铁皮石斛四倍体离体诱导和鉴定及生理特性研究

以铁皮石斛原球茎为材料,经不同质量浓度的秋水仙素(C₂₂H₂₅O₆)和0.02 g·mL^-1二甲基亚砜(DMSO)混合水溶液处理后进行组织培养,通过对变异株进行形态学、细胞学及流式细胞仪鉴定,以期获得稳定的四倍体植株并分析其生理特性。结果表明:用2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理铁皮石斛原球茎36 h后,植株诱导率达20%;诱导四倍体植株在形态上明显矮化、茎秆粗壮、叶片变小增厚、气孔直径增大;细胞遗传学观察发现,四倍体植株染色体2n=4x=76,二倍体植株染色体2n=2x=38;流式细胞仪分析显示,DNA相对含量四倍体为400,二倍体仅为200;四倍体植株叶片中叶绿素含量、可溶性蛋白、可溶性糖含量均高于二倍体,分别为5.03、3.59、2.98 mg·g^-1;四倍体叶片中主要抗氧化酶POD和SOD活性均显著高于二倍体,分别为9.08、180.4 U·mg^-1,且四倍体植株明显降低了MDA含量累积。研究认为,2.0 g·L^-1秋水仙素和0.02 g·mL^-1 DMSO混合水溶液处理原球茎36 h可提高诱导成功率、降低嵌合体比例,此组合为诱导四倍体较佳诱导条件。     

氮离子注入对蒙古黄芪多倍体诱导的影响

利用二倍体蒙古黄芪种子为材料,以低能氮离子束为诱变源,将化学诱导与物理诱变相结合,探索出一套高效的多倍体诱导新方法。研究结果表明：氮离子注入种子后表现出明显的生物学效应;氮离子注入与秋水仙素联合诱导黄芪多倍体的效果很明显。氮离子注入剂量为2.6×10¹⁶ N⁺/cm²,秋水仙素浓度为100 mg·L^-1,培养5 d诱导率最高为44.4%;氮离子注入剂量为5.2×10¹⁶ N⁺/cm², 秋水仙素浓度为150 mg·L^-1,培养10d的诱导率最高为46.2%;二者均高于对照组秋水仙素浓度为100 mg·L^-1培养15 d的最高诱导率13.9%。利用细胞染色体计数鉴定多倍体为四倍体。     

Ginkgo biloba responds to herbivory by activating early signaling and direct defenses

Background

Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses.

Methodology/Principal Findings

Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca²⁺]_cyt) and H₂O₂ production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca²⁺]_cyt and H₂O₂. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides.

Conclusions/Significance

By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this “living fossil” plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.     

大花蕙兰多倍体的高效诱导

以大花蕙兰‘红瀑布’无菌苗丛芽为材料、秋水仙素为诱变剂,采用不同的处理浓度、时间诱导大花蕙兰体细胞加倍。通过形态学和细胞学观察、统计等方法对其进行倍性鉴定。结果表明：秋水仙素浓度0.05%,处理时间24 h的条件下,诱导率高达28.2%;多倍体苗外部形态、叶绿体数目、气孔数目和大小与二倍体差异大,加倍后的细胞核明显变大,染色体倍数增加。     

