Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

High-resolution mapping of a new brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in rice (Oryza sativa L.)

Brown planthopper (BPH) is a destructive insect pest of rice in Asia. Identification and the incorporation of new BPH resistance genes into modern rice cultivars are important breeding strategies to control the damage caused by new biotypes of BPH. In this study, a major resistance gene, Bph18(t), has been identified in an introgression line (IR65482-7-216-1-2) that has inherited the gene from the wild species Oryza australiensis. Genetic analysis revealed the dominant nature of the Bph18(t) gene and identified it as non-allelic to another gene, Bph10 that was earlier introgressed from O. australiensis. After linkage analysis using MapMaker followed by single-locus ANOVA on quantitatively expressed resistance levels of the progenies from an F₂ mapping population identified with marker allele types, the Bph18(t) gene was initially located on the subterminal region of the long arm of chromosome 12 flanked by the SSR marker RM463 and the STS marker S15552. The corresponding physical region was identified in the Nipponbare genome pseudomolecule 3 through electronic chromosome landing (e-landing), in which 15 BAC clones covered 1.612 Mb. Eleven DNA markers tagging the BAC clones were used to construct a high-resolution genetic map of the target region. The Bph18(t) locus was further localized within a 0.843-Mb physical interval that includes three BAC clones between the markers R10289S and RM6869 by means of single-locus ANOVA of resistance levels of mapping population and marker-gene association analysis on 86 susceptible F₂ progenies based on six time-point phenotyping. Using gene annotation information of TIGR, a putative resistance gene was identified in the BAC clone OSJNBa0028L05 and the sequence information was used to generate STS marker 7312.T4A. The marker allele of 1,078 bp completely co-segregated with the BPH resistance phenotype. STS marker 7312.T4A was validated using BC₂F₂ progenies derived from two temperate japonica backgrounds. Some 97 resistant BC₂F₂ individuals out of 433 screened completely co-segregated with the resistance-specific marker allele (1,078 bp) in either homozygous or heterozygous state. This further confirmed a major gene-controlled resistance to the BPH biotype of Korea. Identification of Bph18(t) enlarges the BPH resistance gene pool to help develop improved rice cultivars, and the PCR marker (7312.T4A) for the Bph18(t) gene should be readily applicable for marker-assisted selection (MAS). K. K. Jena and J. U. Jeung contributed equally to this study.     

Pesticide-induced susceptibility of rice to brown planthopper Nilaparvata lugens

The effects of rice plants treated with various pesticides (jingganmycin, bisultap and methamidophos) on feeding, survival rates and population growth of Nilaparvata lugens Stål (Homoptera: Delphacidae), susceptibility of the treated rice plants and amounts of free amino acids and sucrose were studied. Experiments indicated that the effects of the tested pesticides were dependent on nymphal age, pesticide and their dose and time after application. Jingganmycin at 75 g a.i. ha^–1 significantly increased the N. lugens population. Both jingganmycin and bisultap increased the survival rate of N. lugens nymphs. The feeding rate of the insects was also affected by the pesticide application, but the effect varied between nymphal age and time after application and lasted no longer than 15 d. Results clearly indicated that pesticide application increased the susceptibility of rice plants to N. lugens. Although the free amino acids in rice plants did not change with the pesticide treatments, the concentration of sucrose significantly decreased 5 d after application and the C/N ratio significantly decreased in jingganmycin treated plants 5 d and 10 d after application.     

