Disentangling the roles of history and local selection in shaping clinal variation of allele frequencies and gene expression in Norway spruce (Picea abies)

Understanding the genetic basis of local adaptation is challenging due to the subtle balance among conflicting evolutionary forces that are involved in its establishment and maintenance. One system with which to tease apart these difficulties is clines in adaptive characters. Here we analyzed genetic and phenotypic variation in bud set, a highly heritable and adaptive trait, among 18 populations of Norway spruce (Picea abies), arrayed along a latitudinal gradient ranging from 47°N to 68°N. We confirmed that variation in bud set is strongly clinal, using a subset of five populations. Genotypes for 137 single-nucleotide polymorphisms (SNPs) chosen from 18 candidate genes putatively affecting bud set and 308 control SNPs chosen from 264 random genes were analyzed for patterns of genetic structure and correlation to environment. Population genetic structure was low (F(ST) = 0.05), but latitudinal patterns were apparent among Scandinavian populations. Hence, part of the observed clinal variation should be attributable to population demography. Conditional on patterns of genetic structure, there was enrichment of SNPs within candidate genes for correlations with latitude. Twenty-nine SNPs were also outliers with respect to F(ST). The enrichment for clinal variation at SNPs within candidate genes (i.e., SNPs in PaGI, PaPhyP, PaPhyN, PaPRR7, and PaFTL2) indicated that local selection in the 18 populations, and/or selection in the ancestral populations from which they were recently derived, shaped the observed cline. Validation of these genes using expression studies also revealed that PaFTL2 expression is significantly associated with latitude, thereby confirming the central role played by this gene in the control of phenology in plants.     

GENOME‐WIDE INVESTIGATION OF REPRODUCTIVE ISOLATION IN EXPERIMENTAL LINEAGES AND NATURAL SPECIES OF NEUROSPORA: IDENTIFYING CANDIDATE REGIONS BY MICROARRAY‐BASED GENOTYPING AND MAPPING

Inherent incompatibilities between genetic components from genomes of different species may cause intrinsic reproductive isolation. In evolution experiments designed to instigate speciation in laboratory populations of the filamentous fungus Neurospora, we previously discovered a pair of incompatibility loci (dfe and dma) that interact negatively to cause severe defects in sexual reproduction. Here we show that the dfe‐dma incompatibility also is a significant cause of genetic isolation between two naturally occurring species of Neurospora (N. crassa and N. intermedia). The strong incompatibility interaction has a simple genetic basis (two biallelic loci) and antagonistic epistasis occurs between heterospecific alleles only, consistent with the Dobzhansky–Muller model of genic incompatibility. We developed microarray‐based, restriction‐site associated DNA (RAD) markers that identified ～1500 polymorphisms between the genomes of the two species, and constructed the first interspecific physical map of Neurospora. With this new mapping resource, the approximate genomic locations of the incompatibility loci were determined using three different approaches: genome scanning, bulk‐segregant analyses, and introgression. These population, quantitative, and classical genetics methods concordantly identified two candidate regions, narrowing the search for each incompatibility locus to only ～2% of the nuclear genome. This study demonstrates how advances in high‐throughput, genome‐wide genotyping can be applied to mapping reproductive isolation genes and speciation research.     

Disproportionate numbers of rDNA loci in diploid and polyploid testis nuclei of gerris najas detected by fluorescence in situ hybridization

The replication state of rDNA in testes nuclei undergoing polyploidization by classical-type endomitosis was investigated in Gerris najas (Heteroptera) by means of fluorescence in situ hybridization. The number of just one rDNA locus per haploid genome was determined by in situ hybridization on meiotic nuclei. Additionally, DNA measurements of spermatids and testes nuclei were performed. Although regular duplication levels of nuclear DNA were found within the limits of the accuracy of the method, these did evidently not apply to the ribosomal genes. The comparison of the number of rDNA signals with the DNA content of 106 testis nuclei revealed drastic variations of the number of rDNA loci between individual nuclei with similar DNA content. Polyploid nuclei of the testis epithelium showed too low numbers of rDNA loci in relation to those expected from the levels of ploidy, while cyst cell nuclei displayed increased numbers of rDNA loci. The results indicate that the ribosomal genes are either underreplicated, or in part eliminated, during the endomitotic cycles of epithelium cell nuclei, but amplified in the cyst cell nuclei, probably already at their diploid stage.     

Expression Profiling and Validation of Potential Reference Genes During <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> Embryogenesis

Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, α-Tub, EF1-α, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.     

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.     

Identification of differentially expressed genes in primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease that affects exocrine glands. To study the molecular mechanism and identify crucial genes/pathways in pSS pathogenesis, the microarray-based whole-genome gene expression profiles from salivary glands of patients with pSS and non-sicca controls were retrieved. After normalization and subsequent batch effect adjustment, significance analysis of microarrays method was applied to five available datasets, and 379 differentially expressed genes (DEGs) were identified. The 300 upregulated DEGs were enriched in Gene Ontology terms of immune and inflammatory responses, including antigen processing and presentation, interferon-mediated signaling pathway, and chemotaxis. Previously reported pSS-associated genes, including HLA-DRA, TAP2, PRDM1, and IFI16, were found to be significantly upregulated. The downregulated DEGs were enriched in pathways of salivary secretion, carbohydrate digestion and absorption, and starch and sucrose metabolism, implying dysfunction of salivary glands during pathogenesis. Next, a protein-protein interaction network was constructed, and B2M, an upregulated DEG, was shown to be a hub, suggesting its potential involvement in pSS development. In summary, we found the activation of pSS-associated genes in pathogenesis, and provide clues for salivary glands dysfunction. Experimental investigation on the identified DEGs in this study will deepen our understanding on pSS.     

川楝内生放线菌的分离及多样性研究

【目的】分离四川省各个地区川楝内生放线菌并研究其物种多样性。【方法】应用7种选择性分离培养基分离样品根、茎、叶、树皮和果实中的内生放线菌,采用16SrRNA基因RFLP分析代表菌株多样性。【结果】研究共获得403株内生放线菌。不同地点、不同植株部位、不同培养基分离得到的内生放线菌数目均有差异。广元采集的样品分离得到的数目最多,为86株;最少的是绵阳,仅有12株。从植物表皮中分离到148株放线菌,占获得菌株总数的36.7%;而从果中分离到31株,仅占获得菌株总数的7.6%;虽然从根部分离到的数量也很少,但是其出菌率却是最高的。5号和3号培养基的分离效果最为理想。16S rRNA基因RFLP分析结果显示所有供试菌株在68%的相似性上聚在一起,在84%的相似水平上分成了10个遗传类型。代表菌株的16SrRNA基因序列测定及系统发育分析结果表明:分离得到的放线菌包括4个属,分别是链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。其中,链霉菌是优势类群,占代表菌株数目的比例高达91%,而稀有放线菌的比例只有9%。【结论】研究发现的川楝内生放线菌主要属于链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。