ORIGIN OF FRAGARIA POLYPLOIDS. II. UNREDUCED AND DOUBLED-UNREDUCED GAMETES

Offspring from natural hybrids between octoploid Fragaria chiloensis (2n = 56) and diploid F. vesca (2n = 14) backcrossed under natural conditions to F. chiloensis were studied. The natural F₁ hybrids themselves were of three kinds: (1) The expected pentaploids which resulted from the union of normally reduced gametes of diploid F. vesca and octoploid F. chiloensis; (2) A hexaploid F₁ hybrid which resulted from the union of an unreduced gamete from diploid F. vesca with a normally reduced gamete from octoploid F. chiloensis; and (3) A 9-ploid F₁ hybrid which probably arose from the union of an unreduced gamete of the octoploid F. chiloensis with a normally reduced gamete of diploid F. vesca. The progenies that resulted from the natural backcrossing of each of the three sorts of F₁ hybrids to F. chiloensis were as follows: The pentaploid F₁ hybrids (2n = 35) yielded mostly 9-ploid offspring from unreduced 5X gametes; a relatively high percentage of 14-ploid plants arising from doubled-unreduced 10 X gametes and a few 2N = ±46 aneuploids from reduced gametes. The hexaploid F₁ hybrid (2n = 42) on backcrossing yielded over 50% 10-ploid offspring with the rest 2n = ±50 aneuploids from reduced gametes. The 9-ploid F₁ hybrid (2n = 63) on backcrossing yielded mostly aneuploids normally distributed about a modal 2n = 59 chromosome class resulting from a 31 chromosome gamete, with a few 2n = 56 and 2n = 63 euploids. The 9-ploids may facilitate diploid Å octoploid introgression. Screening of the open-pollinated offspring from F. chiloensis revealed almost 2% 12-ploid (2n = 84) offspring from the union of the reduced and unreduced F. chiloensis gametes. The probable genomic constitution of the observed novel ploidy levels and those that theoretically may be generated from the known hybrids are presented. The origin of the existing polyploids from diploids through simple unreduction is postulated.     

In vitro induction and identification of autotetraploid of <Emphasis Type="Italic">Bletilla striata (Thunb.) Reichb.f</Emphasis>. by colchicine treatment

Polyploidy breeding has proved to be a valuable approach for acquiring the high yield superior varieties in medicinal plants. An effective protocol for obtaining Bletilla striata autotetraploid is in vitro induction of protocorms with colchicine. The protocorms of B.striata were soaked in different concentrations of colchicine solution [0.05, 0.1 and 0.2% (w/v)] for 12, 24, 36, 48 and 60 h, and the ploidy of the seedlings was identified by chromosome counting and flow cytometry analysis. The results showed that the optimal condition for induction of autotetraploid of B. striata protocorms was treated with 0.2% colchicine for 36 h with the induction rate reached as high as 26.7%. In addition, the morphological and anatomical characteristics were observed and compared between the diploid and tetraploid plants. And we found that the features of tetraploid plants were significantly different from diploid plants, such as tetraploid plants possessed thicker and deeper green leaves, larger stomata and more chloroplast number, which could be used as simple and efficient parameters for screening tetraploid. This study laid a foundation for breeding superior varieties of B. striata.     

Photosynthetic responses to soil water stress in summer in two Japanese urban landscape tree species (<Emphasis Type="Italic">Ginkgo biloba</Emphasis> and <Emphasis Type="Italic">Prunus yedoensis</Emphasis>): effects of pruning mulch and irrigation management

Key message

Stomatal regulation involves beneficial effects of pruning mulch and irrigation on leaf photosynthesis in Prunus yedoensis and Ginkgo biloba under moderate drought. G. biloba showed conservative water use under drought.

Abstract

Leaf photosynthesis is highly sensitive to soil water stress via stomatal and/or biochemical responses, which markedly suppress the growth of landscape trees. Effective irrigation management to maintain leaf photosynthesis and information on species-specific photosynthetic responses to soil water stress are essential for the sustainable management of landscape trees in Japan, in which summer drought often occurs. In order to investigate effective irrigation management, we used plants with moderate soil water stress as controls, and examined the effects of daily irrigation and pruning mulch on leaf photosynthesis in container-grown Ginkgo biloba and Prunus yedoensis, which are the first and second main tall roadside trees in Japan. Stomatal conductance was significantly increased by pruning mulch and daily irrigation, with similar increases in leaf photosynthesis being observed in P. yedoensis and G. biloba. In order to obtain information on species-specific photosynthetic responses to soil water stress, we compared the responses of leaf photosynthesis and leaf water status to reductions in soil water content (SWC) between the two species. G. biloba maintained a constant leaf water potential, leaf water content, maximum carboxylation rate, and electron transport rate with reductions in SWC, whereas reductions were observed in P. yedoensis. We concluded that pruning mulch and irrigation effectively offset the negative impact of moderate water stress on leaf photosynthesis in summer in P. yedoensis and G. biloba via stomatal regulation, and also that G. biloba maintained its photosynthetic biochemistry and leaf water status better than P. yedoensis under severe water stress.

