Minghui 63 Research Articles

Analysis of anthocyanins and total flavonoids content in functional rice and its recombination inbred lines.

Anthocyanin is one of the flavonoids, which has strong antioxidant properties. Functional rice rich in anthocyanins can not only improve immunity, but also anti-radiation, beauty, anti-aging effect, very popular in the market. In this study, we used Zibaoxiangnuo 1 (ZBXN 1), a functional rice variety which is rich in total flavonoids and anthocyanins, as the experimental material to construct Recombination Inbred Lines (RILs) with Minghui63 (MH63), a variety without anthocyanins. The contents of anthocyanins and total flavonoids of RILs and two parents were determined for three consecutive generations. The average anthocyanin content of parent ZBXN 1 was 319.31 mg/kg, and the anthocyanin inheritance of RIL population was relatively stable, with 10 samples higher than ZBXN 1. In addition, there was no significant difference in the total flavonoids content between the two parents, the total flavonoids content of Z25 in RIL population was 0.33%. Based on these studies, we believe that ZBXN 1 has abundant and stable anthocyanins, which can be used as an intermediate breeding material for breeding high-quality varieties with high anthocyanins, and lay a foundation for breeding more anthocyanin-rich rice varieties.

Individual and combined effects of land use and weeds on Cry1Ab/c protein expression and yield of transgenic cry1Ab/c rice

ABSTRACT Considering the anticipated commercial exploitation of insect-resistant transgenic rice and that the planting area of cultivated rice overlaps with wild rice, simulating an escape of transgenic rice from farmlands and exploring its fitness after entering semi-natural or natural ecosystems through uncontrolled seed dispersal or gene flow are critical to understand the resulting potential long-term environmental risks. The expression of foreign Cry1Ab/c protein and vegetative and reproductive fitness of insect-resistant transgenic rice Huahui1 (HH1) and its parental-line Minghui63 (MH63) were studied under four treatments combining land use and weed competition: farmland and uncultivated land under weed control (F-NW and U-NW, respectively), and farmland and uncultivated land without weed control (F-W and U-W, respectively). The expression of Cry1Ab/c was significantly lower in U-NW, F-W, and U-W than that in the control treatment, F-NW. Except for plant height, key vegetative (tiller number and biomass) and reproductive (grain number and grain weight per plant) growth indices of transgenic HH1 were significantly lower than those of the parental-line MH63 in F-NW and U-NW, indicating a significant fitness cost. In F-W and U-W, vegetative growth indices (plant height, tiller number, and biomass) were similar in HH1 and MH63; however, key reproductive indices including seed-set rate were significantly higher in HH1 than in MH63, indicating significant fitness benefits. Although these results support large-scale cultivation of insect-resistant transgenic rice in China, the ecological risk involved is high in farmland or uncultivated land without weed control (F-W and U-W).

Reference-Guided De Novo Genome Assembly to Dissect a QTL Region for Submergence Tolerance Derived from Ciherang-Sub1.

Global climate change has increased the number of severe flooding events that affect agriculture, including rice production in the U.S. and internationally. Heavy rainfall can cause rice plants to be completely submerged, which can significantly affect grain yield or completely destroy the plants. Recently, a major effect submergence tolerance QTL during the vegetative stage, qSub8.1, which originated from Ciherang-Sub1, was identified in a mapping population derived from a cross between Ciherang-Sub1 and IR10F365. Ciherang-Sub1 was, in turn, derived from a cross between Ciherang and IR64-Sub1. Here, we characterize the qSub8.1 region by analyzing the sequence information of Ciherang-Sub1 and its two parents (Ciherang and IR64-Sub1) and compare the whole genome profile of these varieties with the Nipponbare and Minghui 63 (MH63) reference genomes. The three rice varieties were sequenced with 150 bp pair-end whole-genome shotgun sequencing (Illumina HiSeq4000), followed by performing the Trimmomatic-SOAPdenovo2-MUMmer3 pipeline for genome assembly, resulting in approximate genome sizes of 354.4, 343.7, and 344.7 Mb, with N50 values of 25.1, 25.4, and 26.1 kb, respectively. The results showed that the Ciherang-Sub1 genome is composed of 59–63% Ciherang, 22–24% of IR64-Sub1, and 15–17% of unknown sources. The genome profile revealed a more detailed genomic composition than previous marker-assisted breeding and showed that the qSub8.1 region is mostly from Ciherang, with some introgressed segments from IR64-Sub1 and currently unknown source(s).

