QTL mapping of domestication and diversifying selection related traits in round-fruited semi-wild Xishuangbanna cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L. var. <Emphasis Type="Italic">xishuangbannanesis</Emphasis>)

Key message

QTL analysis revealed 11 QTL underlying flowering time and fruit size variation in the semi-wild Xishuangbanna cucumber, of which, FT6.2 and FS5.2 played the most important roles in determining photoperiod-dependent flowering time and round-fruit shape, respectively.

Abstract

Flowering time and fruit size are two important traits in domestication and diversifying selection in cucumber, but their genetic basis is not well understood. Here we reported QTL mapping results on flowering time and fruit size with F₂ and F_2:3 segregating populations derived from the cross between WI7200, a small fruited, early flowering primitive cultivated cucumber and WI7167, a round-fruited, later flowering semi-wild Xishuangbanna (XIS) cucumber. A linkage map with 267 microsatellite marker loci was developed with 138 F₂ plants. Phenotypic data of male and female flowering time, fruit length and diameter and three other traits (mature fruit weight and number, and seedling hypocotyl length) were collected in multiple environments. Three flowering time QTL, FT1.1, FT5.1 and FT6.2 were identified, in which FT6.2 played the most important role in conferring less photoperiod sensitive early flowering during domestication whereas FT1.1 seemed more influential in regulating flowering time within the cultivated cucumber. Eight consensus fruit size QTL distributed in 7 chromosomes were detected, each of which contributed to both longitudinal and radial growth in cucumber fruit development. Among them, FS5.2 on chromosome 5 exhibited the largest effect on the determination of round fruit shape that was characteristic of the WI7167 XIS cucumber. Possible roles of these flowering time and fruit size QTL in domestication of cucumber and crop evolution of the semi-wild XIS cucumber, as well as the genetic basis of round fruit shape in cucumber are discussed.

     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.     

Movements of narwhals (<Emphasis Type="Italic">Monodon monoceros</Emphasis>) from Admiralty Inlet monitored by satellite telemetry

Twenty-one narwhals tagged in 2003 and 2004 in Admiralty Inlet showed a different summer distributional pattern than previous narwhal-tracking studies from Somerset Island, Eclipse Sound and Melville Bay. The migration of the narwhals tracked from Admiralty Inlet moved out through Lancaster Sound 15 days earlier (P < 0.0001) than the narwhals summering around Eclipse Sound, whereas the Admiralty Inlet narwhals reached the mouths of Eclipse Sound 18 days later (P < 0.0001) than the Eclipse Sound summering population. The winter range of the Admiralty Inlet narwhals overlapped with the winter range of narwhals from Melville Bay and Eclipse Sound in central southern Baffin Bay and Northern Davis Strait, but not with the winter range of narwhals from Somerset Island that wintered further north. Distribution size of range, and population size did not appear to be related. An example of considerable year to year variation between area of summer and winter distribution in the 2 years was believed to be related to the sample size and number of pods of whales tagged, rather than to differences in sex or age classes.     

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]₁₁ microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]₁₁ mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]₁₁ allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.     

MOLECULAR PHYLOGENY OF THE HETEROTROPHIC DINOFLAGELLATES,PROTOPERIDINIUM, DIPLOPSALIS AND PREPERIDINIUM (DINOPHYCEAE), INFERRED FROM LARGE SUBUNIT rDNA1

The genera Protoperidinium Bergh, Diplopsalis Bergh, and Preperidinium Mangin, comprised of species of marine, thecate, heterotrophic dinoflagellates in the family Protoperidinaceae Balech, have had a confused taxonomic history. To elucidate the validity of morphological groupings within the Protoperidinium and diplopsalids, and to determine the evolutionary relationships between these and other dinoflagellates, we undertook a study of molecular phylogeny using the D1–D3 domains of the large subunit (LSU) of the rDNA. Based on morphology, the 10 Protoperidinium species examined belonged to three subgenera and five morphological sections. Two diplopsalid species were also included. Single‐cell PCR, cloning, and sequencing revealed a high degree of intraindividual sequence variability in the LSU rDNA. The genus Protoperidinium appeared to be recently divergent in all phylogenetic analyses. In maximum parsimony and neighbor joining analyses, Protoperidinium formed a monophyletic group, evolving from diplopsalid dinoflagellates. In maximum likelihood and Bayesian analyses, however, Protoperidinium was polyphyletic, as the lenticular, diplopsalid heterotroph, Diplopsalis lenticula Bergh, was inserted within the Protoperidinium clade as basal to Protoperidinium excentricum (Paulsen) Balech, and Preperidinium meunieri (Pavillard) Elbrächter fell within a separate clade as a sister to the Oceanica and Protoperidinium steidingerae Balech. In all analyses, the Protoperidinium were divided into two major clades, with members in the Oceanica group and subgenus Testeria in one clade, and the Excentrica, Conica, Pellucida, Pyriforme and Divergens sections in the other clade. The LSU rDNA molecular phylogeny supported the historical morphologically determined sections, but not a simple morphology based model of evolution based on thecal plate shape.     

Multiple introductions,admixture and bridgehead invasion characterize the introduction history of Ambrosia artemisiifolia in Europe and Australia

Admixture between differentiated populations is considered to be a powerful mechanism stimulating the invasive success of some introduced species. It is generally facilitated through multiple introductions; however, the importance of admixture prior to introduction has rarely been considered. We assess the likelihood that the invasive Ambrosia artemisiifolia populations of Europe and Australia developed through multiple introductions or were sourced from a historical admixture zone within native North America. To do this, we combine large genomic and sampling data sets analysed with approximate Bayesian computation and random forest scenario evaluation to compare single and multiple invasion scenarios with pre‐ and postintroduction admixture simultaneously. We show the historical admixture zone within native North America originated before global invasion of this weed and could act as a potential source of introduced populations. We provide evidence supporting the hypothesis that the invasive populations established through multiple introductions from the native range into Europe and subsequent bridgehead invasion into Australia. We discuss the evolutionary mechanisms that could promote invasiveness and evolutionary potential of alien species from bridgehead invasions and admixed source populations.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.