Evaluation of six commonly used reference genes for gene expression studies in herbicide‐resistant Avena fatua biotypes

Avena fatua of the family Poaceae is one of the most common and economically damaging grass weeds. Resistance to herbicides that inhibit acetyl‐coenzyme A carboxylase and acetolactate synthase activities has recently been detected in A. fatua. The resistance may be due to mutations in the herbicide targets and/or enhanced herbicide metabolism resulting from changes in gene expression, including in genes involved in detoxifying herbicide active ingredients. To analyse gene expression, stable housekeeping genes must be experimentally determined and used for data normalisation. In this study, A. fatua plants were treated with different herbicide types and plant materials were harvested at three time points following treatment. Six candidate reference genes (18S rRNA, ACT, EF1α, GAPDH, TBP, and TUB) were selected, sequenced and analysed by RT‐qPCR. The resulting data were assessed using four algorithms from the RefFinder software to determine gene expression stability. We identified TBP and GAPDH as the most stably expressed A. fatua reference genes following herbicide treatment.     

You cannot fight fire with fire: a demographic model suggests alternative approaches to manage multiple herbicide‐resistant Avena fatua

Multiple herbicide‐resistant (MHR ) weed populations pose significant agronomic and economic threats and demand the development and implementation of ecologically based tactics for sustainable management. We investigated the influence of nitrogen fertiliser rate (56, 112, 168, or 224 kg N ha^?1) and spring wheat seeding density (67.3 kg ha^?1 or 101 kg ha^?1) on the demography of one herbicide susceptible and two MHR Avena fatua populations under two cropping systems (continuous cropping and crop‐fallow rotation). To represent a wide range of environmental conditions, data were obtained in field conditions over 3 years (2013–2015). A stochastic density‐dependent population dynamics model was constructed using the demographic data to project A. fatua populations. Elasticity analysis was used to identify demographic processes with negative impacts on population growth. In both cropping systems, MHR seedbank densities were negatively impacted by increasing nitrogen fertilisation rate and wheat density. Overall, MHR seedbank densities were larger in the wheat‐ fallow compared with the continuous wheat cropping system and seedbank densities stabilised near zero in the high nitrogen and high spring wheat seeding rate treatment. In both cropping systems, density‐dependent seed production was the most influential parameter impacting population growth rate. This study demonstrated that while the short‐term impact of weed management tactics can be investigated by field experiments, evaluation of long‐term consequences requires the use of population dynamics models. Demographic models, such as the one constructed here, will aid in selecting ecologically based weed management tactics, such as appropriate resource availability and modification to crop competitive ability to reduce the impact of MHR .     

Multifunctionalised benzoxazinones in the systems Oryza sativa–Echinochloa crus‐galli and Triticum aestivum–Avena fatua as natural‐product‐based herbicide leads

BACKGROUND: Fifteen novel derivatives of D‐DIBOA, including aromatic ring modifications and the addition of side chains in positions C‐2 and N‐4, had previously been synthesised and their phytotoxicity on standard target species (STS) evaluated. This strategy combined steric, electronic, solubility and lipophilicity requirements to achieve the maximum phytotoxic activity. An evaluation of the bioactivity of these compounds on the systems Oryza sativa–Echinochloa crus‐galli and Triticum aestivum–Avena fatua is reported here. RESULTS: All compounds showed inhibition profiles on the two species Echinochloa crus‐galli (L.) Beauv. and Avena fatua L. The most marked effects were caused by 6F‐4Pr‐D‐DIBOA, 6F‐4Val‐D‐DIBOA, 6Cl‐4Pr‐D‐DIBOA and 6Cl‐4Val‐D‐DIBOA. The IC₅₀ values for the systems Echinochloa crus‐galli–Oryza sativa and Avena fatua–Triticum aestivum for all compounds were compared. The compound that showed the greatest selectivity for the system Echinochloa crus‐galli–Oryza sativa was 8Cl‐4Pr‐D‐DIBOA, which was 15 times more selective than the commercial herbicide propanil (Cotanil‐35). With regard to the system Avena fatua–Triticum aestivum, the compounds that showed the highest selectivities were 8Cl‐4Val‐D‐DIBOA and 6F‐4Pr‐D‐DIBOA. The results obtained for 6F‐4Pr‐D‐DIBOA are of great interest because of the high phytotoxicity to Avena fatua (IC₅₀ = 6 µM , r² = 0.9616). CONCLUSION: The in vitro phytotoxicity profiles and selectivities shown by the compounds described here make them candidates for higher‐level studies. 8Cl‐4Pr‐D‐DIBOA for the system Echinochloa crus‐galli‐Oryza sativa and 6F‐4Pr‐D‐DIBOA for Avena fatua‐Triticum aestivum were the most interesting compounds. Copyright © 2010 Society of Chemical Industry     

