Gapless Genome Assembly of ZH8015 and Preliminary Multi-Omics Analysis to Investigate ZH8015's Responses Against Brown Planthopper Infestation

Accurate genomic information is essential for advancing genetic breeding research in specific rice varieties.This study presented a gapless genome assembly of the indica rice cultivar Zhonghui 8015(ZH8015)using Pac Bio HiFi,Hi-C,and ONT(Oxford Nanopore Technologies)ultra-long sequencing technologies,annotating 43037 gene structures.Subsequently,utilizing this genome along with transcriptomic and metabolomic techniques,we explored ZH8015's response to brown planthopper(BPH)infestation.Continuous transcriptomic sampling indicated significant changes in gene expression levels around 48 h after BPH feeding.Enrichment analysis revealed particularly significant alterations in genes related to reactive oxygen species scavenging and cell wall formation.Metabolomic results demonstrated marked increases in levels of several monosaccharides,which are components of the cell wall and dramatic changes in flavonoid contents.Omics association analysis identified differentially expressed genes associated with key metabolites,shedding light on ZH8015's response to BPH infestation.In summary,this study constructed a reliable genome sequence resource for ZH8015,and the preliminary multi-omics results will guide future insect-resistant breeding research.

Density Restriction Effects of Reproductive Rate of Rice Brown Planthopper Population in Super Rice Yongyou 6

[ Objective ] The paper was to study the density restriction effects of reproductive rate of rice brown planthopper （BPH） population in super indiea and japonica hybrid rice Yongyou 6. [Method] Different amounts of rice brown planthoppor were released to super indiea and japonica hybrid flee Yongyou 6 during fillering, booting and gain filling stage, respectively. The reproductive rate and variation dynamics of rice brown planthoppor in super rice was also investigated and analyzed. [Result] Tillering stage： the population quantity of rice brown planthoppor during booting and gain filling stage gradually increased with the increasing basal amount of its population during tillering stage; when the population density of rice brown planthopper during grain filling stage reached a certain limit （ 〉 250 head/cluster）, the population density during milking stage showed decrease trend with the increasing of such density; the development of rice brown planthoppor population in super rice Yongyou 6 showed the characteristics of fast in middle and slow in two ends. Booting stage： the development of rice brown planthopper pop- ulation from booting to gain filling stage increased with the increasing basal amount of density, and their correlation during milking stage was not significant; flee brown planthopper population still had high reproduction capacity in the middle and late stage of super rice, which had latent risk on rice yield. Gain filling stage ： when population density of rice brown planthopper 〈 70 head/cluster, the development of it population increased with the increase of basal amount of density, which had great threat to yield. [ Conclusion] The result provided theoretical basis for establishment and improvement of monitoring early warning system, cost-ef- fective control of occurrence and damage of rice brown planthopper, and improvement of the sustained control ability against rice brown planthopper.

Pyramiding blast,bacterial blight and brown planthopper resistance genes in rice restorer lines

Rice blast, bacterial blight （BB） and brown planthopper （BPH） are the three main pests of rice. This study investigated pyr-amiding genes resistant to blast, BB and BPH to develop restorer lines. Ten new lines with blast, BB and/or BPH resistance genes were developed using marker-assisted selection （MAS） technique and agronomic trait selection （ATS） method. Only HR13 with resistance genes to blast, BB and BPH was obtained. In addition to blast and BB resistance, four lines （HR39, HR41, HR42, HR43） demonstrated moderate resistance to BPH, but MAS for BPH resistance genes were not conducted in developing these four lines. These data suggested that there were unknown elite BPH resistance genes in the Zhongzu 14 donor parent. A more effective defense was demonstrated in the lines withPi1 andPi2 genes although the weather in 2012 was favorable to disease incidence. Blast resistance of the lines with a single resistance gene,Pita, was easily inlfuenced by the weather. Overal, the information obtained through pyramiding multiple resistance genes on developing the restorer lines is helpful for rice resistance breeding.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Effects of Temperature on Functional Response of Anagrus nilaparvatae Pang et Wang (Hymenoptera:Mymaridae) on the Eggs of Whitebacked Planthopper,Sogatella furcifera Horvth and Brown Planthopper,Nilaparvata lugens Stl

