Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR(TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35 S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.

Effects of N Management on Yield and N Uptake of Rice in Central China

Efficient N fertilizer management is critical for the economic production of rice and the long-term protection of environmental quality. A field experiment was designed to study the effects of N fertilizer management practices on grain yield and N uptake office. The experiment was laid out in the randomized complete block design with four replications in Central China during 2008 and 2009. Five N treatments denoted as No, NIsoA, N15oB, N24OA, and N24oB, respectively, were studied. NO represented no N application and served as a control, N15OA and N15oB indicated the total N application of 150 kg N ha-I but with two different application schedules （A and B） across the early stage of rice growth. Schedule A was applied as follows： 40% basal, 30% at 10 d after transplanting （DAT） and 30% at 36 DAT （nearly at the panicle initiation stage）, while schedule B was as follows： 30% at basal, 20% at 10 DAT, and 50% at 36 DAT. Similarly, N240A and N240B indicated the total N application of 240 kg N ha-1 with schedules A and B as described above. To quantify N uptake from fertilizer and soil, a 15N experiment was also conducted within the main experimental field, with micro-plots. Grain yields were significantly increased as N rates increased from 0 to 240 kg N ha-1. At the same rate, splitting N application as schedule B significantly increased the grain yield, spikelets per panicle, percentage of ripened grain, and 1 000-grain weight, compared with the N application according to schedule A. Mean rice recovery of N fertilizer by ~SN tracing method ranged from 25.39% at N24oA to 34.89% at NIsoB, however, N fertilizer residual rate in the soil ranged from 12.40% at N240A to 16.61% at N^s0w About 31.5 and 28.5% of total uptake of ~SN derived from basal fertilizer was absorbed at panicle initiation and heading stages, respectively. However, 65.6-92.5% of total uptake of 15N derived from topdressing fertilizer was absorbed at the heading stage. Based on yield and nitrogen recovery efficiency, splitting N application according to schedule B at the rate of 240 kg N ha-1 will be more profitable among the tested five N treatments in Central China.

Identification of two Monilia species from apricot in China

Monilinia fructicola, Monilia mumecola and Monilia yunnanensis have been reported as the causal agents of brown rot disease on stone fruits in China. Up to date, these species have been identified from peach and plum, and M. mumecola has also been reported on apricot recently. To investigate whether M. fructicola and M. yunnanensis can cause brown rot disease on apricot in China, 37 isolates were collected from four orchards in Chongqing and Beijing municipalities in 2014. These isolates were divided into two phenotypes according to their distinct colony appearances. Two representative isolates of each phenotype and reference species of M. mumecola from apricot were selected for further analysis. Based on the morphological characterization and molecular identification, the two phenotypes of isolates were identified as M. fructicola and M. yunnanensis, respectively.

Sublethal Effects of Metaflumizone on Plutella xylostella (Lepidoptera: Plutellidae)

Metaflumizone is a novel sodium channel blocker insecticide, which has been registered for controling the diamondback moth （DBM）, Plutella xylostella （Lepidoptera： Plutellidae） in China. It is unavoidable for DBMs to be exposed to sublethal dose of metaflumizone in brassicaceous vegetable field. Sublethal effects of metaflumizone at LC15 （1.06 mg L-1） and LCz5 （2.03 mg L-1） on the biological characteristics were investigated. Pupation rate, pupal period and pupal weight were significantly declined comparing with the control （P=0.05） when third instar larvae were exposed to LC15 and LC25 of metaflumizone. Comparing the control group to LC15 and LC25 treatment groups, there were significant differences in the development periods of eggs, first instar larvae, pupae and total preoviposition period. The peak of age-specific fecundity in LC15 and LC25 treatment groups was lagged behind control group. Mean values of intrinsic rate of increase （rm）, net reproductive rate （R0） and finite rate of increase （λ） observed from the LCl5 and LC25 treatment groups were lower than those from the control group. But mean values of gross reproduction rate （GRR） was not different. The mean generation time （T） of the treatment groups was prolonged. Whether sublethal doses of metaflumizone could stimulate reproduction in the long term needs further investigation on continuous generations.