Simple sequence repeat analysis of genetic diversity among Acetyl‐CoA carboxylase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola populations in Korea

Echinochloa species are amongst the most problematic weeds in rice fields of Korea. The steady reliance on the Acetyl‐CoA carboxylase (ACCase) and acetolactate synthase inhibiting herbicides for control of these weeds has led to resistance to these herbicides. The objective of this study was to assess the genetic diversity among populations of ACCase inhibitor‐resistant and ‐susceptible Echinochloa crus‐galli and E. oryzicola in Korea, to better understand their population structure and possible origins of resistance. Seven simple sequence repeat markers were applied to assess the genetic diversity between resistant and susceptible E. crus‐galli and E. oryzicola from 12 populations in Korea. Genetic diversity was slightly higher in the resistant group. The Unweighted Pair Group Method using Arithmetic algorithm (UPGMA) dendrogram generated two distinct clades. One clade consisted of Echinochloa spp. from three populations, i.e. Anmyeondo, Gimje 4 and Gongju, which are resistant and differentiated from the susceptible populations, and the other clade contained the rest of the populations. Structure modelling supported two clades of UPGMA clustering. Based on these data, we can infer that some resistant populations are greatly differentiated, whereas other resistant biotypes are still building up resistance in rice fields in Korea. Resistance traits will be fixed and continue to spread over time without proper control measures.     

Microsatellite variability of sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides (Cyperaceae) in Kinki,Japan

Schoenoplectus juncoides is one of the most harmful weeds found in East Asian paddy fields. Recent emergence of biotypes that are resistant to the herbicide sulfonylurea (SU) has made weed control difficult. To examine the effect of the evolution of this herbicide resistance on genetic diversity within local populations, we investigated microsatellite variability within and among paddy field populations of S. juncoides in Kinki, Japan. In vivo assay of acetolactate synthase activity and root elongation assay in the presence of SU revealed that of 21 populations, five were sulfonylurea‐susceptible (SU‐S) and eight were completely sulfonylurea‐resistant (SU‐R). The remaining eight populations were a mixture of SU‐S and SU‐R individuals. The average gene diversity for SU‐R populations (H_S = 0.168) was lower than those for SU‐S (H_S = 0.256) and mixed (H_S = 0.209) populations, but the difference was not significant. This indicates that positive selection for SU‐R phenotype did not cause a genome‐wide reduction in genetic diversity. Genetic differentiation among S. juncoides populations was higher than that observed for most weed species studied previously. Although populations in neighbouring paddy fields showed a high level of differentiation, Bayesian clustering analyses suggested that some level of gene flow occurs among them and that the genetic exchange or colonisation between neighbouring populations could contribute to the geographical expansion of the resistant allele.     

Non‐target site resistance to flucarbazone,imazamethabenz and pinoxaden is controlled by three linked genes in Avena fatua

Extensive herbicide usage has led to the evolution of resistant weed populations that cause substantial crop yield losses and increase production costs. The multiple herbicide‐resistant (MHR) Avena fatua populations utilised in this study are resistant to members of all selective herbicide families, across five modes of action, available for A. fatua control in US small grain production, and thus pose significant agronomic and economic threats. Resistance to acetolactate synthase and acetyl‐CoA carboxylase inhibitors is not conferred by known target site mutations, indicating that non‐target site resistance (NTSR) mechanisms are involved. Understanding the inheritance of NTS MHR is of upmost importance for continued agricultural productivity in the face of the rapid increase in resistant weed populations worldwide. As few studies have examined the inheritance of NTSR in autogamous weeds, we investigated the inheritance and genetic control of NTSR in the highly autogamous, allohexaploid species A. fatua. We found that NTSR in MHRA. fatua is controlled by three separate, closely‐linked nuclear genes for flucarbazone‐sodium, imazamethabenz‐methyl and pinoxaden. The single‐gene NTSR inheritance patterns reported here contrast with other examples in allogamous species and illustrate the diversity of evolutionary responses to strong selection.     