Understanding the temperature affecting parasitic efficiency is critical to succeed in utilizing parasitoid as natural enemy in pest management. Laboratory studies were carried out to determine the effects of temperature on parasitoid preference of female Anagrus nilaparvatae Pang et Wang （Hymenoptera：Mymaridae） to the eggs of whitebacked planthopper （WBPH）, Sogatella furcifera Horváth and brown planthopper （BPH）, Nilaparvata lugens Stl to build a composite model describing changes in parasitic response along a temperature gradient （18, 22, 26, 30, 34°C）. The results showed that attack responses of A. nilaparvatae on WBPH and BPH were the best described by a Type II functional response. The two parameters, attack rates （a） and handling times （Th）, of A. nilaparvatae to both eggs were influenced by the temperature. The maximum attack rates to WBPH （1.235） and BPH （1.049） were at 26 and 34°C, respectively, and the shortest handling times to WBPH （0.063） and BPH （0.057） were at 30 and 26°C, respectively. However, the optimal temperature for parasitic efficiency of A. nilaparvatae to WBPH and BPH eggs was both at 26°C, which showed that the present microclimate temperature of the habitat in the paddyfield was beneficial to A. nilaparvatae and indicated that parasitic efficiency of A. nilaparvatae would be impaired by global warming.

Evaluation of Rice Germplasm for Resistance to the Small Brown Planthopper (Laodelphax striatellus) and Analysis of Resistance Mechanism

One hundred and thirty-eight rice accessions were screened for resistance to the small brown planthopper （SBPH） （Laodelphax striatellus Fallen） by the modified seedbox screening test. Twenty-five rice accessions with different levels of resistance to SBPH were detected, accounting for 18.1% of the total accessions, which included 2 highly resistant, 9 resistant and 14 moderately resistant varieties. Compared with indica rice, japonica rice was more susceptible to SBPH. Antixenosis test, antibiosis test and correlation analysis were performed to elucidate the resistance mechanism. The resistant check Rathu Heenati （RHT）, highly resistant varieties Mudgo and Kasalath, and resistant variety IR36 expressed strong antixenosis and antibiosis against SBPH, indicating the close relationship between resistance level and these two resistance mechanisms in the four rice varieties. Antibiosis was the dominant resistance pattern in the resistant varieties Daorenqiao and Yangmaogu due to their high antibiosis but low antixenosis. Dular, ASD7 and Milyang 23 had relatively strong antixenosis and antibiosis, indicating the two resistance mechanisms were significant in these three varieties. The resistant DV85 expressed relatively high level of antixenosis but low antibiosis, whereas Zhaiyeqing 8 and Guiyigu conferred only moderate antibiosis and antixenosis to SBPH, suggesting tolerance in these three varieties. Antibiosis and antixenosis governed the resistance to SBPH in the moderately resistant accession 9311. Antixenosis was the main resistance type in V20A. Tolerance was considered to be an important resistance mechanism in Minghui 63 and Yangjing 9538 due to their poor antibiosis and antixenosis resistance. The above accessions with strong antibiosis or antixenosis were the ideal materials for the resistance breeding.

Improving the resistance of the rice PTGMS line Feng39S by pyramiding blast,bacterial blight,and brown planthopper resistance genes

Knowledge of rice(Oryza sativa L.)genes and various DNA markers can be used in genomic breeding programs aimed at developing improved elite rice cultivars.We used an efficient genomic breeding approach to pyramid four resistance genes(Pi2,Xa23,Bph14,and Bph15)in the popular photoperiod-and thermosensitive genic male sterile(PTGMS)rice line Feng39S.We performed foreground selection for the target genes,followed by recombinant selection and background selection.This process reduced the sizes of the genomic segments harboring the target genes(566.8 kb for Pi2,1143.9 kb for Xa23,774.7 kb for Bph14,and 1574.9 kb for Bph15)and accelerated the recovery of the recurrent parent genome to proportions ranging from 98.77%to 99.16%,thus resulting in four near-isogenic lines.To assemble the four resistance genes in Feng39S,we performed a double-way cross combined with foreground and background selection to generate two improved lines of Feng39S(Pi2+Xa23+Bph14+Bph15)with a recurrent parent genome recovery of 98.98%.The two lines showed agronomic performance,grain quality,and fertility–sterility transition characteristics similar to those of the original Feng39S line.The newly developed PTGMS lines and corresponding hybrid combinations were resistant to various field blast isolates and seven representative isolates of bacterial blight.At the seedling stage,the lines also showed resistance against brown planthopper.This study provides an efficient and accurate genomic breeding approach for introducing desirable traits into PTGMS lines.