RFLP-facilitated investigation of the quantitative resistance of rice to brown planthopper ( Nilaparvata lugens)

Quantitative trait loci (QTLs), conferring quantitative resistance to rice brown planthopper (BPH), were investigated using 160 F₁₁ recombinant inbred lines (RILs) from the Lemont/Teqing cross, a complete RFLP map, and replicated phenotyping of seedbox inoculation. The paternal indica parent, Teqing, was more-resistant to BPH than the maternal japonica parent, Lemont. The RILs showed transgressive segregation for resistance to BPH. Seven main-effect QTLs and many epistatic QTL pairs were identified and mapped on the 12 rice chromosomes. Collectively, the main-effect and epistatic QTLs accounted for over 70% of the total variation in damage scores. Teqing has the resistance allele at four main-effect QTLs, and the Lemont allele resulted in resistance at the other three. Of the main-effect QTLs identified, QBphr5b was mapped to the vicinity of gl1, a major gene controlling leaf and stem pubescence. The Teqing allele controlling leaf and stem pubescence was associated with resistance, while the Lemont allele for glabrous stem and leaves was associated with susceptibility, indicating that this gene may have contributed to resistance through antixenosis. Similar to the reported BPH resistance genes, the other six detected main-effect QTLs were all mapped to regions where major disease resistance genes locate, suggesting they might have contributed either to antibiosis or tolerance. Our results indicated that marker-aided pyramiding of major resistance genes and QTLs should provide effective and stable control over this devastating pest. Received: 10 December 2000 / Accepted: 7 May 2001     

水稻麦黄酮对褐飞虱的抗性潜力

使用石油醚、乙酸乙酯、无水乙醇和水依次对抗性水稻品种IR36乙醇提取物进行萃取,并测试了4种萃取物对褐飞虱的活性。结果发现乙酸乙酯萃取物处理后3d的活性最强,褐飞虱1～2龄若虫和3～4龄若虫的死亡率分别是26．0％和48．0％。乙酸乙酯萃取物经柱层析分离得到麦黄酮。将麦黄酮定量加入人工饲料中饲养褐飞虱3龄若虫15d。结果表明,褐飞虱3、4龄若虫的排蜜露量随着麦黄酮在饲料中浓度（50～500μg／ml）的增加而减少,褐飞虱3、4龄若虫的死亡率却随着麦黄酮在饲料中浓度的增加而显著提高。当饲料中麦黄酮的浓度为500μg／ml时,褐飞虱3、4龄若虫的死亡率分别为58．21％和31．75％。麦黄酮50～500μg／ml浓度处理,褐飞虱3龄和4龄若虫的发育历期和相对生长量与对照之间没有明显的差异,表明麦黄酮有拒食作用。用500μg／ml的麦黄酮溶液涂抹到对褐飞虱敏感的水稻品种TN1植株上对褐飞虱雌成虫有明显的拒食作用和忌避产卵作用。本项研究结果表明水稻源的麦黄酮在水稻对褐飞虱的抗性中有重要的作用。     

RAPD markers linked to brown planthopper Nilaparvatha lugens resistance locus in rice

Brown plant hopper, a major pest in rice causes "hopper burn" in the field. The resistance gene for brown planthopper was mapped by using 20 recombinant inbred lines (RIL's) derived from a cross between resistant line Oryza. officinalis derivative (IR 54742-2-21-12-17-6) and a susceptible rice cultivar ASD 16 using bulked segregant analysis. On an average of 4 loci were amplified and two RAPD primers amplified loci that co-segregated with resistance/susceptibility. The segregating RAPD loci were mapped using Mapmaker programme into 13 groups. The expected and the 95% confidence level were found to be 15.2 and 47.7 cM respectively, confirming the location of the brown planthopper resistant gene on the region of chromosome 4. These RAPD markers will accelerate breeding programme for brown planthopper resistance.     

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F₂ population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F₃ families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.     

