Compared with sole nitrate (NO₃^–) or sole ammonium (NH₄⁺) supply, mixed nitrogen (N) supply may promote growth of maize seedlings.  Previous study suggested that mixed N supply not only increased photosynthesis rate, but also enhanced leaf growth by increasing auxin synthesis to build a large sink for C and N utilization.  However, whether this process depends on N absorption is unknown.  Here, maize seedlings were grown hydroponically with three N forms (NO₃^– only, 75/25 NO₃^–/NH₄⁺ and NH₄⁺ only).  The study results suggested that maize growth rate and N content of shoots under mixed N supply was little different to that under sole NO₃^– supply at 0–3 d, but was higher than under sole NO₃^– supply at 6–9 d.  ¹⁵N influx rate under mixed N supply was greater than under sole NO₃^– or NH₄⁺ supply at 6–9 d, although NO₃^– and NH₄⁺ influx under mixed N supply were reduced compared to sole NO₃^– and NH₄⁺ supply, respectively.  qRT-PCR determination suggested that the increased N absorption under mixed N supply may be related to the higher expression of NO₃^– transporters in roots, such as ZmNRT1.1A, ZmNRT1.1B, ZmNRT1.1C, ZmNRT1.2 and ZmNRT1.3, or NH₄⁺ absorption transporters, such as ZmAMT1.1A, especially the latter.  Furthermore, plants had higher nitrate reductase (NR) glutamine synthase (GS) activity and amino acid content under mixed N supply than when under sole NO₃^– supply.  The experiments with inhibitors of NR reductase and GS synthase further confirmed that N assimilation ability under mixed N supply was necessary to promote maize growth, especially for the reduction of NO₃^– by NR reductase.  This research suggested that the increased processes of NO₃^– and NH₄⁺ assimilation by improving N-absorption ability of roots under mixed N supply may be the main driving force to increase maize growth.

Numbers of vertebrae is an important economic trait associated with body size and meat productivity in animals.  However, the genetic basis of vertebrae number in donkey remains to be well understood.  The aim of this study was to identify candidate genes affecting the number of thoracic (TVn) and the number of lumbar vertebrae (LVn) in Dezhou donkey.  A genome-wide association study was conducted using whole genome sequence data imputed from low-coverage genome sequencing.  For TVn, we identified 38 genome-wide significant and 64 suggestive SNPs, which relate to 7 genes (NLGN1, DCC, SLC26A7, TOX, WNT7A, LOC123286078, and LOC123280142).  For LVn, we identified 9 genome-wide significant and 38 suggestive SNPs, which relate to 8 genes (GABBR2, FBXO4, LOC123277146, LOC123277359, BMP7, B3GAT1, EML2, and LRP5).  The genes involve in the Wnt and TGF-β signaling pathways and may play an important role in embryonic development or bone formation and could be good candidate genes for TVn and LVn.

Milling and appearance quality are important contributors to rice grain quality.  Abundant genetic diversity and a suitable environment are crucial for rice improvement.  In this study, we investigated the milling and appearance quality-related traits in a panel of 200 japonica rice cultivars selected from Liaoning, Jilin and Heilongjiang provinces in Northeast China.  Pedigree assessment and genetic diversity analysis indicated that cultivars from Jilin harbored the highest genetic diversity among the three geographic regions.  An evaluation of grain quality indicated that cultivars from Liaoning showed superior milling quality, whereas cultivars from Heilongjiang tended to exhibit superior appearance quality.  Single- and multi-locus genome-wide association studies (GWAS) were conducted to identify loci associated with milling and appearance quality-related traits.  Ninety-nine significant single-nucleotide polymorphisms (SNPs) were detected.  Three common SNPs were detected using the mixed linear model (MLM), mrMLM, and FASTmrMLM methods.  Linkage disequilibrium decay was estimated and indicated three candidate regions (qBRR-1, qBRR-9 and qDEC-3) for further candidate gene analysis.  More than 300 genes were located in these candidate regions.  Gene Ontology (GO) analysis was performed to discover the potential candidate genes.  Genetic diversity analysis of the candidate regions revealed that qBRR-9 may have been subject to strong selection during breeding.  These results provide information that will be valuable for the improvement of grain quality in rice breeding.