Seed dormancy and soil seedbank of the invasive weed Chenopodium hybridum in north‐western China

Seed dormancy and persistence in the soil seedbank play a key role in timing of germination and seedling emergence of weeds; thus, knowledge of these traits is required for effective weed management. We investigated seed dormancy and seed persistence on/in soil of Chenopodium hybridum, an annual invasive weed in north‐western China. Fresh seeds are physiologically dormant. Sulphuric acid scarification, mechanical scarification and cold stratification significantly increased germination percentages, whereas dry storage and treatments with plant growth regulators or nitrate had no effect. Dormancy was alleviated by piercing the seed coat but not the pericarp. Pre‐treatment of seeds collected in 2012 and 2013 with sulphuric acid for 30 min increased germination from 0% to 66% and 62% respectively. Effect of cold stratification on seed germination varied with soil moisture content (MC) and duration of treatment; seeds stratified in soil with 12% MC for 2 months germinated to 39%. Burial duration, burial depth and their interaction had significant effects on seed dormancy and seed viability. Dormancy in fresh seeds was released from October to February, and seeds re‐entered dormancy in April. Seed viability decreased with time for seeds on the soil surface and for those buried at a depth of 5 cm, and 39% and 10%, respectively, were viable after 22 months. Thus, C. hybridum can form at least a short‐lived persistent soil seedbank.     

Indirect evidence for sexual reproduction in Cercospora beticola populations from sugar beet

Cercospora beticola is the main causal agent of cercospora leaf spot on sugar beet and has a large negative impact on the yield and quality of sugar beet production worldwide. Previous studies have shown that both mating type idiomorphs of C. beticola are present in natural populations, suggesting that C. beticola is heterothallic and may be reproducing sexually. Cercospora beticola isolates are diverse in the morphology of their conidia, onset of disease symptoms and fungicide resistance. To find the source of this diversity and to determine if sexual reproduction occurs in this fungus, C. beticola populations were collected from Western Europe, Iran and New Zealand. The mating types of these isolates were determined and AFLP analyses were used to study the genetic diversity in these populations. The mating type ratios did not deviate significantly from a 1:1 ratio in most of the populations and AFLP analyses showed high levels of genetic variation within and between the populations, with 86·4% of the isolates having unique genotypes. All populations were in significant linkage disequilibrium but levels of disequilibrium were low, and loci from only one primer pair were in significant gametic equilibrium in populations from the Netherlands and Italy. From these results there is the possibility that C. beticola reproduces sexually. High levels of gene flow among the samples from Europe demonstrated a single panmictic European population. This study confirms C. beticola to be a genetically highly diverse species, supporting the assumption that some populations are reproducing sexually.     

台湾乳白蚁4个地理种群遗传分化的研究

采用线粒体COⅡ基因为分子标记对中国南部4个台湾乳白蚁地理种群的遗传分化进行了初步研究。结果显示,4个地理种群共有9个多态位点和8种单倍型;种群间遗传分化统计量F_st与地理距离无显著相关。探讨了台湾乳白蚁种群分化的影响因素和相应的防治对策。     

玉米螟赤眼蜂不同地理种群mtDNA基因序列分析及遗传分化研究

本研究通过对玉米螟赤眼蜂Trichogramma ostriniae不同地理种群的线粒体DNA COⅠ、COⅡ和Cytb基因进行序列分析,研究了我国玉米螟赤眼蜂不同地理种群的遗传分化程度.结果发现,玉米螟赤眼蜂群体内线粒体基因具有丰富的遗传多态性,且各地理种群之间已产生不同程度的遗传分化.中性检测结果表明,玉米螟赤眼蜂进化遵循中性模型,且在过去呈现种群扩张趋势.玉米螟赤眼蜂不同地理种群之间的遗传距离在0.002～0.020之间.种群系统聚类树分支结构和Mantel相关性分析结果表明,玉米螟赤眼蜂各地理种群的遗传距离与地理距离间不具有显著的相关性.     