Foreign Cry1Ab/c Delays Flowering in Insect-Resistant Transgenic Rice via Interaction With Hd3a Florigen.

Genetic modifications in rice, which resulted in insect resistance, have been highly efficacious. However, they have also induced undesirable secondary phenotypes, such as delayed flowering. The molecular mechanisms associated with these unwanted effects remain unclear. Here, we showed that the flowering time for insect-resistant transgenic cry1Ab/c rice Huahui-1 (HH1) was delayed, compared with that for the parental rice Minghui-63 (MH63), cultivated on farmland and saline–alkaline soils. In contrast, the insect-resistant transgenic cry1C^* rice cultivars T1C-19 and MH63 had similar flowering times under the same conditions. We quantified the following: the expression of five major flowering genes in HH1, T1C-19, and MH63; florigen Hd3a protein expression levels in HH1 and MH63; interactions between Cry1Ab/c and the five main flowering proteins; and the effects of E3s ubiquitin ligase-mediated Cry1Ab/c expression on florigen Hd3a. Hd3a transcription was significantly lower in HH1 but not in T1C-19, compared with that in MH63. The results of yeast two-hybrid, complementary bimolecular fluorescence, and co-immunoprecipitation assays revealed that florigen Hd3a interacted with the exogenous Cry1Ab/c expressed in HH1 and not the exogenous Cry1C^* expressed in T1C-19. When Cry1Ab/c, Hd3a, and E3s fusion proteins were transiently co-expressed in tobacco cells, the Hd3a expression level was significantly lower than the level of Cry1Ab/c and Hd3a co-expression. Thus, the downregulation of Hd3a expression and the interaction between Cry1Ab/c and Hd3a interfere with Hd3a protein expression and might cooperatively delay HH1 flowering time. To the best of our knowledge, this study is the first to explain the delay in flowering time in insect-resistant transgenic rice, mediated by interactions between exogenous and endogenous proteins. This information might help elucidate the molecular mechanisms associated with these unwanted phenotypes effects and improve the process of biosafety assessment of transgenic rice.

Cry1C rice doesn\u2019t affect the ecological fitness of rice brown planthopper, Nilaparvata lugens either under RDV stress or not

The potential risks of Bt rice on non-target arthropods (NTAs) should be evaluated and defined before commercial production. Recently, effects of Bt rice on NTAs under abiotic and biotic stress conditions attracted much attention. Here we reported the effects of Bt rice T1C-19 (Cry1C rice) on the non-target herbivore, Nilaparvata lugens (rice brown planthopper, BPH) with or without RDV (rice dwarf virus) infection conditions. BPH showed no feeding and oviposition preference between Bt rice T1C-19 and its non-Bt parental rice Minghui 63 (MH63), as well as between RDV-infected and RDV-free rice plants. Meanwhile, rice type, RDV infection status, and their interaction had little impacts on the survival, development and fecundity of BPH. By comparison with non-Bt control, Bt rice T1C-19 with or without RDV infection had no significant effects on the life-table parameters of BPH including rm, R0, T, DT and λ. Thus, it could be concluded that Bt rice T1C-19 doesn’t affect the ecological fitness of BPH either under RDV stress or not.

Identification and Validation of Aerobic Adaptation QTLs in Upland Rice.

The aerobic adaptation of upland rice is considered as the key genetic difference between upland rice and lowland rice. Genetic dissection of the aerobic adaptation is important as the basis for improving drought tolerance and terrestrial adaptation by using the upland rice. We raised BC1-BC3 introgression lines (ILs) in lowland rice Minghui 63 (MH63) background. The QTLs of yield and yield-related traits were detected based on ILs under the aerobic and lowland environments, and then the yield-related QTLs were identified in a backcrossed inbred population of BC4F5 under aerobic condition. We further verified phenotypes of QTL near-isogenic lines. Finally, three QTLs responsible for increasing yield in aerobic environment were detected by multiple locations and generations, which were designated as qAER1, qAER3, and qAER9 (QTL of aerobic adaptation). The qAER1 and qAER9 were fine-mapped. We found that qAER1 and qAER9 controlled plant height and heading date, respectively; while both of them increased yields simultaneously by suitable plant height and heading date without delay in the aerobic environment. The phenotypic differences between lowland rice and upland rice in the aerobic environment further supported the above results. We pyramided the two QTLs as corresponding molecular modules in the irrigated lowland rice MH63 background, and successfully developed a new upland rice variety named as Zhongkexilu 2. This study will lay the foundation for using aerobic adaptation QTLs in rice breeding programs and for further cloning the key genes involved in aerobic adaptation.

