Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F₈ recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F₈ population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of “false positives” (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.     

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB_313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF₃ support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF₃ through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.     

Morphology,ontogeny, and molecular phylogeny of two novel bakuellid-like hypotrichs (Ciliophora: Hypotrichia), with establishment of two new genera

The morphology, ontogeny and molecular phylogeny of Apobakuella fusca gen. n., sp. n. and Parabistichella variabilis gen. n., sp. n., from south China were investigated. Apobakuella fusca, brown colored, demonstrates bakuellid-like infraciliature, and a similar ontogenesis as the genus Bakuella. It is argued, however, that this species represents a novel genus, Apobakuella, which is characterized by two or more marginal rows on the right, several buccal and parabuccal cirri, and lack of frontoterminal and caudal cirri. Phylogenetic analysis based on SSU rRNA gene sequences supports the close relationship of Apobakuella with Neobakuella and Diaxonella within the core Urostylida. By contrast, Parabistichella variabilis has a dominant frontoventral row, few midventral pairs, a long midventral row, and one marginal row on each side. Its morphogenesis exhibits: (1) partial reorganization of the parental adoral membranelles; (2) over six frontoventral-transverse cirri anlagen; (3) intrakinetal development of the midventral row; and (4) very likely, formation of the frontoventral row from the midventral row anlage. Both the morphological characteristics and the SSU rRNA gene sequences suggest that it is incertae sedis among the basal hypotrichs. Further investigation of key taxa with additional molecular markers is required to reveal a better understanding on the phylogeny of Parabistichella.     

Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

金荞麦果实中有效成分的分析

采用HPLC法对金荞麦果实中的有效成分进行了分析,结果表明:金荞麦果实中含有芦丁、槲皮素和表儿茶素等有效成分,其中主要以芦丁为主,槲皮素、表儿茶素等含量甚微.金荞麦果实中的有效成分主要集中在种子部分,果皮中则不合或含量甚微.     

Polymorphisms in MHC class Ia genes and resistance to IHNV in rainbow trout (Oncorhynchus mykiss)

Infectious haematopoietic necrosis virus (IHNV) is detrimental to the farming of rainbow trout (Oncorhynchus mykiss) and other salmonids in the Northern hemisphere. The major histocompatibility complex (MHC) plays a key role in immune response in invertebrates, as evidenced by the close correlation of MHC polymorphisms with disease resistance/susceptibility. To analyse the correlation between rainbow trout resistance and susceptibility to IHNV and genetic variation in exon 2 of MHC class Ia gene, UBA, we employed two approaches, namely, polymerase chain reaction-single strand conformation polymorphism analysis and cloning/sequencing. From 102 resistant and 82 susceptible individuals, a total of 12 alleles in UBA exon 2 (GenBank: JX136662–JX136673) were identified, including 11 novel alleles. The maximum number of these alleles in a single individual was four, suggesting that UBA exon 2 most likely resides on at least two loci in the genome. Most of the variations in UBA exon 2 were located in the peptide-binding region and were determined to have been subject to positive selection during evolution. Correlation analysis revealed that Onmy-UBA*0111 and Onmy-UBA*0107 are highly associated with IHNV susceptibility (P = 0.001), whereas Onmy-UBA*0101, Onmy-UBA*0102, and Onmy-UBA*0103 are highly related to IHNV resistance (P = 0.000). In addition, the three resistant alleles were predominant in the IHNV disease-resistant population; thus, these molecular markers can be used for anti-IHNV breeding of rainbow trout.     

Indirect and suboptimal control of gene expression is widespread in bacteria

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes’ function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR‐1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory.