Differential niche modification by males and females of a dioecious herb: extending the Jack Sprat effect

Males and females of dioecious plants often differ in morphological, physiological and life‐history traits, probably as a result of their different requirements for reproduction. We found that the growth and reproductive effort of individuals of the dioecious herb Mercurialis annua depended on whether males or females had been growing in the soil previously. This suggests that males and females of M. annua differentially modify the soil in which they are growing. Our study indicates that sexual dimorphism in dioecious plants can give rise to increased environmental heterogeneity as a consequence of sex‐specific niche modification.     

Comparison between four isoaccepting transfer ribonucleic acids and corresponding synthetases in male and female flowers of the dioecious species Mercurialis annua L

Isoaccepting species of leucyl-, valyl-, seryl- and tyrosyl-tRNAs from male and female organs of the dioecious plant Mercurialis annua were compared by co-chromatography on RPC5 columns. Each organ possessed a specific pattern of tRNAs and aminoacyl-tRNA synthetases. Female tRNAs were quantitatively and qualitatively more numerous and the corresponding synthetases were more active. Implications of these data on developmental control are discussed.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。     

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex‐reversed females (neomales). To provide molecular evidence for the proposed system, novel sex‐linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male‐specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female‐specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female‐specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female‐linked SCAR markers can be applied for rapid detection of prawn gender. These sex‐specific SCAR markers and sex‐associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphic DNA)分子标记技术,寻找谭清苏铁（Cycas tanqingii）中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465 (CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域（SCAR）分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings

Female plants of several dioecious angiosperms are commercially valued for production of fruits or seeds, viz. papaya, nutmeg, pistachio, kiwi fruit and jojoba. To make the cultivation profitable it is necessary to grow more female than male plants. To discriminate between male and female plants, sex-specific molecular markers have been identified in a few dioecious species such as Silene and pistachio. However, accurate and convenient sex diagnostic methods for early sexing of seedlings are not available to date. For the first time, we report here a PCR-based Seedling Sex Diagnostic Assay (SSDA) specially designed for early sexing of papaya seedlings. We have developed a male-specific SCAR marker in papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. The potential of this SCAR marker is further exploited to develop a simplified and highly accurate sex diagnostic assay by (1) including an internal PCR control, (2) following a single-step DNA extraction procedure and (3) optimising the PCR conditions to simultaneously amplify male-specific and control bands from the crude leaf extract. This diagnostic approach would be of great commercial significance to papaya growers as well as to seed companies and plant nurseries for early identification of female seedlings of dioecious species. In principle, this experimental design could be easily applied to molecular analysis of any agriculturally important trait for which specific DNA probes could be identified and hence opens new avenues of research in the field of genetic diagnostics of plants.     

Isolation and localization by histoimmunology of isoperoxidases specific for male flowers of the dioecious species Mercurialis annua L.

Specific anodic isoperoxidases of male flowers of the dioecious plant Mercurialis annua were extracted, partially purified, and injected into rabbits. The serum against these specific peroxidases was used after absorption to localize these male isoenzymes during flower development by means of histoimmunology. (Indirect immunoperoxidase method). The peroxidases were characteristic of microsporogenesis and tapetum differentiation. Their synthesis started at an early stage of male organogenesis. They were not observed in other sites of peroxidase activity of male flowers (tunica, endothecium, filament, vascular anatomy) or in female flowers (embryo sac or nucellus). I propose that these isoenzymes constitute an early and specific marker of male organogenesis in higher plants.     

Identification of two markers linked to the sex locus in dioecious <Emphasis Type="Italic">Asparagus officinalis</Emphasis> plants

One hundred decamer primers of random-amplified polymorphic DNA were tested on dioecious Asparagus officinalis plants to identify sex-linked molecular markers. One primer (S368) produced two markers (S368-928 and S368-1178) in female plants. These two DNA markers were identified in 30 male and female plants, respectively, and a S368-928 marker was proved to be linked to the female sex locus. The female-linked S368-928 marker was sequenced and specific primers were synthesized to generate a 928 bp marker of sequence characterized amplified regions (SCAR) in female plants, SCAR₉₂₈. SCAR₉₂₈ could be used to correctly screen homozygous mm female plants of A. officinalis. However, results of Southern blot analysis suggest that the hybridization pattern of S368-928 was presented in both sex plants. This text was submitted by the authors in English.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

Le Infezioni Con “Stubborn” Ottenute per Trauma E Il Grado di Recettività dei Tipi di Grano

Abstract

Dioecy ensures cross pollination but pollen must travel from male to female plants, sometimes covering long distances. The present study concerns the reproductive strategy of two Italian dioecious plants: Mercurialis annua L. and Bryonia dioica L. The former is anemophilous and the latter is entomophilous; the latter has nectar and pollen as rewards. The distribution of pollen grains on the stigma is very different in the two species, reflecting the different types of pollination. In both species there is a number-dependent mechanism of pollen germination on the stigma. This mechanism could enhance male gametophytic competition.     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.     

菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Pollination Ecophysiology of Mercurialis annua L. (Euphorbiaceae), an Anemophilous Species Flowering all Year Round

Mercurialis annua L. is a dioecious anemophilous species thatflowers all year round in central and southern Italy. The flowersof both sexes are dimorphic: the female flower has a vestigialcalyx; the male flower consists only of a calyx that opens atanthesis. The anthers always dehisce after anthesis. The anthesisof male flowers seems to be temperature dependent, whereas antherdehiscence is related to relative humidity. The pollen grainsvary in volume according to the season: they are smaller whenrelative humidity is low and vice versa. They always decreasein volume after anther dehiscence and have the capacity to varyin volume and reach equilibrium with a changing environment.Viability is high, but may drop suddenly during heavy rain orhail that damage the exposed male flowers. The number of pollengrains per stigma varies from 0 to 300. The data is discussedin relation to the type of pollination and environmental characteristics.Copyright1994, 1999 Academic Press Mercurialis annua, dioecism, anthesis, anther dehiscence, pollen volume, pollen viability, anemophilous pollination, pollination ecology     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.