褐飞虱抗药性研究现状

褐飞虱Nilaparvata lugens(Stal)对杀虫剂产生抗药性是其近年来暴发频繁的重要原因。文章综述国内外关于褐飞虱抗药性的研究成果,包括褐飞虱抗性测定方法、抗药性的发展、交互抗性、抗性遗传、抗性机理及抗性治理等。田间褐飞虱种群对新烟碱类药剂产生不同程度的抗药性,其中对吡虫啉产生高水平到极高水平抗性,对氯噻啉和噻虫嗪分别产生中等水平和低水平的抗药性,对呋虫胺和烯啶虫胺仍然处于敏感性阶段。此外,褐飞虱种群对噻嗪酮(昆虫生长调节剂)产生低水平到中等水平抗性。长期大面积使用化学药剂是褐飞虱产生抗药性的重要原因。因此,必须加强褐飞虱的抗性治理,以延缓其抗药性进一步发展。     

水稻品种对褐飞虱的田间抗性及其生态学机制

评价了7个水稻品种(浙粳22、宜香845、中组14、秀水123、二优倍九、感虫对照品种TN1和抗虫对照品种IR36)在田间成株期对褐飞虱Nilaparvata lugens(Stl)的抗性表现,并初步分析了它们的抗性机制。结果表明,在田间网室人工诱发条件下,浙粳22、宜香845、秀水123和中组14等4个品种的抗性水平与抗虫品种IR36相同,均小于3级,表现为抗虫,而二优培九则与感虫品种TN1相同,表现为感虫。在自然发生条件下的水稻分蘖期至乳熟期,宜香845和中组14上褐飞虱数量仅为TN1上的7.84%～24.44%,秀水123和浙粳22则为TN1上的30.19%～60.27%。在4个抗性品种上褐飞虱的产卵量和卵孵化率明显减少,而若虫存活率仅为TN1上的20%～30%。与IR36相同,4个抗性品种上褐飞虱的若虫存活率和种群增长倍数均显著低于TN1上,说明他们对褐飞虱的抗性主要表现为抗生性。     

The multicopper oxidase gene family in the brown planthopper,Nilaparvata lugens

The multicopper oxidase (MCO) family of enzymes includes laccases, ascorbate oxidases, bilirubin oxidases and a subgroup of metal oxidases. On the basis of a bioinformatics investigation, we identified 7 genes encoding putative multicopper oxidase proteins in the genome of the brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae). MCO1 and MCO2 are conserved, while others diverse in insects. Analysis of developmental and tissue-specific expression patterns revealed the following: NlMCO2 was mainly expressed in the integument, and its expression peaked periodically during molting; NlMCO3 was an ovary-specific MCO gene with a high expression level only at the adult stage; NlMCO4 was a salivary gland-specific MCO gene that was expressed at all developmental stages; NlMCO5 only had short-term expression in the middle of the fourth instar stage and was expressed mainly in the gut; NlMCO6 had a developmental expression pattern similar to that of NlMCO2 and was expressed in most N. lugens tissues; and NlMCO1 was expressed in most N. lugens tissues except for the testis, whereas NlMCO7 was mainly expressed in the gut and the Malpighian tube. BPHs injected with double-stranded RNA (dsRNA) targeting NlMCO2 failed to pigment and sclerotize, were colorless and soft-bodied and subsequently died in a short time. Lethal phenotypes were also observed in insects challenged by dsRNA targeting NlMCO6. However, no observable morphological or internal structural abnormality was obtained in the insects treated with dsRNA for NlMCO1, NlMCO3, NlMCO4, NlMCO5 or NlMCO7.     

十六个水稻品种(系)对褐飞虱的抗虫性评价

本文通过苗期抗性鉴定、田间抗虫性鉴定、稻田节肢动物功能团优势度比较及产量测定的方法来评价16个水稻品种(系)对褐飞虱Nilaparvata lugens(Stl)的抗性。结果表明,有12个品种(系)对褐飞虱表现为抗级,2个品系表现为中抗。这14个抗虫-中抗的品种(系)在成株期也表现一定的抗虫性,且与对照TN1之间差异显著。本试验条件下,广占63-3S/华恢15、广两优106、Y58S/华15、广两优476、广占63-4S/华恢15这5个品种(系)不仅抗虫性好,而且产量也较高,分别比TN1和主栽品种扬两优6号高63.49%～66.57%和3.47%～11.57%,具有广泛的开发应用前景。     