Characterization of genes responsive to aerobic conditions by transcriptomic and genomic analyses of upland rice

The adaptation to aerobic environments enables upland rice to produce a sustainable grain yield under suboptimal conditions. In order to understand the molecular mechanisms involved in providing the adaptation of upland rice to aerobic environments, we previously developed introgression lines (ILs) with the irrigated rice variety Minghui63 (MH63) as the recipient parent and the upland rice variety Luyin46 (LY46) as the donor parent for subsequent identification of the relevant genes. In this study, IL-U135 was analyzed in detail because of its adaptation to aerobic conditions. Transcriptome profiling revealed 20 and 8 differentially expressed genes (DEGs) between IL-U135 and MH63 under anaerobic and aerobic conditions, respectively. In contrast, 306 and 188 DEGs were identified between LY46 and MH63 under anaerobic and aerobic conditions, respectively. Gene ontology (GO) analysis indicated that the adaptation of upland rice to aerobic environments is intimately associated with CLV3/ESR-related (CLE) signal transduction pathways, CLAVATA1 kinase activity, and salicylic acid related metabolism biosynthetic pathways. The IL-U135, LY46 and MH63 genomes were resequenced to map the genes responsible for adaptations to aerobic conditions. One the basis of an integrated analysis of the transcriptomic and genomic profiles, we propose that genes encoding an NBS-LRR protein (LOC_Os11g10570) and an ATP-binding protein (LOC_Os11g31480) may contribute to the adaptation of upland rice to aerobic environments. Our results may provide new insights into the molecular mechanism underlying the adaptation of rice to aerobic conditions.

Enhanced yield performance of transgenic cry1C* rice in saline-alkaline soil

ABSTRACTChina has a large area of saline-alkaline land that can be utilized for the cultivation of transgenic rice. Therefore, the growth and reproductive behavior of transgenic rice are not only a problem for production that needs to be resolved, but also an important aspect of environmental risk assessment for saline alkali soil. In the present study, an insect-resistant transgenic cry1C* rice, T1C-19, was grown in farmland and saline-alkaline soils. The transcription and translation of the exogenous cry1C*, and vegetative and reproductive fitness, such as plant height, tiller number, biomass, filled grain number and weight per plant, were assessed. Our findings indicated that the transcription and translation of exogenous cry1C* gene in T1C-19 rice grown in saline-alkaline soil were lower than that grown in farmland; however, the correlation was not significant. The vegetative and reproductive growth abilities of T1C-19 were lower than that of the parental rice, Minghui63 (MH63), in farmland. In alkaline-saline soil, except for tiller number and biomass, there were no significant differences between T1C-19 and MH63 in other vegetative indices. In contrast, the reproductive indices of T1C-19 were significantly higher than those of MH63. The results suggested that T1C-19 had a strong reproductive capacity, and significantly reduced the loss of yield caused by insects, thereby leading to a higher yield than that of MH63 grown in saline-alkaline soils. This may promote the cultivation of saline-alkaline soil to permit farming of T1C-19 in China in the future, despite the possible increased ecological risks.

OsGL6, a conserved AP2 domain protein, promotes leaf trichome initiation in rice

Trichomes are specialized epidermal cells that play crucial roles in resisting environmental stress and enhancing plant development. In Arabidopsis thaliana, the main genes controlling trichome formation have been consecutively identified. However, few genes like this were reported in rice. In this study, we identified the hairy phenotype of indica variety 75-1-127. This was used to construct a segregation population with a cross of hairless variety Minghui63 (MH63) to fine map the trichome formation genes. Genetic analysis indicated that hairy phenotype was controlled by a pair of dominant genes on chromosome 6, which was designated as GLABRA6 (OsGL6). OsGL6 was an allele of HL6 gene whose sequences containing rich variations in genomes. Compared to wild type, the overexpressing transgenic lines revealed that OsGL6 promoted trichome initiation. We found that OsGL6 interacted with serine/threonine protein kinase OSK3 (OSK3) or COP9 signalosome complex subunit 5a (CSN5) in yeast. These results provide potential information for understanding the molecular mechanism of trichome formation in rice.

