A review of new PCR-based genetic markers and their utility to weed ecology

Several recent molecular developments provide new genetic tools for addressing difficult problems in weed ecology. In this review, we describe some of the techniques and the DNA markers they generate [including amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs) or microsatellites, and intron-polymerase chain reaction (intron-PCR)], contrast their relative advantages and disadvantages, and discuss how they might be used in weed research. As these new markers generally reveal higher levels of variation than other techniques, they promise to improve our understanding of breeding systems, assist in determining the origin(s) of invaders, help to resolve taxonomic boundaries and relationships between closely related taxa and enable the identification of phenotypic linked markers. In addition, compared with other techniques, some of these markers may be more cost-effective, less technically demanding and more reliable. Regardless of the marker adopted, all genetic studies will benefit from careful consideration of experimental design and the formulation of testable hypotheses with practical outcomes.     

Breeding for resistance: breeding for Plum pox virus resistant apricots (Prunus armeniaca L.) in Spain

Plum pox virus , Dideron type (PPV-D), was first detected in Spain in 1984. Since then, it has spread among Japanese plum trees and apricot trees has been extremely rapid in the main producing areas. In Spain, breeding for resistance was the only efficient method for controlling the disease on apricot. Two breeding programmes are currently producing new hybrids resistant to the disease. The main problem encountered by both programmes is the difficult procedure needed for screening the trait that delays the programmes. Nevertheless, more than 8000 seedlings have been produced, two new varieties have been released and several advanced selections are under study. The procedure for screening sharka resistance has varied by techniques and cultivars used which has resulted in different hypotheses about inheritance of the trait. Additionally, several studies on mapping and molecular markers in progress could provide markers for molecular assisted selection that can increase the breeding efficiency.     

Genomics‐informed design of loop‐mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level

The objective of this study was to develop a rapid, sensitive detection assay for the quarantine pathogen Xanthomonas arboricola pv. pruni, causal agent of stone fruit bacterial spot, an economically important disease of Prunus spp. Unique targets were identified from X. arboricola pv. pruni genomes using a comparative genomics pipeline of other Xanthomonas species, subspecies and pathovars, and used to identify specific diagnostic markers. Loop‐mediated isothermal amplification (LAMP) was then applied to these markers to provide rapid, sensitive and specific detection. The method developed showed unrivalled specificity with the 79 tested strains and, in contrast to previously established techniques, distinguished between phylogenetically close subspecies such as X. arboricola pv. corylina. The sensitivity of this test is comparable to that of a previously reported TaqMan^? assay at 10³ CFU mL^?1, while the unrivalled speed of LAMP technology enables a positive result to be obtained in <15 min. The developed assay can be used with real‐time fluorescent detectors for quantitative results as well as with DNA‐staining dyes to function as a simplified strategy for on‐site pathogen detection.     

植物病原菌SSR标记开发与利用

SSR标记具有共显性、高多态性、位点专化性、稳定性好、操作技术简单等特点,是十分理想的分子标记。随着一些简单、经济和高效的SSR分离技术的发展,SSR标记已逐渐在植物病原菌中被开发和利用。本文综述了植物病原菌中SSR标记的主要开发策略及其应用进展。     

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches?? broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70?M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。     