Wheat grain yield is generally sink-limited during grain filling.  The grain-filling rate (GFR) plays a vital role but is poorly studied due to the difficulty of phenotype surveys.  This study explored the grain-filling traits in a recombinant inbred population and wheat collection using two highly saturated genetic maps for linkage analysis and genome-wide association study (GWAS).  Seventeen stable additive quantitative trait loci (QTLs) were identified on chromosomes 1B, 4B, and 5A.  The linkage interval between IWB19555 and IWB56078 showed pleiotropic effects on GFR₁, GFR_max, kernel length (KL), kernel width (KW), kernel thickness (KT), and thousand kernel weight (TKW), with the phenotypic variation explained (PVE) ranging from 13.38% (KW) to 33.69% (TKW).  198 significant marker-trait associations (MTAs) were distributed across most chromosomes except for 3D and 4D.  The major associated sites for GFR included IWB44469 (11.27%), IWB8156 (12.56%) and IWB24812 (14.46%).  Linkage analysis suggested that IWB35850, identified through GWAS, was located in approximately the same region as QGFRmax2B.3-11, where two high-confidence candidate genes were present.  Two important grain weight (GW)-related QTLs colocalized with grain-filling QTLs.  The findings contribute to understanding the genetic architecture of the GFR and provide a basic approach to predict candidate genes for grain yield trait QTLs.

Carex rigescens (Franch.) V. Krecz is a wild turfgrass perennial species in the Carex genus that is widely distributed in salinised areas of northern China.  To investigate genome-wide salt-response gene networks in C. rigescens, transcriptome analysis using high-throughput RNA sequencing on C. rigescens exposed to a 0.4% salt treatment (Cr_Salt) was compared to a non-salt control (Cr_Ctrl).  In total, 57 742 546 and 47 063 488 clean reads were obtained from the Cr_Ctrl and Cr_Salt treatments, respectively.  Additionally, 21 954 unigenes were found and annotated using multiple databases.  Among these unigenes, 34 were found to respond to salt stress at a statistically significant level with 6 genes up-regulated and 28 down-regulated.  Specifically, genes encoding an EF-hand domain, ZFP and AP2 were responsive to salt stress, highlighting their roles in future research regarding salt tolerance in C. rigescens and other plants.  According to our quantitative RT-PCR results, the expression pattern of all detected differentially expressed genes were consistent with the RNA-seq results.  Furthermore, we identified 11 643 simple sequence repeats (SSRs) from the unigenes.  A total of 144 amplified successfully in the C. rigescens cultivar Lüping 1, and 69 of them reflected polymorphisms between the two genotypes tested.  This is the first genome-wide transcriptome study of C. rigescens in both salt-responsive gene investigation and SSR marker exploration.  Our results provide further insights into genome annotation, novel gene discovery, molecular breeding and comparative genomics in C. rigescens and related grass species.

Alfalfa (Medicago sativa L.) is an important forage crop and is also a target of many fungal diseases including Fusarium spp.  As of today, very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against Fusarium spp. and specifically against Fusarium proliferatum, the causal agent of alfalfa root rot.  In this study, we used a proteomic approach to identify inducible proteins in alfalfa during a compatible interaction with F. proliferatum strain YQC-L1.  Samples used for the two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry were from roots and leaves of alfalfa cultivar AmeriGraze 401+Z and WL656HQ.  Plants were grown in hydroponic conditions and at 4 days post inoculation with YQC-L1.  Our disease symptom assays indicated that AmeriGraze 401+Z  was tolerant to YQC-L1 infection while WL656HQ was highly susceptible.  Analysis of differentially expressed proteins found in the 2-DE was further characterized using the MASCOT MS/MS ion search software and associated databases to identify multiple proteins that might be involved in F. proliferatum resistance.  A total of 66 and 27 differentially expressed proteins were found in the roots and leaves of the plants inoculated with YQC-L1, respectively.  These identified proteins were placed in various categories including defense and stress response related metabolism, photosynthesis and protein synthesis.  Thirteen identified proteins were validated for their expressions by quantitative reverse transcription (qRT)-PCR.  Our results suggested that some of the identified proteins might play important roles in alfalfa resistance against Fusarium spp.  These finding could facilitate further dissections of molecular mechanisms controlling root rot disease in alfalfa and potentially other legume crops.   