Fitness of F1 hybrids between stacked transgenic rice T1c-19 with cry1C*/bar genes and weedy rice

Compared to single-trait transgenic crops, stacked transgenic plants may be more prone to become weedy, and transgene flow from stacked transgenic plants to weedy relatives may pose a potential environmental risk because these hybrids could be more advantageous under specific environmental conditions. Evaluation of the potential environmental risk caused by stacked transgenes is essential for assessing the environmental consequences caused by crop-weed transgene flow. The agronomic performance of fitness-related traits was assessed in F1+ (transgene positive) hybrids (using the transgenic line T1c-19 as the paternal parent) in monoculture and mixed planting under presence or absence glufosinate pressure in the presence or absence of natural insect pressure and then compared with the performance of F1– (transgene negative) hybrids (using the non-transgenic line Minghui 63 (MH63) as the paternal parent) and their weedy rice counterparts. The results demonstrated that compared with the F1– hybrids and weedy rice counterparts, the F1+ hybrid presented higher performance (P<0.05) or non-significant changes (P>0.05) under natural insect pressure, respectively, lower performance (P<0.05) or non-significant changes (P>0.05) in the absence of insect pressure in monoculture planting, respectively. And compared to weedy rice counterparts, the F1+ hybrid presented higher performance (P<0.05) or non-significant changes (P>0.05) in the presence or absence of insect pressure in mixed planting, respectively. The F1+ hybrids presented non-significant changes (P>0.05) under the presence or absence glufosinate pressure under insect or non-insect pressure in monoculture planting. The all F1+ hybrids and two of three F1– hybrids had significantly lower (P<0.05) seed shattering than the weedy rice counterparts. The potential risk of gene flow from T1c-19 to weedy rice should be prevented due to the greater fitness advantage of F1 hybrids in the majority of cases.

Hd1 function conversion in regulating heading is dependent on gene combinations of Ghd7, Ghd8, and Ghd7.1 under long-day conditions in rice

Ghd7, Ghd8, Ghd7.1, and Hd1 are all pleiotropic genes regulating heading date, plant height, and yield in rice. Early studies showed that Hd1 promoted heading under short-day conditions (SD) and delayed heading under long-day conditions (LD). Recent studies found that Hd1 also promoted heading in some genetic backgrounds under LD. In this study, we developed a series of near-isogenic lines for Ghd7, Ghd8, Ghd7.1, and Hd1 in the Zhenshan 97 (ZS97) and Minghui 63 (MH63) backgrounds and recorded their heading dates. In the ZS97 background, Ghd7 alone triggered the conversion of Hd1 function from promoting to suppressing heading under LD. Ghd8 alone and Ghd7.1 alone did not promote but enhanced Ghd7-mediated Hd1 function conversion. In the MH63 background, Ghd7 alone and Ghd7.1 alone did not convert Hd1 function under LD, but they jointly promoted Hd1 function conversion. Conversion of Hd1 function occurred only under LD but not SD. Transcript analysis showed that downregulation or upregulation of Ehd1 and Hd3a by Hd1 in the lines under LD was determined by genetic background. In summary, multiple gene combinations of Ghd7, Ghd7.1, and Ghd8 and unknown genes caused conversion of Hd1 function. Moreover, the different gene combinations had a big difference in photoperiod sensitivities. These findings lay a solid foundation for further unveiling the molecular mechanism of Hd1 function conversion and provide guidance for heading date improvement in rice.

Identification and Analysis of Micro-Exon Genes in the Rice Genome.

