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.

Identification of Major Locus Bph35 Resistance to Brown Planthopper in Rice

An introgression line RBPH660,derived from wild rice Oryza rufipogon,showed stable resistance to brown planthopper(BPH).Segregation analysis indicated BPH resistance of RBPH660 was controlled by multiple genes/QTLs.By using the bulked segregant analysis(BSA)-seq method,two genomic regions harboring QTLs resistance to BPH were identified from 1.20 to 16.70 Mb on chromosome 4 and from 10.20 to 12.60 Mb on chromosome 9 in RBPH660,respectively.A major resistance locus,designated as Bph35 accounting for 51.27%of the phenotypic variation with a LOD score of 42.51,was mapped to the candidate region of chromosome 4 between In Del(insertion-deletion)markers PSM16 and R4 M13.For fine mapping of Bph35,one simple sequence repeat and three newly developed In Del markers were used to screen the recombinants.Finally,the Bph35 locus was delimited in the region from 6.28 to 6.93 Mb and there were 18 predicted protein-encoding genes with a total of 114 non-synonymous single nucleotide polymorphism(SNP)variant sites between the resistant and susceptible parents.Out of these genes,Os04 g0193950,encoding a putative NB-ARC(nucleotidebinding adaptor shared by APAF-1,R proteins and CED-4)and LRR(leucine-rich repeat)domain protein with nine non-synonymous SNP substitutions in its coding sequence regions,might be the candidate gene for Bph35.These findings would facilitate the map-based cloning of the Bph35 gene and development of resistant varieties against BPH in 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.

A method for raising brown planthopper (BPH), Nila-parvata lugens (Stl)

To get mass ppoulation of the BPH, agood method for rasing BPH isneeded. In this paper, a convenientmethod was recommended.BPH population used in the ex-periment was provided by Jiangsu A-cad of Agri Sci. Six raising BPHmethods, i. e, with rice buds (RB),rice seedling (RS), seedling soillesscultured (SSC), mature rice (MR),and rice stem cultured in nutrient

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by Two-dimensional Electrophoresis

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. Two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

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.

Induced defense responses in rice plants against small brown planthopper infestation

The small brown planthopper(SBPH), Laodelphax striatellus Fallén(Homoptera: Delphacidae), is a serious pest of rice(Oryza sativa L.) in China. To understand the mechanisms of rice resistance to SBPH, defense response genes and related defense enzymes were examined in resistant and susceptible rice varieties in response to SBPH infestation. The salicylic acid(SA) synthesis-related genes phenylalanine ammonia-lyase(PAL), NPR1, EDS1 and PAD4 were induced rapidly and to a much higher level in the resistant variety Kasalath than in the susceptible cultivar Wuyujing 3 in response to SBPH infestation. The expression level of PAL in the Kasalath rice at 12 h post-infestation(hpi) increased 7.52-fold compared with the un-infested control, and the expression level in Kasalath was 49.63, 87.18, 57.36 and 75.06 times greater than that in Wuyujing 3 at 24, 36, 48 and 72 hpi, respectively. However, the transcriptional levels of the jasmonic acid(JA) synthesis-related genes LOX and AOS2 in resistant Kasalath were significantly lower than in susceptible Wuyujing 3 at 24, 36, 48 and 72 hpi. The activities of the defense enzymes PAL, peroxidase(POD), and polyphenol oxidase(PPO) increased remarkably in Kasalath in response to SBPH infestation, and were closely correlated with the PAL gene transcript level. Our results indicated that the SA signaling pathway was activated in the resistant Kasalath rice variety in response to SBPH infestation and that the gene PAL played a considerable role in the resistance to SBPH.

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.

Donors for Resistance to Brown Planthopper Nilaparvata lugens(St?l) from Wild Rice Species

Out of 1 989 wild accessions sown in seed boxes for screening, only 1 003 wild accessions with good germination were screened against brown planthopper(BPH), Nilaparvata lugens(St?l) under greenhouse conditions. The collection comprised of accessions from 11 wild species and African cultivated rice. The germplasm was screened for BPH following standard seed box screening technique in the greenhouse. As many as 159 accessions were identified as resistant during the year 2012 based on one year screening. A selected set of BPH resistant accessions were screened again during 2013. Based on the two years screening, seven accessions of O. nivara(AA), one accession of O. officinalis(CC), seven accessions of O. australiensis(EE), five accessions of O. punctata(BB and BBCC) and nine accessions of O. latifolia(CCDD) were confirmed to be resistant to BPH. So far no BPH resistance genes have been identified and designated from O. nivara and O. punctata, hence these may act as new sources of resistance.

LncRNAs are potentially involved in the immune interaction between small brown planthopper and rice stripe virus

Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-coding RNAs(lnc RNA) have been reported to block virus replication in hosts. However, the function of lnc RNAs in RSV infection and replication is still unknown. Here, we aimed to study regulatory mechanisms of lnc RNA in an immune system during RSV infection. First, lnc RNA genes were predicted from SBPH transcriptomes using a bioinformatics pipeline based on characteristics of lnc RNA. We identified 4 786 lnc RNA genes corresponding to 5 790 transcripts in SBPH from an RNA-Seq dataset of 15 transcriptomes. Differential expression analysis indicated that 3, 11, and 25 lnc RNA genes were highly expressed in gut, salivary gland, and ovary, respectively, of viruliferous SBPH(Student’s t-test, P<0.05). We randomly selected eight lnc RNAs for expression validation using quantitative real-time PCR, confirming the differential expression of these lnc RNAs between viruliferous and non-viruliferous SBPH. In summary, we present evidence that the expression of lnc RNA genes was induced by RSV infection, suggesting that RSV might be involved in the antivirus immune system in SBPH and participate in regulating the RSV replication mechanism. These data provide helpful information for future investigations of the interaction between lncRNA and RSV.

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.

Expression Analysis of Genes Related to Rice Resistance Against Brown Planthopper, Nilaparvata lugens

Brown planthopper(BPH) is an insect species that feeds on the vascular system of rice plants. To examine the defence mechanism of rice plants against BPH, the pathogenesis-related genes(PR1a, PR2, PR3, PR4, PR6, PR9, PR10a, PR13, PR15 and PRpha), signaling molecule synthesis genes(AOS, AXR, ACO and LOX), antioxidant-related genes(CAT, TRX, GST and SOD) and lignin biosynthesis-related genes(CHS, CHI and C4H) were investigated in a resistant rice variety. AOS, PR6,PR9 and PR15 genes showed significantly increased relative expression levels at 24.38-, 19.17-, 14.71-, and 12.74-fold compared to the control. Moderate increased relative expression levels of lignin biosynthesis-related gene(C4H), pathogenesis-related genes(PR4, PR10a and PRpha), and antioxidant-related gene(GST) were found, while CHI, LOX, SOD, TRX1 and AXR showed decreased relative expression levels. It was thus clearly shown that wound-induced response genes were activated in rice plants after BPH attacks through AOS activation. Jasmonic acid signaling molecule may activate PR6, PR15, GST and CAT subsequently increasing their expression for H_2O_2 detoxification. PR6 were expressed at the highest relative level among the PR genes. These genes therefore have also a considerable synergistic role with the other genes against BPH by interfered their digestion tract system.

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.

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.

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.

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.

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.