     

Evolutionary History of the HAP2/GCS1 Gene and Sexual Reproduction in Metazoans

The HAP2/GCS1 gene first appeared in the common ancestor of plants, animals, and protists, and is required in the male gamete for fusion to the female gamete in the unicellular organisms Chlamydomonas and Plasmodium. We have identified a HAP2/GCS1 gene in the genome sequence of the sponge Amphimedon queenslandica. This finding provides a continuous evolutionary history of HAP2/GCS1 from unicellular organisms into the metazoan lineage. Divergent versions of the HAP2/GCS1 gene are also present in the genomes of some but not all arthropods. By examining the expression of the HAP2/GCS1 gene in the cnidarian Hydra, we have found the first evidence supporting the hypothesis that HAP2/GCS1 was used for male gamete fusion in the ancestor of extant metazoans and that it retains that function in modern cnidarians.     

Focus Issue on the Plant Physiology of Global Change: Production of Diploid Male Gametes in Arabidopsis by Cold-Induced Destabilization of Postmeiotic Radial Microtubule Arrays

Whole-genome duplication through the formation of diploid gametes is a major route for polyploidization, speciation, and diversification in plants. The prevalence of polyploids in adverse climates led us to hypothesize that abiotic stress conditions can induce or stimulate diploid gamete production. In this study, we show that short periods of cold stress induce the production of diploid and polyploid pollen in Arabidopsis (Arabidopsis thaliana). Using a combination of cytological and genetic analyses, we demonstrate that cold stress alters the formation of radial microtubule arrays at telophase II and consequently leads to defects in postmeiotic cytokinesis and cell wall formation. As a result, cold-stressed male meiosis generates triads, dyads, and monads that contain binuclear and polynuclear microspores. Fusion of nuclei in binuclear and polynuclear microspores occurs spontaneously before pollen mitosis I and eventually leads to the formation of diploid and polyploid pollen grains. Using segregation analyses, we also found that the majority of cold-induced dyads and triads are genetically equivalent to a second division restitution and produce diploid gametes that are highly homozygous. In a broader perspective, these findings offer insights into the fundamental mechanisms that regulate male gametogenesis in plants and demonstrate that their sensitivity to environmental stress has evolutionary significance and agronomic relevance in terms of polyploidization.The spontaneous formation of polyploid species through whole-genome duplication is a major force driving diversification and speciation in plant evolution (Wang et al., 2004). The redundant genomic material produced by polyploidization provides genotypic plasticity that facilitates adaptation and confers enhanced competitiveness compared with diploid progenitors (Adams and Wendel, 2005a, 2005b; Leitch and Leitch, 2008). Molecular analyses suggest that the genomes of most angiosperms (more than 90%) retain evidence of one or more ancient genome-wide duplication events (Cui et al., 2006). Moreover, recently, Wood et al. (2009) established that up to 15% of angiosperm and 31% of gymnosperm speciation events were accompanied by polyploidization. Polyploidization in plants is also commercially beneficial. Many important crop species including wheat (Triticum aestivum), potato (Solanum tuberosum), tobacco (Nicotiana tabacum), coffee (Coffea arabica), and numerous fruit varieties are polyploid (Bretagnolle and Thompson, 1995). Although several mechanisms can yield polyploids, it is thought that most polyploid plants are formed by the spontaneous production and fusion of diploid (2n) gametes (Bretagnolle and Thompson, 1995; Ramsey and Schemske, 1998). However, despite the evolutionary and agricultural significance of sexual polyploidization in plants (Ramanna and Jacobsen, 2003), the molecular mechanism underlying 2n gamete formation in natural populations is poorly understood.Several cytological defects lead to diploid gamete formation in both male and female reproductive lineages. In some species, premeiotic and postmeiotic genome doubling events are reported, but diploid gametes typically result from a defect in one of the two meiotic divisions, a phenomenon referred to as “restitution” (Bretagnolle and Thompson, 1995; Ramsey and Schemske, 1998). Meiotic restitution mechanisms are categorized into three classes: (1) omission of one of the meiotic cell divisions; (2) alterations in meiosis I (MI) or meiosis II (MII) spindle morphology; or (3) defects in meiotic cytokinesis (Ramanna and Jacobsen, 2003). Additionally, depending on the genetic makeup of the resulting 2n gametes, meiotic restitution mechanisms can be further subdivided into two classes: first division restitution (FDR) and second division restitution (SDR). In FDR, the sister chromatids disjoin and segregate to opposite poles, yielding 2n gametes that largely retain the heterozygosity of the parental plant. In SDR, sister chromatids do not disjoin in MII and segregate to the same pole, generating highly homozygous 2n gametes (Köhler et al., 2010).Several genes governing 2n gamete formation have been identified and characterized in potato, maize (Zea mays), and Arabidopsis (Arabidopsis thaliana; Consiglio et al., 2004; Brownfield and Köhler, 2011). Mutations in Arabidopsis DYAD/SWITCH1 and maize ARGONAUTE104 (AGO104) and AM1 induce a complete loss of MI and, consequently, convert the meiotic cell cycle into a mitotic one (Ravi et al., 2008; Pawlowski et al., 2009; Singh et al., 2011). Lesions in Arabidopsis OSD1/GIG1 and TAM/CYCA2;1, two proteins involved in progression of the meiotic cell cycle, cause a complete loss of MII, generating highly homozygous 2n gametes in both male and female meiosis (d’Erfurth et al., 2009, 2010). Spindle-based meiotic restitution mechanisms have been reported in both Arabidopsis jason and atps1 mutants and in the potato ps mutant, in which parallel, fused, and tripolar spindles in male MII lead to the formation of FDR 2n spores (Mok and Peloquin, 1975; d’Erfurth et al., 2008; De Storme and Geelen, 2011). Disruption of postmeiotic male cytokinesis, which is regulated by a mitogen-activated protein kinase (MAPK) kinase signaling pathway, also results in polyploid gametes. Mutations in TES/STUD/AtNACK2, MKK6/ANQ1, and MPK4, three main components of the cytokinetic MAPK signaling cascade, induce a complete loss of cytokinesis following male meiosis, generating fully restituted tetraploid pollen grains (Hulskamp et al., 1997; Spielman et al., 1997; Soyano et al., 2003; Zeng et al., 2011).Despite progress on understanding cytological mechanisms and genetic factors governing the formation of 2n gametes in natural populations, less is known about the environmental factors involved. There is evidence that 2n gamete production can be stimulated by both biotic and abiotic stresses, such as nutritional deprivation, wounding, disease, herbivory, and temperature stress (Ramsey and Schemske, 1998). In Lotus tenuis, temperature stresses, and in particular high temperatures, increase the level of parallel spindle-driven 2n gamete production (Negri and Lemmi, 1998). Similarly, in rose (Rosa spp.), short periods of high temperature (48 h at 30°C–36°C) can induce cytomixis and parallel and tripolar spindles at male metaphase II, generating dyads and triads at the end of male sporogenesis (Pécrix et al., 2011). Low-temperature environments can also stimulate 2n gamete formation. For example, Solanum phureja grown in cool field environments produces more restituted spores compared with lines grown under normal conditions (McHale, 1983). Similarly, in Datura spp. and Achillea borealis, unreduced pollen formation is higher at low temperatures (Ramsey and Schemske, 1998; Ramsey, 2007). Recently, Mason et al. (2011) demonstrated that cold stress significantly stimulates 2n pollen production in some interspecific Brassica spp. hybrids. Temperature-induced diploid gamete formation is not restricted to plants. Low temperatures have also been shown to stimulate the formation of 2n spores in some animal species, particularly among fish and amphibians (Bogart et al., 1989; Mable et al., 2011). Moreover, ecological population studies have demonstrated that polyploid plant and animal species occur more frequently at higher altitudes and at latitudes closer to the poles (Beaton and Hebert, 1988; Barata et al., 1996; Dufresne and Hebert, 1998), leading to the suggestion that cold climates stimulate the production of polyploid gametes.In this study, we demonstrate that short periods of cold stress induce a development-specific production of meiotically restituted spores in Arabidopsis, which thereby constitutes an ideal model system to identify potential cytological and molecular factors involved in stress-induced sexual polyploidization. Using a combination of cytological and genetic approaches, we reveal the cytological basis for cold-induced meiotic restitution and additionally demonstrate that restituted binuclear and polynuclear spores spontaneously develop into diploid and polyploid pollen grains. We also use pollen tetrad-based segregation analysis to monitor the genetic makeup of cold-induced 2n gametes and Arabidopsis mutants to examine the potential role of some candidate regulators (e.g. TAM/CYCA1;2 and MKK2) in the sensitivity of male meiosis to low-temperature stress.     

