Changes in bacterial community and abundance of functional genes in paddy soil with cry1Ab transgenic rice

A field experiment involving cry1Ab transgenic rice(GM) and its parental non-cry1Ab rice(M) has been on-going since 2014. The diversity of the bacterial communities and the abundance of the microbial functional genes which drive the conversion of nitrogen in paddy soil were analyzed during the growth period of rice in the fifth year of the experiment, using 16 S rRNAbased Illumina Mi Seq and real-time PCR on the amoA, nirS and nirK genes. The results showed no differences in the alpha diversity indexes of the bacterial communities, including Chao1, Shannon and Simpson, between the fields cultivated with line GM and cultivar M at any of the growth stages of rice. However, the bacterial communities in the paddy soil with line GM were separated from those of paddy soil with cultivar M at each of the growth stages of rice, based on the unweighted Uni Frac NMDS or PCoA. In addition, the analyses of ADONIS and ANOSIM, based on the unweighted Uni Frac distance, indicated that the above separations between line GM and cultivar M were statistically significant(P<0.05) during the growth season of rice. The increases in the relative abundances of Acidobacteria or Bacteroidetes, in the paddy soils with line GM or cultivar M, respectively, led to the differences in the bacterial communities between them. At the same time, functional gene prediction based on Illumina Mi Seq data suggested that the abundance of many functional genes increased in the paddy soil with line GM at the maturity stage of rice, such as genes related to the metabolism of starch, amino acids and nitrogen. Otherwise, the copies of bacterial amo A gene, archaeal amo A gene and denitrifying bacterial nir K gene significantly increased(P<0.05 or 0.01) in the paddy soil with line GM. In summary, the release of cry1Ab transgenic rice had effects on either the composition of bacterial communities or the abundance of microbial functional genes in the paddy soil.

Transcriptome and metabolome profiling of unheading in F1 hybrid rice

Heading date is a crucial agronomic trait.However,rice usually delays heading due to the photoperiod,temperature,hormones or age.The present research was conducted to analyze the mechanism cotrlling heading date in F,hybrid rice.We constructed two test-crossing populations using two introgression lines(ILs),P20 and P21 coming from SH527/FH838 as the male parent,respectively,and male sterile line Jin23A as the female parent.Meanwhile,the F,hybrids of H20,obtained by mating P20 with Jin23A and having no heading,and H21,from the crossing between P21 and Jin23A having normal heading,were both observed under long days.Here,we analyzed the photoperiodic response of F,hybrids by transcriptome and metabolome profiling.The greater differences displayed in the transcriptome and the metabolome were caused by photoperiod(exogenous)instead of genes(endogenous).The coping mechanism resulted from long days(LD)in H20,leading to differences in the circadian rhythm and glutathione metabolism relative to other samples.The circadian oscillator and GSH/GSSG cycle typically regulate ROS homeostasis,and both of them are responsible for modulating ROS in H20 under LD condition.Both circadian rhythm genes and the reported genes related to heading date function via the DHD1/OsMFT1-Ehd1-RFT1-OsMADS14/OsMADS18 pathway and the glutathione metabolism pathway by regulating oxidative reduction processes.Both pathways are involved in the heading process and they interacted through the oxidative reduction process which was induced by photoperiod regulation,and all of them collectively modulated the heading process.The results of this study will be helpful for unraveling the mechanism of F,hybrid responses to unheading under LD condition.

Field Evaluation of Growth and Yield of Two Local Rice Varieties (Tox-728-1 and Madjitolngar) in Response to Indogenous Mycorrhizal Inoculation in South-Chad

This study was carried out to find out how dependent are two local rice varieties (Magitolngar and Tox-728-1) to inoculation with selected endogenous arbuscular mycorrhizal fungi (AMF) in a field where they were isolated. The multi-indigenous endomycorrhiza spores previously isolated and identified were the active ingredient in the production of bioinoculants used for this purpose. Spores massively multiplied from the rhizosphere of each rice variety in each of the four locally collected soils substrates were harvested to constitute 08 AMF inoculants (Kema = T1;Lama = T2;Latox = T3;Ndjatox = T4;Koloma = T5;Kolotox = T6;Ndjama = T7;Ketox = T8). These inoculants were field tested on the two rice varieties at Kelo, under a complete randomized block design, comprising 10 treatments (8 inoculants, 01 positive control = T9, 01 negative control = T10), each of which was repeated thrice. The analysis of data indicates that AMF-inoculated plants were taller, developed more tillers/plant, and produced more rice grains/panicle than non-AMF-inoculated plants for both studied rice varieties. The rice variety Madjitolngar yielded more grains (7.5 t/ha) than the Tox-728-1 variety (5.8 t/ha). Moreover, inoculants Koloma (T1), Latox (T3) and Kolotox (T6) on the one hand, Koloma (T1) and Ketox (T8) on the other hand, were best suited for the improvement of growth and yield of the rice varieties Madjitolngar and Tox-728-1 respectively, tested under field conditions at Kelo. In this study, the two rice varieties have shown a dependency to endomycorrhizal symbiosis at Kelo, and therefore, an industrial-scale production of efficient endomycorrhal inoculants is necessary to sustainably boost the productivity of this important crop in Chad.

Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

利用CRISPR/Cas9基因编辑技术构建水稻OsSLR1敲除突变体

GA是种子萌发的促进因子,能解除种子休眠和刺激萌发,GA的信号通路可以通过对DELLA蛋白的去抑制作用来完成的。通过对不同物种的DELLA蛋白功能的研究,发现不同物种的DELLA蛋白的功能具有高度保守性,本实验主要研究水稻中的DELLA蛋白SLR1,利用CRISPR/Cas9基因编辑技术构建OsSLR1基因敲除突变株。首先,通过对OsSLR1的结构和功能域进行分析比对,最终选择3个靶点;然后,利用重组法构建CRISPR/Cas9敲除载体;最后,通过农杆菌介导法,将敲除载体转染成熟的日本晴水稻愈伤,待培育出幼苗,通过PCR和Western Blot进行鉴定。最终筛选得到1株Os SLR1敲除突变株系slr1-1,通过观察突变体萌发表型,发现slr1-1突变体第2天发芽率显著高于日本晴,而且slr1-1突变体的生长速度显著快于日本晴。通过不同浓度PAC对slr1-1突变体萌发表型的影响,发现水稻的DELLA蛋白SLR1敲出的突变体种子萌发不受PAC抑制,表明OsSLR1是通过调控GA信号途径影响水稻种子的萌发,详细的调控机制和互作蛋白等还有待深入的研究。通过对SLR1调控GA信号通路控制水稻的萌发的研究,提高水稻的萌发率,保持优良的水稻种子萌发力,为解决水稻种植上的出芽率不齐等问题提供新的品种。

Pyramiding of the dep1-1 and NAL1NJ6 alleles achieves sustainable improvements in nitrogen-use efficiency and grain yield in japonica rice breeding

Rice is one of the most important cereal crops in the world, and a substantial increase in grain yield is necessary for food security. However, high yields of semidwarf modern rice varieties are heavily dependent on the application of mineral nitrogenous fertilizer (Tilman et al., 2002;Sun et al., 2014). Nitrogen (N)-insensitive sponses associated with reduced N-use efficiency (NUE) is a major characteristic of the green revolution varieties (GRVs), in which the growth-inhibiting protein SLENDER RICE1 (SLR1) accumulates (Li et al., 2018). Unfortunately, increasing the level of N fertilizer use to reach the full yield potential of GRVs is subject to diminishing returns, quite apart from its deleterious effect on the environments (Rahn et al., 2009;Liu et al., 2015). Therefore, there is an urgent need to develop new rice GRVs that increase NUE while maintaining their high yields. Recently, several genes (e.g., DEP1, OsNRTl.lB, OsNRT2.3b, ARE1 and GRF4) responsible for improved NUE have been identified in rice (Sun et al.. 2014;Hu et al., 2015;Fan et al., 2016;Wang et al., 2018;Li et al., 2018). More importantly, boosting the activity of the transcription factor GRF4 overcomes the ability of SLR1 to prevent the GRF4-GIF1 interaction, which in turn promotes the coordinated expression of the genes involved in N assimilation and carbon fixation and consequently enhances the NUE of rice GRVs, thereby improving our ability to grow crops sustainably (Li et al., 2018). However, current understanding of the genetic basis for improving NUE remains at the level of identification of a number of quantitative trait loci (QTLs), without any understanding of the nature of the gene products.

Targeted mutagenesis in rice using CRISPR-Cpf1 system

Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.

Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems

The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.

The pleiotropic ABNORMAL FLOWER AND DWARF1 affects plant height, floral development and grain yield in rice

Moderate plant height and successful establishment of reproductive organs play pivotal roles in rice grain production. The molecular mechanism that controls the two aspects remains unclear in rice. In the present study,we characterized a rice gene, ABNORMAL FLOWER AND DWARF1（AFD1） that determined plant height, floral development and grain yield. The afd1 mutant showed variable defects including the dwarfism, long panicle, low seed setting and reduced grain yield. In addition, abnormal floral organs were also observed in the afd1 mutant including slender and thick hulls, and hull-like lodicules.AFD1 encoded a DUF640 domain protein and was expressed in all tested tissues and organs. Subcellular localization showed AFD1-green fluorescent fusion protein（GFP） was localized in the nucleus. Meantime, our results suggested that AFD1 regulated the expression of cell division and expansion related genes.