Effect of Nitrogen on Water Content, Sap Flow, and Tolerance of Rice Plants to Brown Planthopper, Nilaparvata lugens

Water content (WC) and sap flow from leaf sheath of rice plants with varying nitrogen levels at different growth stages, and fluctuations in relative water content (RWC) of rice plants being damaged by brown planthoppcr (BPH), Nilaparvata lugens were determined in the laboratory, and the tolerance of rice plants to BPH at different nitrogen regimes was evaluated in the greenhouse at International Rice Research Institute (IRRI), the Philippines. The results indicated that both WC and RWC were increased significantly, as the amount of sap flow from rice plants was reduced statistically, with the increase of nitrogen content in rice plants. RWC in rice plants applied with high nitrogen fertilizer decreased drastically by the injury of BPH nymphs, while the reduced survival duration of rice plants with the increase of nitrogen content was recorded. These may be considered to be one of the important factors in increasing the susceptibility to BPH damage on rice plants applied with nitrogen fertilizer.

Transmission of Rice Black-Streaked Dwarf Virus from Frozen Infected Leaves to Healthy Rice Plants by Small Brown Planthopper (Laodelphax striatellus)

In order to preserve virus for identifying the resistance of rice varieties against rice black-streaked dwarf disease, a simple and reliable method was developed, through which virus-free small brown planthopper （SBPH） acquired rice black-streaked dwarf virus （RBSDV） from frozen infected leaves and the virus was transmitted to healthy rice plants. The experimental results showed that SBPH could obtain RBSDV from frozen infected rice leaves and the virus could be transmitted to a susceptible rice variety. For the ability to acquire RBSDV and transmit the virus to healthy plants by SBPH, there was no significant difference between frozen infected leaves and in vitro infected leaves. The novel method could be applied to identification of rice variety resistance to rice black-streaked dwarf disease, facilitating the breeding process for rice black-streaked dwarf disease resistance.

Understanding Brown Planthopper Resistance in Rice: Genetics, Biochemical and Molecular Breeding Approaches

Brown planthopper(BPH,Nilaparvata lugens St自I)is the most devastating pest of rice in Asia and causes significant yield loss annually.Around 37 BPH resistance genes have been identified so far in indica,African rice varieties along with wild germplasms such as Oryza officinalis,O.minuta,O.nivara,O.punctata,O.rufipogon and O,latifolia.Genes/QTLs involved in BPH resistance,including Bph1,bph2/BPH26,Bph3,Bph6,bph7,BPH9,Bph12,Bph14,Bph15,Bph17,BPH18,bph19,Bph20,Bph21(t),Bph27,Bph27©Bph28(t),BPH29,QBph3,QBph4,QBph4.2,Bph30,Bph32,Bph33,Bph35 and Bph36,have been fine-mapped by different researchers across the globe.The majority of genes/QTLs are located on rice chromosomes 1,3,4,6,11 and 12.Rice plants respond to BPH attack by releasing various endogenous metabolites like proteinase inhibitors,callose,secondary metabolites(terpenes,alkaloids,flavonoid,etc.)and volatile compounds.Besides that,hormonal signal pathways mediating(antagonistic/synergistic)resista nee responses in rice have been well studied.Marker-assisted breedi ng and genome editi ng techniq ues can also be adopted for improving resista nee to novel BPH biotypes.

High Temperature Modifies Resistance Performances of Rice Varieties to Brown Planthopper,Nilaparvata lugens(Stl)

To investigate the effect of temperature on the resistance characteristics of dce varieties with different resistance genes to brown planthopper （BPH）, Nilaparvata lugens （Stal）, the resistances of IR26 （Bphl） and IR36 （bph2） to BPH population in Hangzhou, China were monitored in greenhouse during September in 2007 and 2008 by using the standard seedling screening techniques （SSST） developed by the International Rice Research Institute （IRRI）. Furthermore, the changes in resistance of IR26 and IR36 to BPH, soluble sugar and oxalic acid contents in 25-day-old rice plants of susceptible variety TN1 and resistant varieties IR26 and IR36 were detected at five temperatures （22℃, 25℃, 28℃, 31℃ and 34℃）. IR26 completely lost resistance both in greenhouse and at the five tested temperatures. IR36 still had moderate resistance at natural temperature, but its resistance decreased gradually when the temperature increased from 25℃ to 34℃, and fully lost its resistance at 31℃ and 34℃. The highest durable resistance of IR26 and IR36 were recorded at 25℃. The soluble sugar content in plants of the three tested rice varieties increased with temperature increase, and the oxalic acid content increased with the temperature increase at first, maximized at 25℃, and then declined. Two-way ANOVA indicated significant effects of temperature and rice variety on contents of soluble sugar and oxalic acid in rice plants

Content Variations of the Secondary Compounds in Rice Plants and Their Influence on Rice Resistance to Brown Planthopper,Nilaparvata lugens

Content variations of the four components in the plants of rice ASD7, IR36 （resistant to brown planthopper） and TN1 （susceptible to brown planthopper）, and their influence on rice resistance to brown planthopper （BPH, Nilaparvata lugens） were investigated. The resistance diversity of rice to BPH biotype II at different plant ages was chiefly attributed to the content variability of the secondary compounds. The contents in the leaf sheath were the lowest as compared with other parts of rice plant. It might explain the reason that BPH has a preference for feeding on leaf sheath aggregately from chemical point of view.

