cancer syndromes using exome sequencing.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Exome sequencing identifies novel and recurrent mutations in <Emphasis Type="Italic">GJA8</Emphasis> and <Emphasis Type="Italic">CRYGD</Emphasis>associated with inherited cataract

Background

Inherited cataract is a clinically important and genetically heterogeneous cause of visual impairment. Typically, it presents at an early age with or without other ocular/systemic signs and lacks clear phenotype-genotype correlation rendering both clinical classification and molecular diagnosis challenging. Here we have utilized trio-based whole exome sequencing to discover mutations in candidate genes underlying autosomal dominant cataract segregating in three nuclear families.

Results

In family A, we identified a recurrent heterozygous mutation in exon-2 of the gene encoding γD-crystallin (CRYGD; c.70C > A, p.Pro24Thr) that co-segregated with `coralliform' lens opacities. Families B and C were found to harbor different novel variants in exon-2 of the gene coding for gap-junction protein α8 (GJA8; c.20T > C, p.Leu7Pro and c.293A > C, p.His98Pro). Each novel variant co-segregated with disease and was predicted in silico to have damaging effects on protein function.

Conclusions

Exome sequencing facilitates concurrent mutation-profiling of the burgeoning list of candidate genes for inherited cataract, and the results can provide enhanced clinical diagnosis and genetic counseling for affected families.

     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Xanthium italicum</Emphasis>, <Emphasis Type="Italic">Xanthium strumarium</Emphasis> and <Emphasis Type="Italic">Arctium lappa</Emphasis> as new hosts for <Emphasis Type="Italic">Diaporthe helianthi</Emphasis>

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Genomewide analysis of <Emphasis Type="Italic">ABCB</Emphasis>s with a focus on <Emphasis Type="Italic">ABCB1</Emphasis> and <Emphasis Type="Italic">ABCB19</Emphasis> in <Emphasis Type="Italic">Malus domestica</Emphasis>

The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon–intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots of M9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.