本研究从历史数据以及公开文章中收集了57个与大豆种子可溶性糖含量相关的数量性状位点(QTLs)。通过meta、overview和共线性分析来细化QTL区间，共得到8个共有QTL。使用染色体片段代换系(CSSLs)群体对这些共有QTL进行验证，选择了两个包含共有QTL和有导入片段的品系：其中一个与共有QTL区间相关的一个品系可溶性糖含量较高，另一个品系可溶性糖含量较低。在种子发育的早期、中期和晚期对这两个品系进行转录组测序，分别鉴定出158个、109个和329个差异表达基因。通过重测序数据和共有QTL区间分析，在野生大豆遗传导入片段中鉴定出3个候选基因Glyma.19G146800、Glyma.19G122500和Glyma.19G128500。通过对两个CSSL亲本SN14和ZYD00006的序列比对，发现Glyma.19G122500编码序列发生单核苷酸多态性(SNP)突变，导致氨基酸序列发生非同义突变，影响了蛋白质结构。基于这一SNP，我们开发了竞争性等位基因特异性PCR (KASP)标记，并将其用于CSSL品系的鉴定。这些结果为进一步鉴定大豆可溶性糖相关基因及进一步育种奠定了基础。

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 

Received  11 November, 2017    Accepted  28 January, 2018

The increasing atmospheric carbon dioxide concentration, caused by fossil fuel combustion and deforestation, plays an important role in plant growth and development. Wheat, as a major staple crop, adapts to climate change by tuning its inherent molecular mechanism, which is not well understood. The present study employed the RNA-Seq method to generate transcriptome profiles of the wheat Norin 10 in response to elevated CO₂ in comparison with ambient CO₂. The 10 895 787 high-quality clean reads of Norin 10 were assembled de novo using Trinity (without a reference genome) resulting in a total of 18 206 candidate transcripts with significant BLAST matches. GO enrichment analysis of Norin 10 at different CO₂ concentrations showed that some functional genes related to plastids, precursor metabolites, and energy, thylakoid and photosynthesis were apparently enriched at elevated CO₂ (550 μmol mol^–1) in contrast to that at ambient CO₂ (400 μmol mol–1); these findings were further confirmed by RT-PCR analysis. The findings demonstrated the specific effects of elevated CO₂ during long-term period in free air CO2 enrichment (FACE) on transcriptome response of the high yielding wheat variety, Norin 10, which has a large spike.

    Quorum sensing (QS) is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fistulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers (AI) both in vivo and in vitro and to clone the luxS gene that encoded autoinducer-2 (AI-2) synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber (NDF) and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones (AHLs) activity was determined using gas chromatography-mass spectrometer (GC-MS) analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of luxS in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of luxS.

Nitrogen (N) is one of the macronutrients required for plant growth, and reasonable application of N fertilizers can increase crop yields and improve their quality. However, excessive application of N fertilizers will decrease N use efficiency and also lead to increases in N2O emissions from agricultural soils and many other environmental issues. Research on the effects of different N fertilizer management practices on wheat yields and N2O emissions will assist the selection of effective N management measures which enable achieving high wheat yields while reducing N2O emissions. To investigate the effects of different N management practices on wheat yields and soil N2O emissions, we conducted field trials with 5 treatments of no N fertilizer (CK), farmers common N rate (AN), optimal N rate (ON), 20% reduction in optimal rate+dicyandiamide (ON80%+DCD), 20% reduction in optimal rate+nano-carbon (ON80%+NC). The static closed chamber gas chromatography method was used to monitor N2O emissions during the wheat growing season. The results showed that there were obvious seasonal characteristics of N2O emissions under each treatment and N2O emissions were mainly concentrated in the sowing- greening stage, accounting for 54.6–68.2% of the overall emissions. Compared with AN, N2O emissions were decreased by 23.1, 45.4 and 33.7%, respectively, under ON, ON80%+DCD and ON80%+NC, and emission factors were declined by 22.2, 66.7 and 33.3%, respectively. Wheat yield was increased significantly under ON80%+DCD and ON80%+NC by 12.3 and 11.9%, respectively, relative to AN while there was no significant change in yield in the ON treatment. Compared with ON, overall N2O emissions were decreased by 29.1 and 13.9% while wheat yields improved by 18.3 and 17.9% under ON80%+DCD and ON80%+NC, respectively. We therefore recommend that ON80%+DCD and ON80%+NC be referred as effective N management practices increasing yields while mitigating emissions.

Mung bean (Vigna radiata L.) has the potential to establish symbiosis with rhizobia, and symbiotic association of soil micro flora may facilitate the photosynthesis and plant growth response to elevated [CO2]. Mung bean was grown at either ambient CO2 400 μmol mol–1 or [CO2] ((550±17) μmol mol–1) under free air carbon dioxide enrichment (FACE) experimental facility in North China. Elevated [CO2] increased net photosynthetic rate (Pn), water use efficiency (WUE) and the non-photochemical quenching (NPQ) of upper most fully-expanded leaves, but decreased stomatal conductance (Gs), intrinsic efficiency of PSII (Fv´/Fm´), quantum yield of PSII (ΦPSII) and proportion of open PSII reaction centers (qP). At elevated [CO2], the decrease of Fv´/Fm´, ΦPSII, qP at the bloom stage were smaller than that at the pod stage. On the other hand, Pn was increased at elevated [CO2] by 18.7 and 7.4% at full bloom (R2) and pod maturity stages (R4), respectively. From these findings, we concluded that as a legume despite greater nutrient supply to the carbon assimilation at elevated [CO2], photosynthetic capacity of mung bean was still suppressed under elevated [CO2] particularly at pod maturity stage but plant biomass and yield was increased by 11.6 and 14.2%, respectively. Further, these findings suggest that even under higher nutrient acquisition systems such as legumes, nutrient assimilation does not match carbon assimilation under elevated [CO2] and leads photosynthesis down-regulation to elevated [CO2].

Soybean is a major cash crop in the world, and its oil content was one of the very important traits. Therefore, the study of gene mapping for oil content in soybean is very important for breeding application. At present, at least 130 QTL loci for soybean oil content have been published; however, the mapping results of oil content were dispersed and a coalescent public map should be established to integrate the published QTLs, and to more efficiently mine genes based on the metaanalysis method of the bioinformatics tools. This study was to construct an integrated map of QTLs for soybean oil content and accelerate the application of bioinformation resource related to oil content improvement in the practice of soybean breeding. We collected information of 130 QTLs reported over the past 20 yr for soybean oil content and used the Software BioMercator 2.1 to project QTLs from their own maps onto a reference map, which was an early-integrated map constructed by Song (2004) for oil-content quantitative trait loci (QTLs) in soybean. Gene mining was performed based on the meta-analysis by running the local ver. GENSCAN and InterProScan. The confidence interval of QTLs was efficaciously narrowed using the meta-analysis method, and 25 consensus QTLs were mapped on the reference map. Using a local version of GENSCAN, 12 805 sequences in the consensus QTL intervals were predicted. With BLAST, these predicted sequences were aligned to gene sequences from the International Protein Index database using InterProScan locally. Thirteen predicted genes were in the class of the geme ontology (GO) accession (0006631), which were involved in the fatty acid metabolic process. These genes were analyzed using BLAST at the NCBI website to examine whether they were related to oil content. Six genes were found in the oil-synthesis pathway. Twenty-five consensus QTLs and six genes were found in the oil-synthesis pathway. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, and these genes will facilitate the researches on the gene mining of oil synthesis and molecular breeding in soybean.