Insecticidal Activity of Piperine Isolated from Piper sarmentosum

In order to clarify the insecticidal active ingredients of Piper sarmentosum,one active ingredient was isolated from ethanol extract of P. sarmentosum by bioassay-guided fractionation method. Its chemical structures were identified to be piperine by MS,1H NMR,13C NMR. The insecticidal activity of piperine and ethanol extract of P. sarmentosum against Aleurodicus dispels Russell were tested by leaf dip method. The results showed that piperine and ethanol extract of P. sarmentosum exhibited strong insecticidal activity against adults and nymphs of A. dispels; LC50values against adults were 28. 59 and 224. 31 mg/L,and LC50values against nymphs were 65. 91 and 336. 68 mg/L,respectively. There was no significant difference between piperine and azadirachtin against adults and nymphs of A.dispels. Therefore,piperine might be one of the main insecticidal ingredients of P. sarmentosum. In addition,piperine and ethanol extract showed ovicidal activity with different mode of action,piperine reduced the survival rate of newly hatched nymphs while ethanol extract impacted hatch of eggs.

Synthesis,Crystal Structure and Biological Activity of 8-((4-((2,3-diaminopyridin-4-yl)oxy)-3-fluorophenyl)-amino)-2-(4-fluorophenyl)-3-methyl-2,7-naphthyridin-1(2H)-one

The new title compound 8-（（4-（（2,3-diaminopyridin-4-yl）-oxy）-3-fluorophenyl）-amino）-2-（4-fluorophenyl）-3-methyl-2,7-naphthyridin-1（2H）-one（C26H20F2N6O2, Mr = 486.48） has been prepared and determined by single-crystal X-ray diffraction. The crystal is of monoclinic, space group P21/c with a = 15.365（3）, b = 13.144（2）, c = 11.863（2）, β= 108.882（3）°, Z = 4, V = 2267.0（7）3, Dc = 1.425 g/cm3, F（000） = 1008, μ = 0.105 mm-1, MoKa radiation（λ = 0.71073）, R = 0.0480 and wR = 0.1294 for 3197 observed reflections with I 〉 2σ（I）. X-ray diffraction analysis reveals that the region C（substituents of 8-amino group and 3-methyl group on the 2,7-naphthyridin-1（2H）-one ring） of compound 6 are effectively planar. Intramolecular and intermolecular hydrogen bonds together with π···π interations are found in the structure. In addition, compound 6 shows potent c-Met and c-Kit kinase inhibition activities.

Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants

Dear Editor CRISPR gene-editing technology has successfully generated targeted mutations in rice and many other plant species （Ma et al., 2015）. Assessment of heritability and phenotypic stability of CRISPR-edited plants requires the elimination of the CRISPR construct. The presence of the CRISPR construct makes it difficult to distinguish the mutations transmitted from the previous generation from newly generated mutations by the CRISPR construct at the current generation. The existence of the CRISPR construct also greatly increases the risk of off-target effects.

Changes in DNA Methylation Pattern at Two Seedling Stages in Water Saving and Drought-Resistant Rice Variety after Drought Stress Domestication

Recent studies revealed that DNA methylation plays an important role in plant growth and development. In this study, a water-saving and drought-resistant rice variety Huhan 3 was subjected to drought stress from tillering to grain-filling stages in six successive growth cycles. The variations in DNA methylation pattern between the original generation （Go） and the sixth generation （G6） were analyzed by using methylation sensitive amplification polymorphism method. The results revealed that the methylated loci accounted for 34.3% to 34.8% of the total loci. Among these methylated loci, 83.1% to 84.8% were full- and hyper-methylated and 15.2% to 16.9% were hemi-methylated. The DNA methylation level decreased from the three-leaf to four-leaf stages in Huhan 3. Differentially methylated loci （DML） between generations or/and between different developmental stages accounted for 4.0% of the total loci, most of which were only related to plant development （57.9%）. Compared to Go, the DNA methylation pattern of G8 changed after drought domestication, at the three-leaf stage, de-methylation accounting for 59.1%, while at the four-leaf stage, re-methylation for 47.9%. Genome-wide alternations of DNA methylation were observed between the two seedling stages, and DML mainly occurred on the gene＇s promoter and exon region. The genes related to DML involved in a wide range of functional biology and participated in many important biological processes.

Transcriptome datasets supply basic gene information for RNAi pest management and gene functional studies in Nephotettix cincticeps(Uhler)

RNA interference （RNAi） technology has the potential to be used in pest management in crop production. Here, the transcriptome of Nephotettix cincticeps （Uhler） was deeply sequenced to investigate the systematic RNAi mechanism and candidate genes for dsRNA feeding. In our datasets, a total of 81 225 transcripts were obtained with the length from 150 bp to about 4.2 kb. Almost all the genes related to the RNAi core pathway were proved to be present in N. cincticeps transcriptome. Two transcripts that respectively encode a systemic interference defective （SID） were identified in our da- tabase, indicating that the systematic RNAi pathway can function effectively in N. cincticeps. Our datasets not only supply basic gene information for the studies of gene expression and functions in N. cincticeps, such as the control genes for gene expression analysis, but also provide candidate genes for RNAi pest management, such as the genes that encode P450 monooxygenase, V-ATPase and chitin synthase.

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis （Lepidoptera： Crambidae）

Two transgenic rice lines （T2A-1 and T1C-19b） expressing cry2A and crylC genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis （Walker） （Lepidoptera： Crambidae）. The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a crylAb/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A- 1 〉 Huahui I 〉 T1C-19b among rice lines, and leaf 〉 stern 〉 root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar： higher level at early stages than at later stages with an exception that high Cry 1C level in T 1 C- 19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the j ointing stage with an exception for T1 C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest crylC is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

Comparative effect of allopolyploidy on transposable element composition and gene expression between Gossypium hirsutum and its two diploid progenitors

An allopolyploidization event formed allotetraploid Gossypium species from an A-genome diploid species and a D-genome diploid species. To explore the responses of transposable elements(TEs) to allopolyploidy, we assembled parallel TE datasets from G. hirsutum, G. arboreum and G. raimondii and analyzed the TE types and the effects of TEs on orthologous gene expression in the three Gossypium genomes.Gypsy was the most abundant TE type and most TEs were located $500 bp from genes in all three genomes. In G. hirsutum, 35.6% of genes harbored TE insertions, whereas insertions were more frequent in G. arboreum and G. raimondii. G. hirsutum had the highest proportion of uniquely matching 24-nt small interfering RNAs(siRNAs) that targeted TEs. TEs,particularly those targeted by 24-nt siRNAs, were associated with reduced gene expression, but the effect of TEs on orthologous gene expression varied substantially among species. Orthologous gene expression levels in G. hirsutum were intermediate between those of G. arboreum and G. raimondii, which did not experience TE expansion or reduction resulting from allopolyploidization. This study underscores the diversity of TEs co-opted by host genes and provides insights into the roles of TEs in regulating gene expression in Gossypium.

A de novo transcriptomic analysis to reveal functional genes in Apolygus lucorum

The widespread planting of genetically engineered cotton producing the CrylAc toxin has led to significantly reduced pesticide applications since 1997. However, conse- quently, the number of green mirid bugs （GMB）, Apolygus lucorum （Meyer-Diir） has in- creased. So far the GMB, instead of the cotton bollworm Helicoverpa armigera （Hiibner）, has become the major pest in the transgenic Bt cotton field and has influenced cotton yield. Disproportionately, only a few studies on GMB at molecular level have been re- ported. Libraries from both third instar nymphs and adults were sequenced using Illumina technology, producing more than 106 million short reads and assembled into 63 029 uni- genes of mean length 597 nt and N50 813 nt, ranging from 300 nt to 9771 nt. BLASTx analysis against Nr, Swissprot, GO and COG was performed to annotate these unigenes. As a result, 26 478 unigenes （42.01%） matched to known proteins and 107 immune-related, 320 digestive-related and 53 metamorphosis-related genes were detected in these annotated unigenes. Additionally, we profiled gene expression using mapping based differentially expressed genes （DEGs） strategy between the two developmental stages： nymph and adult. The results demonstrated that thousands of genes were significantly differentially ex- pressed at different developmental stages. The transcriptome and gene expression data provided comprehensive and global gene resources of GMB. This transcriptome would improve our understanding of the molecular mechanisms of various underlying biological characteristics, including development, digestion and immunity in GMB. Therefore, these findings could help elucidate the intrinsic factors of the GMB resurgence, offering novel pest management targets for future transgenic cotton breeding.

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity AiMasc in BmN cells and found that/z'Masc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.