玉米抗粗缩病遗传及分子标记研究

Maize rough dwarf disease caused by Rice black-streaked dwarf virus (RBSDV) is transmitted by planthopper in China. Identification and development of resistant hybrids are complicated because of the inconsistencies in viral disease pressure every year. Marker-assisted selection can provide means for main-taining virus resistance alleles even in the absence of disease. In this paper a F₂ segregation population was constructed to identity the molecular markers linked to the resistance gene using a cross between a resistant and a susceptible parents (Qi319×Ye107). Fifteen-day-old seedlings of F₂ population were exposed to small brown planthoppers carrying RBSDV for 3 days in specific inoculation chamber. The inoculated plants were transplanted to screenhouse after removing the insects completely. In plant maturity stage the disease resistance of all the individuals were visually assessed. The results showed that 17, 8, 11, 51 and 122 plants were scaled from 0-4 respectively, in which 0 means no symptoms and 4 represents highly susceptible. Chi-square test demonstrated that the segregation ratio of phenotype was 1∶15 (resistant: susceptible) or 1∶6∶9 (resistant∶moderate∶susceptible) in the F₂ population, indicating RBSDV resistance of maize was controlled by two recessive genes. The F₂ individuals DNA were extracted and 261 SSR (simple sequence repeat) primers derived from maize genome ten chromosomes were selected from maize GDB database to construct genetic linkage map. The linkage map consisted of 71 polymorphic SSR markers, spanning a genetic distance of 996.6 cM with an average interval of 14.0 cM between adjacent markers. The resistant and susceptible gene pools were set up for BSA (bulked segregant analysis) and 6 polymorphism markers were obtained with BSA-SSR method between the two pools. The F₂individuals were further analyzed with 6 polymorphism markers. Chi-square test showed that phi 051, umc1407 and umc1432, mapped on chromosome 7 and 10, exhibited segregation distortion significantly and very significantly in susceptible individuals. These three SSR markers were identified as potential markers linked to the resistant loci.     

小麦品种(系)抗白粉病基因推导及分子标记鉴定

利用基因推导法和分子标记对我国主要麦区的小麦品种(系)进行了抗白粉病基因的鉴定。结果表明,南30-10等15个品种(系)含有Pm8,新麦2号等9个品种(系)含有Pm4,中植4号等9个品种(系)含有Pm21,郑麦113含有Pm4b+5b,杨09-111和新紫1号含有Pm2+mld。研究发现,基因推导和分子标记相结合,可大大提高小麦品种(系)抗白粉病基因鉴定结果的准确性,鉴定结果可为抗病育种和品种布局及白粉病的防治提供依据。     

基于转录组的小豆SSR分子标记开发及其应用

为了开发小豆SSR(Simple Sequence Repeats)分子标记,2019—2020年,利用微卫星识别工具MISA和软件Primer 3搜索小豆资源QH1转录组测序得到的Unigene,获得3 045个SSR分子标记,通过对标记的重复基序特点分析,发现标记的重复基序以单核苷酸重复、二核苷酸重复和三核苷酸重复为主,分别占标记总数的41.2%、26.4%和25.6%。利用可以锚定到小豆基因组并能成功设计引物的标记,构建了包括1 505个SSR分子标记的物理图谱。从以上1 505个标记中,随机选取分布于小豆11条染色体上的132个SSR标记进行有效性验证,发现有效扩增的标记为118个,有效率为89.4%。通过多态性标记筛选,获得6个多态性标记,利用这6个标记对36份小豆品种进行聚类分析,发现供试小豆材料可以分为4个类群。说明开发的SSR分子标记能够分析小豆的遗传背景差异,为小豆种质资源鉴定、遗传多样性分析以及优良性状基因定位研究提供可用的分子标记。     

Single Cystosorus Isolate Production and Restriction Fragment Length Polymorphism Characterization of the Obligate Biotroph Spongospora subterranea f. sp. subterranea

ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment length polymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.     

Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses

Two molecular genetic screening techniques, RAPD (random amplified polymorphic DNAs) and ISSR (inter-simple sequence repeats), were applied to detect the level and pattern of genetic diversity of Monochoria vaginalis, a common weed of rice fields, in seven populations from southern China. Among these populations, 116 bands were amplified by 18 RAPD primers, of which 34 bands (29.31%) were polymorphic, and 14 ISSR primers produced 111 bands with 87 polymorphic bands (78.38%). Within each population, a relatively low level of genetic diversity was detected by both RAPD and ISSR analyses, with a mean genetic diversity (H) of 0.0348 and 0.0551 respectively. Analysis of molecular variance of the data from the RAPD and ISSR markers detected that the majority of total genetic variation existed among populations (73.50% and 76.70% respectively) and only minor genetic variation within populations (26.50% and 23.30% respectively). Cluster analysis divided the seven populations into two groups, indicating that the genetic relationships among populations have relatively low correlation with their geographical distribution (Mantel test; r = 0.45 and 0.48 respectively). Our results indicated that both RAPD and ISSR markers were effective and reliable for accurately assessing the degree of genetic variation of M. vaginalis. Comparing the two techniques, ISSR markers were more efficient than the RAPD assay. The Mantel test gave r = 0.16, suggesting no correlation between these two molecular markers.     