基于mtCOI基因序列的我国云南省和缅甸、柬埔寨草地贪夜蛾种群遗传多样性分析

为制定有效的草地贪夜蛾防控措施,分别自缅甸、柬埔寨和我国云南省采集4、2和8个种群共542个草地贪夜蛾样品,基于mtCOI基因序列分析这14个种群的遗传多样性指数、遗传分化系数及基因流。结果表明,缅甸草地贪夜蛾种群的单倍型多样性指数和平均核苷酸差异数分别介于0.273～0.396和4.643～6.727之间,高于我国云南省的草地贪夜蛾种群,分别介于0.047～0.214和0.791～3.636之间;除ZT种群、LS种群和TO种群外,其它11个种群的Fu’s F均为显著正值,表明缅甸、柬埔寨和我国云南省的草地贪夜蛾种群未经历种群扩张事件;在14个种群中,LS种群与其它种群分化较明显,TK种群、MY种群、CP种群、MS种群和KY种群有效迁入个数和有效迁出个数之和较高,分别为699.41、682.50、855.76、684.56和701.31,推测这5个种群在草地贪夜蛾基因交流中起着类似"中转站"的作用。     

内蒙古亚洲小车蝗种群遗传多样性和遗传分化的ISSR分析

亚洲小车蝗Oedaleus asiaticus Bei-Bienko是我国北方草原和农牧交错区的主要害虫。为评价内蒙古地区亚洲小车蝗种群的遗传多样性和遗传分化,应用ISSR标记方法对内蒙古15个亚洲小车蝗种群遗传多样性及遗传分化进行了分析。结果表明,7条引物扩增出85条ISSR条带,均为多态性条带。多态性比例（P）、Nei''s遗传多样性指数（H）和香农多样性指数（I）分别为82.59%、0.2319和0.3421,表明亚洲小车蝗种群具有较高的遗传多样性。基因流（Nm）和基因分化系数（Gst）分别为1.2298和0.3352,表明亚洲小车蝗不同地理种群具有明显的遗传分化。遗传距离与地理距离呈极显著正相关关系。表明地理距离和地形差异可能是形成亚洲小车蝗种群遗传分化的主要原因。     

基于线粒体和初级共生菌基因序列的荻草谷网蚜6个地理种群的遗传多样性研究

荻草谷网蚜Sitobion miscanthi (Takahashi)(在中国长时间学名误用为麦长管蚜Sitobion avenae Fabricius)是我国小麦的主要害虫,为小麦蚜虫优势种。该蚜虫因繁殖速度快、发育历期短、适应能力强且能随着气流远距离迁飞等特性,对小麦安全生产构成严重威胁。目前关于蚜虫种群遗传结构的研究主要基于线粒体基因开展。然而,初级共生菌布赫纳氏菌Buchnera aphidicola几乎存在于所有蚜虫中,其在研究蚜虫种群遗传结构多样性方面的潜在作用报道很少。本研究基于前期收集的荻草谷网蚜6个不同地理种群(苏州、武汉、昆明、廊坊、泰安、银川),对其线粒体COⅠ基因与初级共生菌B.aphidicola的两个单拷贝基因gnd与trpA进行PCR扩增、测序和群体遗传结构分析。根据对3个遗传标记基因聚类分析,荻草谷网蚜6个不同地理种群分为3组,分别为银川种群、苏州种群和其他种群(COⅠ: FCT= 0.364 2,P <0.05;gnd:FCT= 0.403 3,P <0.05;trpA:FCT= 0.222 9,P <0.05),3组间存在显著的遗传差异。银川种群和泰安种群间有相同的稀有单倍型H8(trpA),苏州种群和武汉种群也有相同的稀有单倍型H2与H23 (COⅠ),3组之间仍存在基因交流。因此我们推测在4、5月份东南季风盛行的情况下,银川种群迁入了外来虫源。蚜虫线粒体基因和初级共生菌基因的遗传结构分析结果基本一致,表明共生菌基因在研究蚜虫种群遗传结构和多样性具有潜在应用意义。综上所述,基于线粒体和共生菌基因序列的荻草谷网蚜不同地理种群的遗传结构解析可为蚜虫的迁飞提供分子证据。     

陕西省小麦条锈菌群体遗传结构分析

 利用TP-M13-SSR自动荧光检测技术,对陕西省小麦条锈菌群体遗传结构进行了分析。研究结果显示,在物种水平上,陕西省小麦条锈菌群体Nei’s基因多样性指数（H）为0.18、Shannon 信息指数（I）为0.29,表明该省条锈菌群体遗传多样性较为丰富。同时,条锈菌群体遗传多样性在地区之间也存在明显的差异,在7个条锈菌群体中,宁强种群遗传多样性相对较高（H为0.18,I为0.28）。AMOVA分析显示,陕西省小麦条锈菌在地区间、种群间和群体内的遗传分化分别为：12.26%、10.88%和76.86%,表明陕西省小麦条锈菌群体存在一定的遗传分化,但遗传变异主要发生在群体内部。     