诱导水稻抗褐飞虱的化学激发子筛选

【目的】化学激发子具有毒性低、不易产生抗药性等特点,因此开发基于化学激发子的害虫防控技术,能够降低农药的使用量,促进农业生产的可持续发展。本研究旨在筛选能够诱导水稻产生对褐飞虱Nilaparvata lugens抗性的化学激发子。【方法】将茉莉酸(JA)、茉莉酸甲酯(MeJA)、水杨酸(SA)、水杨酸甲酯(MeSA)、油菜素内酯(BR)、苯甲酸苄酯(BB)、独脚金内酯(SLs)、萘乙酸(NAA)、吲哚丁酸(IBA)、草酸钠(SO)、硅酸钾(PS)和叶枯唑(Bis)12种化合物以根吸或叶鞘涂抹处理水稻,测定这些化合物处理水稻后褐飞虱的卵孵化率和24 h总产卵量。【结果】12种候选化合物中,仅茉莉酸甲酯、油菜素内酯和苯甲酸苄酯对褐飞虱卵孵化率和24 h总产卵量有显著影响。5 mg/L MeJA根吸处理水稻使褐飞虱的卵孵化率显著降低(达58.8%,降低了20.1%),而0.5 mg MeJA叶鞘涂抹处理水稻同时降低了褐飞虱的卵孵化率(达53.3%,降低了35.4%)和24 h总产卵量(达203.5粒/株,降低了15.6%),且其涂抹的浓度越高,褐飞虱的卵孵化率和24 h总产卵量越低。5 mg/L BR根吸处理水稻可以显著降低褐飞虱的卵孵化率(达59.5%,降低了22.1%),但是不影响褐飞虱的24 h总产卵量;褐飞虱的卵孵化率随BR处理浓度增高而降低,其浓度为20 mg/L时,褐飞虱的卵孵化率下降了41.8%。5 mg/L BB根吸处理水稻可以显著降低褐飞虱的24 h总产卵量(达100.3粒/株,降低了26.2%)。体外试验结果表明,MeJA和BR处理对褐飞虱卵孵化率无明显影响,说明两者对褐飞虱卵无直接毒害作用。【结论】化合物茉莉酸甲酯、油菜素内酯和苯甲酸苄酯可以提高水稻对褐飞虱的抗性,其中茉莉酸甲酯和油菜素内酯具有化学激发子的作用。     

Herbivory-induced glucose transporter gene expression in the brown planthopper,Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.     

虫害诱导的水稻挥发物对褐飞虱的驱避作用

水稻叶片在遭受害虫的危害时,释放一些特异性的挥发性物质。这些挥发性信息物质在调节植物、植食性昆虫及其天敌的相互关系中有重要作用。利用“Y”型嗅觉仪研究了褐飞虱对虫害诱导的11种水稻挥发物（浓度为2 Μl 化合物溶解在100 Μl丙酮溶液中）的行为反应。结果表明,(E)-2-己烯醛、(E)-2-己烯-1-醇、-2-庚醇和水杨酸甲酯等4种化合物对褐飞虱成虫有显著的驱避作用,而(Z)-3-己烯-1-醇、-2-庚酮、柠檬烯、罗勒烯、芳樟醇、-β-子丁香烯和橙花叔醇等7种物质对褐飞虱成虫的选择行为无显著影响。浓度梯度的实验表明,浓度(不同体积的待测化合物用100 Μl丙酮稀释)较低的(E)-2-己烯醛（0.5 Μl、1 Μl）和芳樟醇（05 Μl、1 Μl、5 Μl）对褐飞虱成虫的选择行为无显著影响,而较高浓度的(E)-2-己烯醛（5 Μl、10 Μl）和芳樟醇（10 Μl）对褐飞虱成虫均有明显的驱避作用。     