小麦抗条锈品种遗传多样性的SSR分析

［目的］ 利用SSR标记分析我国几大小麦产区主栽品种中抗锈品种的遗传多样性,为小麦抗条锈育种亲本材料的选择提供参考。［方法］ 以当前条锈菌优势小种接种成株期小麦,从几大小麦产区主栽品种中筛选出抗条锈品种。然后利用SSR标记对筛选出的抗锈品种的遗传多样性进行分析。［结果］ 27对SSR引物在上述抗锈品种中共检测到104个等位变异,平均为3.85个;引物的多态信息含量（PIC）在0.210~0.712之间,平均为0.455;抗锈品种间遗传相似系数平均为0.723,表明筛选出的抗锈品种遗传多样性较低,亲缘较近。［结论］ 聚类分析的结果将抗锈品种分为了4个类群,类群的分布与亲缘的远近和品种的地域有一定的相关性。     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

转基因昆虫在害虫防治上的应用前景

对转基因昆虫的发展历史,转基因昆虫的技术要点,特别是载体和标记基因进行了系统阐述,同时对转基因昆虫的应用方向和生物安全性及其管理做了简要介绍。     

Development of molecular markers and probes based on TEF-1α, β-tubulin and ITS gene sequences for quantitative detection of Fusarium oxysporum f. sp. ciceris by using real-time PCR

Fusarium wilt (Fusarium oxysporum f. sp. ciceris) causes significant yield losses in chickpea worldwide. Faster, reliable and more specific molecular detection techniques were developed for the detection of Fusarium oxysporum f. sp. ciceris (Foc). The sequences obtained from multiple alignments of target genes, namely, translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS), were used to design Foc-specific markers/probes. One set of TEF-1α-based molecular marker, namely, SPα-F and R, two sets of β-tubulin-based markers, namely, SPβ1-F and R, and SPβ2-F and R, and one set of ITS gene, namely, SPT-F and R, were developed for the detection and quantification of Foc from diverse samples. The specificity and sensitivity of the designed molecular markers were evaluated through conventional and real-time PCR assays which differentiated the Foc from closely related species of Fusarium and other plant pathogens. In conventional PCR, the minimum detection limits of the markers ranged from 12.5 pg to 100 pg for genomic DNA of Foc and 0.5 ng to 10 ng for infected plant samples. In real-time PCR assay, the minimum detection limits of the markers ranged from 0.001 pg to 0.25 pg for genomic DNA of Foc and from 0.04 pg to 1.5 pg for the infected plant samples. Thus, the markers designed in the present study were found to be specific for Foc and can be used consistently for the detection and identification of Foc isolates. The probes developed from the two sets of markers, namely, SPα and SPβ2, also showed specificity with Foc.     

Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.     

Validation of molecular markers for resistance among Pakistani chickpea germplasm to races of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis>

DNA markers in chickpea, targetting resistance genes for different races of Fusarium oxysporum f.sp. ciceris (Foc), have been identified in chickpea, but validation of these markers is essential for effective use in resistance breeding. In view of this, different simple sequence repeats (SSR) markers were analysed in Pakistani germplasm including induced mutants and some local lines. Most of the SSR markers showed good correlation with phenotypic evaluation of genotypes to different races of Foc and may be used effectively in resistance breeding, except those markers for race 3. Markers for race 3 showed deviations from phenotypic data and the reason might be that race 3 is actually Fusarium proliferatum as reported recently and resistance to this race might involve some other major resistance genes. Poor correlation of markers with foc-3 on LG2 in our study and a recent report of independent segregation of foc-2 and foc-3 in near isogenic lines suggested that linkage distances among different resistance genes need further investigation. Moreover three Pakistani mutant lines (97477, CM444/92 and CM368/93) depicted high levels of resistance to Foc races and can be deployed as a valuable source in resistance breeding programmes.     