Restriction site‐associated DNA sequencing allows for the rapid identification of simple sequence repeat markers in Echinochloa crus‐galli

Echinochloa crus‐galli is a serious weed worldwide. Microsatellite markers (simple sequence repeats, SSRs) are important molecular markers that are used widely for studying genetic diversity in plants. However, a limited number of SSRs is available for E. crus‐galli. The restriction site‐associated DNA (RAD) sequencing approach was combined with Illumina DNA sequencing for the rapid and mass detection of SSRs in E. crus‐galli. The RAD tags were generated from the genomic DNA of E. crus‐galli and were sequenced in order to produce 6921.6 Mb of high‐quality sequences with 45.1% guanine–cytosine content. In total, 3081 putative SSRs were detected, of which 82.2% were dinucleotide motif‐repeats. AT was the most frequent motif, accounting for 35.0% of the SSRs. In order to test the validity of the SSRs that were developed here, eight SSRs that were selected from putative SSRs were used to study the genetic diversity and structures of 20 E. crus‐galli populations that had been collected from rice fields in eastern China. Ninety‐seven alleles were amplified from the eight microsatellite loci among the 20 E. crus‐galli populations. These populations showed low genetic diversity and were classified on the basis of their genetic structures into three distinct groups that corresponded to the three regions of population sampling. The SSRs that were identified in this study represent a valuable resource for studying the genetic diversity, population biology and evolution of E. crus‐galli.     

Segregation of non‐target‐site resistance to herbicides in multiple‐resistant Alopecurus myosuroides plants

Non‐target‐site resistance (NTSR) comprises a set of mechanisms conferring resistance to multiple modes of action. Investigation of the number of loci involved in NTSR will aid in the understanding of these resistance mechanisms. Therefore, six different multiple herbicide‐resistant Alopecurus myosuroides plants with different herbicide history were crossed in two generations with a susceptible wild type. Seeds from the backcrossing generation were studied for their segregation rate for resistance to five herbicides with four different modes of action (HRAC groups C₂, A, B and K₃). Taking into account that NTSR is a set of quantitative traits, the numbers of loci controlling NTSR were estimated using a normal mixture model fitted by the NLMIXED procedure of SAS. Each herbicide was controlled by a different number of loci comparing the six plants. In most of the cases, chlorotoluron resistance was controlled by one locus, whereas resistance to fenoxaprop‐P‐ethyl needed one or two loci. Resistance to pinoxaden was in all plants conferred by two loci. Cross‐resistance of fenoxaprop‐P‐ethyl and pinoxaden was found in all backcrossings, indicating that at least one of the two loci is responsible for both resistances. Resistance to mesosulfuron + iodosulfuron was conferred by a minimum of two loci. Results indicated that a minimum of five different loci can be involved in a multiple NTSR plant. Furthermore, the plant‐specific accumulation of NTSR loci was demonstrated. Such behaviour should be taken into account when evaluating the development and further spread of herbicide resistance.     

福建省烟粉虱不同地理种群遗传结构特征

为明确福建省烟粉虱种群遗传结构特征,基于福建省烟粉虱不同地理种群中40个代表性的线粒体COI基因序列,分析了种群遗传多样性、遗传分化及分子变异情况,并构建了单倍型系统发育树与网络图。结果显示:在590 bp长度的mt COI基因序列中有效位点558个,其中187个核苷酸位点存在变异;序列核苷酸中A、T、C、G含量分别为42.32%、24.36%、20.25%、13.06%,其中A+T的含量为66.68%,表现出明显的A+T偏向性;共检测出11个单倍型,其中Hap3、4、7、9、11为特殊单倍型;种群多样性指数为0.838,核苷酸多样性指数为0.093,表明遗传多样性水平较高;AMOVA分析表明种群遗传变异主要来自种群内,总种群遗传分化系数仅为0.027,种群遗传分化较低。表明福建烟粉虱种群基因交流未受地理距离明显影响,种群遗传分化不显著。