Molecular Characterization and Functional Analysis of Krüppel-homolog 1(Kr-h1) in the Brown Planthopper, Nilaparvata lugens(Stl)

The brown planthopper, Nilaparvata lugens（Stl）, is the most serious insect pest of rice. It has developed high resistance to traditional insecticides because of their intensive use. Juvenile hormone（JH） analogs have been used successfully to control this species and other pest insects. However, the molecular mechanism of JH signaling is not well understood. Krüppel-homolog 1（Kr-h1） is a transcription factor involved in the JH pathway. In this study, the Kr-h1 cDNA was cloned and characterized from N. lugens by rapid amplification of cDNA ends（RACE） and reverse transcription PCR（RT-PCR）. Its spatial and temporal expression profiles were examined by real-time quantitative PCR, and its function was also studied by RNA interference（RNAi）. The open reading frame of NlKr-h1 is 1 833 bp encoding for 611 amino acids. The protein contains eight conserved zinc-finger motifs. NlKr-h1 was expressed at all life stages, with the highest mRNA level in the 4-day embryo. NlKr-h1 mRNA levels rose during each nymphal molt after the 2nd instar. In the adults, the mRNA level in males was significantly higher than that in females of either the macropterous or brachypterous type. The highest expression was observed in the female midgut. NlKr-h1 was activated by juvenile hormone III（JH III） in the 3rd-5th instar nymphs. Disruption of Nlkr-h1 expression by RNAi caused stunted wing development and malformations of both male and female external genitalia. Our findings suggest that Kr-h1 may be a useful target for pest insect management.

Genetic Homozygosis of Resistance of Common Wild Rice (Oryza rufipogon) against Brown Planthopper

[ Objective] The stability of resistance heredity of common wild rice against brown planthopper was studied to screen wild rice germplasm with stable resistance, so as to provide resources for rice breeding with resistance against brown planthopper. [ Method] A total of 1 591 accessions of common wild rice, collected from 46 proterozoic growth locations of Guangxi, were screened to analyze resistance against brown planthopper, and genetic homozygosis in their offspring was detected using bag-selfing or anther culture methods. [ Result] The results showed that most of common wild rice germplasm were heterozygotic, and growth habit and awn of their first self-bred generation showed segregation. Among 1 591 accessions of common wild rice, only 30 accessions showed third to fifth grades of resist- ance against brown planthopper, and most of them showed fifth grade of resistance. Among selfing offspring of 11 resistant materials, the populations from Z1 to Z3 showed segregation of resistant genes, while some materials showed stable resistance in Z4. Out of 11 accessions of materials, resistance of five accessions increased from third grade to first, second and third grades; four accessions increased from fifth grade to first, second and third grades ; two accessions increased from fifth to third grade. Anther culture results showed that 125 accessions of callus from independent origins were obtained after induction by in vitro culture of 6 000 anthers; eight clusters of diploid and two clusters of haploid green seedlings were differentiated, with induction rate of callus as 2.0% and differentiation rate of green seed- lings as 6.4%. Out of eight offspring of anther culture, six showed fifth grade of resistance against brown planthopper and two showed third grade of resistance. [ Conclusion] The stable highly-resistant resources could be obtained and resistance level of common wild rice could be enhanced through bag-selfing and multigeneration identification of resistance against brown planthopper. Anther culture is beneficial to promote genetic homozygosis of resistance of common wild rice against brown planthopper and shorten the screening time.

Tolerance evaluation of rice varieties to the brown planthopper,Nilaparvata lugens feeding

The brown planthopper,Nilaparata lugens(Stl)(BPH)is one of the most important insect pests of rice in China and other east-southern Asian countries.Untilization of rice resistance varieties is one of the most econnomic and effective ways for

Bacterial Community in Different Populations of Rice Brown Planthopper Nilaparvata lugens(Stl)

The structures of bacterial communities in the brown planthopper （BPH） Nilaparvata lugens （St^l） from different geographic and resistant virulent populations were analyzed by using denatured gradient gel electrophoresis （DGGE）. Results showed that the bacterial communities in BPH nymph from the first to the fifth instars varied with nymphal growth and development. The bacterial communities in the first-instar BPH nymph were similar to those in adults. Nine geographic BPH populations were divided into three groups based on the cluster analysis of DGGE fingerprint. The first group was from the Philippines; the second group was from Thailand and Hainan, Yunnan and Zhejiang provinces of China; and the third group was from Vietnam and Guangxi, Hunan and Jiangxi provinces of China. BPH populations adapted to different resistant rice varieties. The BPH populations from Mudgo （with resistant gene Bphl） and ASD7 （with resistant gene bph2） differed with those of the susceptible rice variety TNI.