Development of specific markers for identification of Indian isolates of Fusarium oxysporum f.sp. ricini

Fusarium oxysporum f. sp. ricini (F. o. ricini) is a ubiquitous soil borne pathogen which causes wilt disease on castor (Ricinus communis L). Rapid and reliable detection of the pathogen is essential for undertaking appropriate and timely disease management measures. Identification based on cultural, morphological characteristics and pathogenicity tests are time-consuming and laborious. Traditional methods are now being increasingly replaced by molecular detection techniques, which are much faster and more specific. In this study we have identified two RAPD markers of 1100?bp and 1350?bp in size which can be amplified by OPJ-14 and OPK-12 primers respectively for detection of F. o. ricini. These two fragments were fully sequenced and two pairs of SCAR primers (For-J14 Fwd/Rev and For-K12 Fwd/Rev) were designed. The specific primer pairs amplified a single band from all F. o. ricini isolates and there was no amplification from another thirteen Fusarium species / subspecies tested. These results clearly demonstrate that the designed SCAR primer pairs can be used consistently to detect F. o. ricini isolates, isolated from the diseased samples or soil samples. To our knowledge this is the first report on generation of SCAR markers for identification of Indian F. o. ricini isolates.     

丽草蛉滞育预蛹的重要生化物质变化分析

丽草蛉是多种农林害虫的重要捕食性天敌昆虫,能以预蛹进行兼性滞育越冬,该属性对延长丽草蛉的产品货架期、增加产品储备量及促进产品运输具有重要意义。本研究分别测定了丽草蛉非滞育预蛹（1日龄）和滞育预蛹（1日龄、7日龄）体内的总蛋白质、脂类、糖类及醇类等主要生化物质的含量,以及脂肪酶、海藻糖酶、抗氧化酶（超氧化物歧化酶、过氧化物酶、过氧化氢酶）等关键酶的活性变化,比较了非滞育和滞育丽草蛉预蛹体内重要生化物质的差异。结果表明：滞育和非滞育丽草蛉的生理生化特征显著不同,滞育预蛹显著积累蛋白质、脂类、甘油三酯、糖原等能源物质以及甘油、海藻糖等低温保护物质,并且滞育个体的抗氧化防御能力显著增强,这些生理生化变化有助于提高丽草蛉的抗逆性,满足滞育维持期及滞育解除后恢复发育的能量需求,保证滞育个体的发育和存活。结果为解析丽草蛉滞育的生理机制奠定了基础。     

Resource assessment and clutch size in the bean weevil,Acanthoscelides obtectus

Bean weevil (Acanthoscelides obtectus (Say)) females were found to use seeds (discrete resource patches) differentially when different sizes were offered in multiple-choice tests. Females, either as a group or as individuals, laid significantly (two to six times) more eggs on large seeds than on those of five times smaller mass. In contrast, seed shape (flattened or spherical) did not contribute to clutch-size adjustment. Thus, A obtectus females seem to measure only relative seed size when a comparison is possible. Nevertheless, females overload seeds with eggs and this can result in larval competition, so that, whereas resource size assessment and a robust egg-load adjustment indicate a trade-off between resource use and female fitness, it does not seem to provide much benefit for the progeny in stored dry beans. Several features, eg the use of oviposition markers and its consequences, may counterbalance the possible negative effects. It is assumed that, due to life cycle differences, females in the bean field may realise different fitness gains in comparison with those living in stores.