Meiosis in males of the brown planthopper, Nilaparvata lugens

Meiosis in spermatocytes was studied among the three biotypes of the brown planthopper, Nilaparvata lugens (Stål). The chromosome number, morphology, and behavior of the biotypes were similar. The autosomes divided normally, reductional for the first division and equational for the second. The X chromosome had an unstable segregation behavior; it may segregate equationally or reductionally during the first division depending on its chromatid's orientation. Metaphase I plates contained 14 autosomal bivalents and an X chromosome (14_II + X). The co-oriented sister chromatids of the X chromosome moved together to either one of the poles during anaphase I, while auto-oriented sister chromatids segregated to opposite poles. Because of the anomalous segregation behaviour of the X chromosome, metaphase II plates may both contain 14_I + X chromatid, or one with only 14_I and the other with 14_I + X chromosome. Male N. lugens has a diploid chromosome number of 29.

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Zusammenfassung Die Meiose in Spermatozyten wurde in drei Biotypen von Nilaparvata lugens (Stål) studiert. Die Anzahl der Chromosomen, die Morphologie und das Verhalten der Biotypen waren ähnlich. Die Autosomen teilten in einer normalen Weise, reduzierend für die erste und äquatorial für die zweite Teilung. Das X-Chromosom hatte ein unstabiles Teilungsverhalten; es kann sich äquatorial oder aber auch reduktional trennen, je nachdem wie sich sein Chromatid orientiert hat. Die Ebenen der Metaphase I enthalten 14 autosome Bivalente und ein X-Chromosom (14_II + X). Die co-orientierten Schwester-chromatiden des X-Chromosoms bewegen sich während der Anaphase I zusammen zu einem der beiden Pole, während auto-orientierte Schwester-chromatiden sich nach entgegengesetzten Polen trennen. Wegen des ungewöhnlichen Teilungsverhaltens des X-Chromosoms, können beide Metaphase II Ebenen 14_I und X Chromatid enthalten, oder aber eine Ebene enthält 14_I und die andere 14_I + X.

     

惠州地区褐飞虱对几种药剂的抗药性监测

2009年采用稻茎浸渍法测定广东省惠州地区褐飞虱Nilaparvata lugens(Stl)种群对吡虫啉、噻嗪酮、异丙威、丁烯氟虫腈、烯定虫胺和毒死蜱等杀虫剂的敏感性,测定结果表明:当地褐飞虱种群对吡虫啉产生了极高水平抗性(抗性倍数为422.2倍),对噻嗪酮、异丙威产生了中等水平抗性(抗性倍数分别为11.0和14.0倍),对丁烯氟虫腈仍处于敏感性降低(抗性倍数为3.7倍),对烯定虫胺和毒死蜱敏感(抗性倍数<3倍)。基于褐飞虱对这6种药剂抗性的明显差异,对田间治理褐飞虱合理使用药剂进行了讨论。     

Detection and analysis of QTLs for resistance to the brown planthopper, Nilaparvata lugens, in a doubled-haploid rice population

 We used a mapping population of 131 doubled-haploid lines, produced from a cross between an improved indica rice variety (IR64) and a traditional japonica variety (Azucena), to detect quantitative trait loci (QTLs) for resistance to the brown planthopper (BPH), Nilaparvata lugens. We evaluated the parents and mapping population with six tests that measure varying combinations of the three basic mechanisms of insect host plant resistance, i.e., antixenosis, antibiosis, and tolerance. To factor-out the effect of the major resistance gene Bph1 from IR64, the screening was done with two BPH populations from Luzon Island, The Philippines, that are almost completely adapted to this gene. A total of seven QTLs associated with resistance were identified, located on 6 of the 12 rice chromosomes. Individual QTLs accounted for between 5.1 and 16.6% of the phenotypic variance. Two QTLs were predominantly associated with a single resistance mechanism: one with antixenosis and one with tolerance. Most of the QTLs were derived from IR64, which has been shown to have a relatively durable level of moderate resistance under field conditions. The results of this study should be useful in transferring this resistance to additional rice varieties. Received: 10 May 1998 / Accepted: 4 June 1998