Expression of FAMeT and JHE Influenced by Kruppel homolog-1 Gene from Brown Planthopper

The characteristics of zinc finger of Kruppel homolog-1 （N/kr-hl） gene from brown planthopper and its effect on expression of FAMET and JHE were studied in the paper, in order to understand the regulation role of Nlkr-hl. The evolutionary tree of each zinc finger in functional area of Nlkr-hl was analyzed via MEGAS. 0 software, and expression status of FAMeT and JHE after interference of N/kr-hl was determined using qPCR technology. The results showed that the functional area of N/kr-hl mainly included eight zinc fingers, and the conservativeness of Znl and Zn8 was relatively lower. In the brown planthopper with N/b＇-hl gene knockout, the expression levels of FAMeT and JHE increased. The paper provided the theoretical basis for understanding molecular mechanism of juvenile hormone signaling pathway and biological control of brown planthopper.

Breeding of Maintainer Line and Sterile Line Brown Planthopper Resistant Rice Using Marker-assisted Selection

Guangxi common wild rice variety brown planthopper highly resistant introgression line HS204 was taken as antigen donor material,hybridization,backcrossing,and molecular marker-assisted selection methods were adopted,to select maintainer line and sterile line materials,so as to provide excellent material foundation for resistance breeding of hybrid rice. Through the marker-assisted selection,it obtained 4 pieces of homozygous resistance gene maintainer line intermediate materials( 100B,101B,102B and 103B),and 2 pieces of resistant sterile line materials( 100A and 103A). All 10 combinations that have testcross with highly resistant sterile 100A showed higher level of brown planthopper resistance: 100A/R2586,100A/KR838,and 100A/KR527 had high resistance level,the others had low to intermediate resistance;100A/KR527,100A/R2586,100A/Minghui 63,100A/Fuhui 838 and 100A/Gui 99 combinations had yield per plant significantly higher than the control group( Teyou 7118),increasing by 14. 45%-49. 26%. The obtained resistant lines are expected to provide a better gene source for the breeding of resistant sterile lines of hybrid rice and the obtained resistant sterile lines can be directly used in the selection of three-line hybrid rice.

Disaster Prevention and Mitigation Technologies of Rice Virus Diseases Spread by Small Brown Planthopper (SBPH)

Rice stripe virus disease （RSVD） and rice black-streaked dwarf virus disease （RBSDVD） are two epidemic diseases in Yancheng City, Jiangsu Province in the last decade. The authors investigated the disaster regularity, prevention and control technology of RSVD and RBSDVD systematically. The occurrence and virus transmission of SBPH and disaster regularity of virus diseases were studied; the resistance of some rice varieties was cleared; the effects of physical and agricultural measures such as insect net blocking, appropriate late sowing and plowing on controlling occurrence and virus transmission of SBPH were figured out; a hatch of chemical agents were screened, providing efficient and harmless pesticides for effective control against SBPH and prevention against virus diseases. A set of disaster control and mitigation technologies was proposed in this paper, which was practical, sustainable, and easy to operate for the local planting patterns.

Cloning and characterization of one putative muscle actin gene only expressed at the larval stages from brown planthopper, Nilaparvata lugens

An actin gene （BPH-Actin3） from the important rice pest brown planthopper, Nilaparvata lugens, was cloned and gene expression was characterized at different development stages. The gene was 1461bp with an open reading frame of l l31bp coding for a 376 amino acids protein, with 330 nucleotides of the 3＇-untranslation region （3＇-UTR）. The amino acid sequence deduced from the nucleotide sequence showed higher similarities to other insect muscle actins （94-95%） than those to non-muscle actins （87-93%）. The 3＇-UTR contains several AU-rich elements （AREs） AUUUA/UAAAU and one extended ARE UAAAAAU, which may function in regulating mRNA decay. Northern blot and RT-PCR studies showed BPH-Actin3 expressed at brown planthopper full larval stages with the highest mRNA levels at 3rd and 4th instar stages, but not expressed at egg and adult stages. Because the 3rd and 4th instars are the key development stages for brown planthopper wing-form determination, it was thought BPH-Actin3 might play important roles in brown planthopper wing development.