Micro-exons are a kind of exons with lengths no more than 51 nucleotides. They are generally ignored in genome annotation due to the short length, whereas recent studies indicate that they have special splicing properties and important functions. Considering that there has been no genome-wide study of micro-exons in plants up to now, we screened and analyzed genes containing micro-exons in two indica rice varieties in this study. According to the annotation of Zhenshan 97 (ZS97) and Minghui 63 (MH63), ~23% of genes possess micro-exons. We then identified micro-exons from RNA-seq data and found that >65% micro-exons had been annotated and most of novel micro-exons were located in gene regions. About 60% micro-exons were constitutively spliced, and the others were alternatively spliced in different tissues. Besides, we observed that approximately 54% of genes harboring micro-exons tended to be ancient genes, and 13% were Oryza genus-specific. Micro-exon genes were highly conserved in Oryza genus with consistent domains. In particular, the predicted protein structures showed that alternative splicing of in-frame micro-exons led to a local structural recombination, which might affect some core structure of domains, and alternative splicing of frame-shifting micro-exons usually resulted in premature termination of translation by introducing a stop codon or missing functional domains. Overall, our study provided the genome-wide distribution, evolutionary conservation, and potential functions of micro-exons in rice.

Effects of straw leachates from Cry1C-expressing transgenic rice on the development and reproduction of Daphnia magna

The transgenic rice line T1C-19 provides high resistance to lepidopteran pests because of the synthesis of the Bacillus thuringiensis (Bt) insecticidal protein Cry1C. It thus shows good prospect for commercial planting in China. Species of Cladocera, an order of aquatic arthropods commonly found in aquatic ecosystems such as rice paddies, might be exposed to the insecticidal protein released from Bt-transgenic rice-straw residues. For the study reported herein, we used Daphnia magna (water flea) as a representative of Cladocera to evaluate whether aquatic arthropods are adversely affected when exposed to Bt rice-straw leachates. We exposed D. magna to M4 medium containing various volume percentages of medium that had been incubated with T1C-19 rice straw or rice straw from its non-transformed near-isoline Minghui 63 (MH63) for 21 days. Compared with pure M4 medium (control), the fitness and developmental and reproduction parameters of D. magna decreased significantly when exposed to rice-straw leachates; conversely, no significant differences between the T1C-19 and MH63 rice-straw leachate treatments were observed, indicating that the Bt rice straw leachate did not adversely affect this non-target species.

Phloem transport capacity of transgenic rice T1c-19 (Cry1C*) under several potassium fertilizer levels.

Genetic modification of Cry-proteins from Bacillus thuringiensis (Bt) is a common practice in economically important crops to improve insecticide resistance and reduce the use of pesticides. However, introduction of these genes can have unintended side effects, which should be closely monitored for effective breeding and crop management. To determine the potential cause of these negative effects, we explored assimilate partitioning in the transgenic Bt rice line T1c-19 (Cry1C*), which was compared with that of its wild-type counterpart Minghui 63 (MH63) under different potassium fertilization application treatment conditions. In a pot experiment, 0, 0.4, and 0.6 g K2O was applied per kg of dry soil to determine the phloem transport characteristics of the two rice lines. We used a variety of assessment indicators ranging from morphological to physiological aspects, including the number of large and small vascular bundles in the neck internode at the heading stage, the diameter and bleeding intensity of the neck internode at the filling stage, and the content and apparent ratio of transferred non-structural carbohydrates (NSC) in the culm and sheath from the heading to maturing stages. The K utilization and grain yield at the maturing stage were also concerned. Results presented that the mean setting rate and grain yield of T1c-19 (Cry1C*) decreased by 22.3% and 26.2% compared to those in MH63, respectively. Compared to MH63, the K concentration and accumulation were significantly higher in the culms and leaves, but significantly lower in grain of T1c-19 (Cry1C*). T1c-19 (Cry1C*) had less apparent NSC efflux in the culm and sheath, fewer small vascular bundles, and a smaller diameter and bleeding intensity of the neck internode than MH63. In addition, linear correlation analysis indicated that there were positive correlations among grain yield, setting rate, the apparent NSC efflux in the culm and sheath, number of small vascular bundles, and the neck internode diameter and bleeding intensity. These unintended effects may directly or indirectly be caused by insertion of exogenous Bt (Cry1C*) gene, which should be further considered in the future breeding of transgenic crops.