Creating a crop-heterogeneous system by intraspecific mixtures of different rice varieties can substantially reduce blast diseases. Such variety mixtures provide an ecological approach for effective disease control, maintaining high yields with the minimum fungicide applications. Whether such an approach is universally applicable for random rice variety combinations and what is the variation pattern of the diseases under intercropping still remains unclear. We conducted two-year large-scale field experiments involving 47 rice varieties/lines and 98 variety-combinations to compare the occurrence of rice blast in monoculture and intercropping plots at multiple sites. In the experiments, the plant height of the selected traditional varieties was about 30 cm taller, and their life cycle was 10 days longer, than that of the improved rice varieties. The monoculture included either traditional or modern rice varieties grown in separate plots. The intercropping included both traditional and modern rice varieties planted together in the same plots. Results from the field experiments under natural disease conditions demonstrated significant reduction for rice blast disease in intercropping plots, compared with that in monoculture plots. For traditional varieties, the average blast incidence reduced from ~26% in monoculture to ~10% in intercropping, and the disease severity reduced from ~17 in monoculture to ~5 in intercropping. For modern varieties, the average blast incidence reduced from ~19% in monoculture to ~10% in intercropping, and the severity from ~10 in monoculture to ~4 in intercropping. Traditional rice varieties (~72%) had a much greater increase in the efficiency of disease control than modern varieties (~60%). In addition, substantially lower values of variance in the blast incidence and severity was detected among the variety combinations in intercropping plots than in monoculture plots. Based on these results, we conclude that the intercropping or mixture of rice varieties greatly reduces the occurrence and variation of rice blast disease in particular variety combinations, which makes the intercropping system more stable and consistent for disease suppression on a large scale of rice cultivation.

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase (N6AMT) is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens (Nlu-N6AMT) and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenineMlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding dsRNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

    Alfalfa is widely grown and is one of the most important forage crops in the world, but its growth and biomass production are markedly reduced under salt and drought stress, particularly during the early seedling stage. To identify the mechanisms behind salt and drought responsiveness at the alfalfa seedling stage, the proteins expressed were analyzed under no-treatment, 200 mol L^–1 NaCl and 180 g L^–1 PEG treatment conditions during the seedling stage. Out of more than 800 protein spots detected on two-dimensional electrophoresis (2-DE) gels, 35 proteins showed statistically significant responses (P<0.05) to NaCl and PEG stress, which were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and 32 proteins were positively identified. The identified proteins were divided into seven functional categories: photosynthetic metabolism, protein biosynthesis, folding and assembly, carbohydrate metabolism-associated proteins, stress defense related protein, metabolism of nucleic acid, other function categories and unknown proteins. Our results suggested that these proteins may play roles in alfalfa adaptation to salt and drought stress. Further study of these proteins will provide insights into the molecular mechanisms of abiotic stress and the discovery of new candidate markers in alfalfa.

One concern about the use of transgenic plants is their potential risk to natural enemies. In this study, using the eggs of the rice brown planthopper, Nilaparvata lugens, as a food source, we investigated the effects of Cry1Ab rice on the biological characteristics and functional response of an important predator Cyrtorhinus lividipennis. The results showed that the survival ability (adult emergence rate and egg hatching rate), development (egg duration, nymphal developmental duration), adult fresh weight, adult longevity and fecundity of C. lividipennis on Bt rice plants were not significantly different compared to those on non-Bt rice plants. Furthermore, two important parameters of functional response (instantaneous search rate and handling time) were not significantly affected by Bt rice. In conclusion, the tested Cry1Ab rice does not adversely impact the biological character and functional response of C. lividipennis.

Pasteurella multocida, a Gram-negative nonmotile coccobacillus, is the causative agent of fowl cholera, bovine hemorrhagic septicemia, enzoonotic pneumonia and swine atropic rhinitis. Two filamentous hemagglutinin genes, fhaB1 and fhaB2, are the potential virulence factors. In this study, an inactivation fhaB1 mutant of P. multocida in avian strain C48-102 was constructed by a kanamycin-resistance cassette. The virulence of the fhaB1 mutant and the wild type strain was assessed in chickens by intranasal and intramuscular challenge. The inactivation of fhaB1 resulted in a high degree of attenuation when the chickens were challenged intranasally and a lesser degree when challenged intramuscularly. The fhaB1 mutant and the wild type strain were investigated their sensitivity to the antibody-dependent classical complement-mediated killing pathway in 90% convalescent chicken serum. The fhaB1 mutant was serum sensitive as the viability has reduced between untreated serum and heat inactivated chicken serum (P<0.007). These results confirmed that FhaB1 played the critical roles in the bacterial pathogenesis and further studies were needed to investigate the mechanism which caused reduced virulence of the fhaB1 mutant.