Sex-Specific Posttranslational Regulation of the Gamete Fusogen GCS1 in the Isogamous Volvocine Alga Gonium pectorale

Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to male) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (females) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor.     

DNA fingerprints of living fossil Ginkgo biloba by using ISSR and improved RAPD analysis

In this study, we have aimed to genetically characterize Ginkgo biloba. Nine G. biloba samples from different places of China were collected, and DNA was extracted from the leaves of these samples for inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analysis. ISSR analysis showed high genetic variation among the nine varieties of G. biloba; the polymorphism and similarity coefficients were 87% and 0.40–0.84, respectively. RAPD analysis also showed 93% polymorphism, and the similarity coefficients ranged from 0.44 to 0.87. Persistent genetic isolation that developed for millions of years might influence the genetic variability between the samples of G. biloba. This study generates a genetic map of G. biloba, and reports the highly variable intra-species genetic characteristics of this living fossil among different geographical locations of China. Our study also suggests that ISSR and the improved RAPD markers are useful molecular tools for the genetic characterization of plants.     

残存银杏群落的结构及种群更新特征

银杏是现存裸子植物中最古老的孑遗植物,也是银杏纲植物现存的唯一种,但其自然群落和种群生态学特征至今尚未明晰。对分布于贵州省和重庆市交界的大娄山区7个地点的残存银杏群落的物种组成、垂直结构、主要组成种的种群大小级结构等群落结构特征以及银杏种群更新特征进行了研究。林木层共记录到82个种,其中常绿针叶树3种,常绿阔叶树和落叶阔叶树分别为37种和42种;各群落中相对优势度均以落叶阔叶树为高,银杏在7个样方中为顶极优势种,其中在6个样方中为单优势种。银杏群落林木层在垂直结构上一般可分为3层,银杏为乔木层的主要成分。各个群落中银杏种群的大小级结构均为逆-J型或多峰型,更新连续,而其余种类多为单峰型。不同微地形单元上银杏具有不同的萌枝特性,沟谷生境中银杏的萌枝率显著高于下部边坡和崖锥,有萌个体率的差异是主要的贡献因子,而银杏的萌枝能力与主干的胸径无显著关联。残存银杏群落为典型的发育在不稳定立地上的落叶阔叶林地形顶级群落,银杏通过萌枝维系种群的长期存留和群落的相对稳定。群落中银杏、红豆杉(Taxus chinensis)、红椿(Toona ciliata)和川黄檗(Phellodendron chinense)等多种珍稀濒危植物共存,有必要从群落层次上加强对残存银杏群落的保护。     

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Biogeography and karyotypes of freshwater planarians (Platyhelminthes, Tricladida, Paludicola) in southern Brazil

In the Tricladida (Platyhelminthes), the incidence of different biotypes identified by several ploidy levels is very common. Planarians collected in the State of Rio Grande do Sul were identified using cytogenetics. Different species distributions were observed with respect to Rio Grande do Sul's geomorphology, which could have been caused by their different microhabitats. Girardia tigrina and G. anderlani consisted of diploid and triploid individuals, whereas G. schubarti showed diploids, triploids, and mixoploids; for all these species, individuals of different ploidies were sympatric. Only for diploid G. anderlani were B chromosomes observed. These B chromosomes seem to have an irregular segregational behavior during mitosis, and possibly also during meiosis. However the processes (e.g., selection, mutation) of maintaining 2n, 3n, and 2n/3n individuals within natural populations of G. schubarti remain to be clarified.