Alternative functions of Hd1 in repressing or promoting heading are determined by Ghd7 status under long-day conditions

Previous studies suggested that Hd1 promoted heading under short-day conditions (SD) and delayed heading under long-day conditions (LD). However in this study, Hd1 was demonstrated to consistently promote heading date in Zhenshan 97 (ZS97) background by upregulating Ehd1, Hd3a and RFT1 expression under both SD and LD. While the high photoperiod sensitivity of Hd1 was observed in Minghui 63 (MH63) background, with heading being suppressed in LD but promoted in SD. Comparative analysis of two sets of near isogenic lines of Hd1 in MH63 and ZS97 backgrounds indicated that the alternative functions of Hd1 in promoting or suppressing heading under LD are dependent on the previously cloned flowering repressor gene Ghd7. The interaction between proteins Ghd7 and Hd1 occurred through binding of the CCT domain of Ghd7 to the transcription-activating domain of Hd1, resulting in suppression of Ehd1 and florigen gene expression. The involvement of the transcription-activating domain of Hd1 in this protein-protein interaction probably blocked or weakened its transcriptional activity. These findings suggest that Hd1 alone essentially acts as a promoter of heading date, and the protein interaction between Ghd7 and Hd1 determines photoperiod sensitivity and integrated Hd1-mediated and Ehd1-mediated flowering pathways in rice.

Soil nematode community varies between rice cultivars but is not affected by transgenic Bt rice expressing Cry1Ab or Cry1Ab/Cry1Ac

Insecticidal crystal (Cry) proteins produced by transgenic Bacillus thuringiensis (Bt) rice that enter the soil via root exudation and plant residues may be harmful to non-target soil organisms. We conducted a 3-year field investigation to determine if soil nematode abundance and diversity were affected by exposure to two transgenic Bt rice cultivars, compared to their non-transgenic near isolines. Near isolines were Kemingdao (KMD-Bt) expressing the single Cry1Ab gene and its non-Bt near isoline Xiushui-11 (XSD), as well as Huahui-1 (HH-Bt) expressing the fused Cry1Ab/Cry1Ac gene and its non-Bt near isoline Minghui-63 (MH). Nematode variables including community composition, abundance, trophic groups, and most of the common genera differed significantly between the rice cultivars. However, these nematode variables were similar under transgenic Bt rice and its non-Bt near isoline, although higher Shannon’s index value and Pielou’s index value were found in soils planted with Bt rice than the non-Bt near isoline. During this 3-year field study, gene modification (single Cry1Ab gene and fused Cry1Ab/Cry1Ac gene) supports a more uniform distribution of nematode species but had no effect on soil nematode abundance and community composition. We conclude that continuous cultivation of KMD-Bt and HH-Bt rice varieties for 3 years is not detrimental to soil nematode communities under field conditions.

Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data.

Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.

Non-target effects of Bt transgenes on grain yield and related traits of an elite restorer rice line in response to nitrogen and potassium applications

Using a transgenic Bacillus thuringiensis (Bt) crop is reportedly one of the most effective ways to improve insect resistance; however, the insertion of external Bt genes may affect the non-target growth characteristics of crop. Field experiments were conducted to investigate the yield performance and physiological traits in an elite restorer rice line, Minghui63 (MH63) with Cry1C*, Cry2A* or Cry1Ab/Ac genes under three fertilizer applications [zero nitrogen fertilizer (N0), zero potassium fertilizer (K0) and recommended nitrogen and potassium fertilizer (NK)]. In comparison with MH63, MH63 (Cry1C*) experienced declining grain yields of 15.3–31.0% under different fertilizer applications because of the decreased setting rates associated with the poorer matter transport-related traits. The grain yield of MH63 (Cry2A*) was averagely 18.4% lower than MH63 in only the N0 treatment due to the accelerated leaf senescence, which was correlated with the higher ratios of the Bt protein content to the soluble protein content (BTC/SPC) in the MH63 (Cry2A*) leaves at the filling stage (FS). The yield performance of MH63 (Cry1Ab/Ac) was identical to MH63. In addition, no significant differences were recorded between Bt-MH63 and MH63 regarding N and K assimilation. Hence, our results suggest that the Cry1C* and Cry2A* transgenes affect the yield performance and related traits of MH63 in response to nitrogen and/or potassium applications.

Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields.

As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect non-target organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs.