Avian infectious bronchitis (IB) is a highly contagious infectious disease caused by infectious bronchitis virus (IBV), which is prevalent in many countries worldwide and causes serious harm to the poultry industry.  At present, many commercial IBV vaccines have been used for the prevention and control of IB; however, IB outbreaks occur frequently.  In this study, two new strains of IBV, SX/2106 and SX/2204, were isolated from two flocks which were immunized with IBV H120 vaccine in central China.  Phylogenetic and recombination analysis indicated that SX/2106, which was clustered into the GI-19 lineage, may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine.  Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage, which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine.  The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120.  Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death, and H120 immunization could not provide effective protection against the two IBV isolates.  It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously, with a mortality rate up to 60%.  Considering the continuous mutation and recombination of the IBV genome to produce new variant strains, it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics.

This study investigated the effects of dioscorea opposite waste (DOW) on the growth performance, blood parameters, rumen fermentation and rumen microbiota of weaned lambs.  Sixty healthy weaned Small-Tailed Han lambs (male, (22.68±2.56) kg initially) were used as the experimental animals.  Four levels of concentrate: 0 (control, CON), 10% (DOW1), 15% (DOW2) and 20% (DOW3), were replaced with DOW in the basal diet as experimental treatments.  The results showed that lambs fed the DOW2 diet had a higher (P<0.05) dry matter intake (DMI) than the other groups.  There was no significant difference (P>0.05) among DOW groups in average daily weight gain (ADG), and replacing concentrate with DOW linearly or quadratically increased (P<0.05) the ADG, while lambs fed the DOW2 diet showed greater (P<0.05) ADG than the CON group.  The relative plasma concentration of growth hormone (GH), insulin like growth factor-1 (IGF-1) and insulin were affected by DOW, replacing concentrate with DOW linearly or quadratically (P<0.05) enhanced the plasma concentration of GH, IGF-1 and insulin, which was significantly higher (P<0.05) in the DOW2 group than in the CON, DOW1 and DOW3 groups.  In addition, the DOW treatment showed a lower (P<0.05) concentration of blood urea nitrogen (BUN) than the CON group.  Replacing concentrate with DOW quadratically decreased (P<0.05) the ruminal ammonia nitrogen (NH₃-N) and increased (P<0.05) the total of volatile fatty acids (TVFAs) at 0 and 4 h after feeding as well as linearly decreased (P<0.05) the NH₃-N at 8 h after feeding.  Replacing concentrate with DOW linearly decreased (P<0.05) the propionate and increased the aceate before feeding, and linearly decreased (P<0.05) propionate and quadratically increased (P<0.05) the aceate at 4 and 8 h after feeding.  Lambs fed the DOW2 diet increased the phylum Firmicutes and genera Succiniclasticum and Ruminococcus_1 groups, whereas decreased (P<0.05) the relative abundance of phylum Deferribacteres and genera intestinimonas and Ruminiclostridium.  In summary, replacing the concentrate with 15% DOW was beneficial for improving the rumen fermentation and ADG by increasing the DMI and modulating the rumen microbial community.

Early bacterial colonization and succession within the gastrointestinal tract have been suggested to be crucial in the development of host immunity.  In this study, we have investigated the changes in live weight and concentrations of selected serum parameters in relation to their fecal bacterial communities as determined by high throughput sequencing of the 16S rRNA gene over the same period in lambs.  The results showed that lambs’ growth performance, the serum parameters, fecal bacterial community and fecal bacterial functions were all affected (P<0.05) by age of the lambs.  Similarity within age groups of fecal microbiota was lower in the preweaning period and increased sharply (P<0.05) after weaning at 60 days.  The similarity between the samples collected from birth to 90 days of age and those collected at 120 days of age, increased (P<0.05) sharply after 30 days of age.  Some age-associated changes in microbial genera were correlated with the changes in concentrations of immune indicators, including negative (P<0.05) correlations between the relative abundance of Lachnospiraceae UCG-010, Eubacterium coprostanoligenes group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-009, Ruminococcaceae UCG-013, Ruminiclostridium 6, Ruminococcaceae UCG-008, and Oscillibacter with serum concentrations of lipopolysaccharide (LPS), D-lactate dehydrogenase (DLA), immunoglobulin (IgA, IgM, and IgG), and cytokines (interleukin-1β (IL-1β), IL-6, IL-12, and IL-17), tumor necrosis factor-α (TNF-α), and the relative abundance of these genera increased from 45 days of age.  In conclusion, these results suggested that the age-related abundances of particular genera were correlated with serum markers of immunity in lambs, and there might be a critical window in the period from birth to 45 days of age which provide an opportunity for potential manipulation of the fecal microbial ecosystems to enhance immune function.

Cassava is an important tropical cash crop.  Severe drought stresses affect cassava productivity and quality, and cause great economic losses in agricultural production.  Enhancing the drought tolerance of cassava can effectively improve its yield.  Long non-coding RNAs (lncRNAs) are present in a wide variety of eukaryotes.  Recently, increasing evidence has shown that lncRNAs play a critical role in the responses to abiotic stresses.  However, the function of cassava lncRNAs in the drought response remains largely unknown.  In this study, we identified a novel lncRNA, DROUGHT-INDUCED INTERGENIC lncRNA (DIR).  Gene expression analysis showed that DIR was significantly induced by drought stress treatment, but did not respond to abscisic acid (ABA) or jasmonic acid (JA) treatments.  In addition, overexpression of the DIR gene enhanced proline accumulation and drought tolerance in transgenic cassava.  RNA-seq analysis revealed that DIR preferentially affected drought-related genes that were linked to transcription and metabolism.  Moreover, RNA pull-down mass spectrometry analysis showed that DIR interacted with 325 proteins.  A protein–protein interaction (PPI) analysis found a marked enrichment in proteins associated with the mRNA export and protein quality control pathways.  Collectively, these results suggest that DIR and its interacting proteins that regulate mRNA or protein metabolism are involved in mediating the drought stress response.  Thus, regulating DIR expression has potential for improving cassava yield under drought conditions.

Although fungal communities in the gastrointestinal tract have a significant role in animal health and performance, their dynamics within the tract are not well known.  Thus, this study investigated fungal community dynamics in the rumen and rectum of lambs from birth to 4 mon of age by using IT1S rDNA sequencing technology together with the RandomForest approach to determine age-related changes in the fungal ecology.  The results indicated that gastrointestinal fungal community composition, diversity, and abundance altered (P<0.05) with the increasing age of the lambs.  Two phyla, Ascomycota and Basidiomycota, dominated the samples.  Similarity within age groups of the rumen fungi increased sharply after 45 days of age, while the similarity increased (P<0.05) significantly after 60 days of age in the rectum.  The age-related genera, Acremonium, Microascus, Valsonectria, Myrmecridium, Scopulariopsis, Myrothecium, Saccharomyces, and Stephanonectria, were presented in both ruminal and rectal communities, and their changes in relative abundance were consistent at both sites.  The principal coordinates analysis showed significant differences (P<0.05) between the fungal communities in the rumen and rectum.  Our findings demonstrate that both the age of lambs and the gastrointestinal tract region can affect the composition of these fungal communities, and this provides new insight and directions for future studies in this research area.

The peroxisomal matrix oxidase, catalase and peroxidase are imported peroxisomes through the shuttling receptors, which regulates the cellular oxidative homeostasis and function.  Here, we report that PTS1 shuttling receptor FvPex5 is involved in the localization of PTS1, utilization of carbon sources and lipids, elimination ROS, cell wall stress, conidiation, fumonisin B₁ (FB₁) production, and virulence in maize pathogen Fusarium verticillioides.  Significantly, differential expression of PTS1-, PTS2-, PEX- and FB₁ toxin-related genes in wild type and ΔFvpex5 mutant were examined by RNA-Seq analyses and confirmed by RT-PCR assay.  In addition, different expression of PTS1 and PTS2 genes of the ΔFvpex5 mutant were enriched in diverse biochemical pathways, such as carbon metabolism, nitrogen metabolism, lipid metabolism and the oxidation balance by combining GO and KEGG annotations.  Overall, we showed that FvPex5 is involved in the regulation of genes associated with PTS, thereby affecting the oxidation balance, FB₁ and virulence in F. verticillioides.  The results help to clarify the functional divergence of Pex5 orthologs, and may provide a possible target for controlling F. verticillioides infections and FB₁ biosynthesis.

As a critical food crop, sweetpotato (Ipomoea batatas (L.) Lam.) is widely planted all over the world, but it is deeply affected by Sweetpotato Virus Disease (SPVD).  The present study utilized short tandem target mimic (STTM) technology to effectively up-regulate the expression of laccase (IbLACs) by successfully inhibiting the expression of miR397.  The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation, including phenylalanine ammonialyase (PAL), 4-coumarate-CoAligase (4CL), hydroxycinnamoyl CoA:shikimatetransferase (HTC), caffeicacid O-methyltransferase (COMT), and cinnamyl alcohol dehydrogenase (CAD).  Meanwhile, the activities of PAL and LAC increased significantly, finally leading to increased lignin content.  Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants, reduced the accumulation of SPVD transmitted by Bemisia tabaci (Gennadius), and promoted healthy sweetpotato growth.  The results provide new insights for disease resistance breeding and green production of sweetpotato. 

Soil salinity causes the negative effects on the growth and yield of crops. In this study, two sweet potato (Ipomoea batatas L.) cultivars, Xushu 28 (X-28) and Okinawa 100 (O-100), were examined under 50 and 100 mmol L^–1 NaCl stress. X-28 cultivar is relatively high salt tolerant than O-100 cultivar. Interestingly, real-time quantitative polymerase chain reaction (RT-qPCR) results indicated that sweet potato high-affinity K⁺ transporter 1 (IbHKT1) gene expression was highly induced by 50 and 100 mmol L^–1 NaCl stress in the stems of X-28 cultivar than in those of O-100 cultivar, but only slightly induced by these stresses in the leaves and fibrous roots in both cultivars. To characterize the function of IbHKT1 transporter, we performed ion-flux analysis in tobacco transient system and yeast complementation. Tobacco transient assay showed that IbHKT1 could uptake sodium (Na⁺). Yeast complementation assay showed that IbHKT1 could take up K⁺ in 50 mmol L^–1 K⁺ medium without the presence of NaCl. Moreover, Na⁺ uptake significantly increased in yeast overexpressing IbHKT1. These results showed that IbHKT1 transporter could have K⁺-Na⁺ symport function in yeast. Therefore, the modes of action of IbHKT1 in transgenic yeast could differ from the mode of action of the other HKT1 transporters in class I. Potentially, IbHKT1 could be used to improve the salt tolerance nature in sweet potato.

The information of single nucleotide polymorphisms (SNPs) is quite unknown in sweetpotato.  In this study, two sweetpotato varieties (Xushu 18 and Xu 781) were sequenced by Illumina technology, as well as de novo transcriptome assembly, functional annotation, and in silico discovery of potential SNP molecular markers.  Tetra-primer Amplification Refractory Mutation System PCR (ARMS-PCR) is a simple and sufficient method for detecting different alleles in SNP locus.  Total 153 sets of ARMS-PCR primers were designed to validate the putative SNPs from sequences.  PCR products from 103 sets of primers were different between Xu 781 and Xushu 18 via agarose gel electrophoresis, and the detection rate was 67.32%.  We obtained the expected results from 32 sets of primers between the two genotypes.  Furthermore, we ascertained the optimal annealing temperature of 32 sets of primers.  These SNPs might be used in genotyping, QTL mapping, or marker-assisted trait selection further in sweetpotato.  To our knowledge, this work was the first study to develop SNP markers in sweetpotato by using tetra-primer ARMS-PCR technique.  This method was a simple, rapid, and useful technique to develop SNP markers, and will provide a potential and preliminary application in discriminating cultivars in sweetpotato.

Litter uniformity, which is usually represented by within-litter weight coefficient of variation at birth (CVB), could influence litter performance of sows and the profitability of pig enterprises. The objective of this study was to characterize CVB and its effect on other reproductive traits in Large White sows. Genetic parameters and genetic correlation of the reproductive traits, including CVB, within-litter weight coefficient of variation at three weeks (CVT), total number born (TNB), number born alive (NBA), number born dead (NBD), gestation length (GL), piglet mortality at birth (M₀), piglet mortality at three weeks (M₃), total litter weight at birth (TLW0), and total litter weight at three weeks (TLW₃) were estimated for 2 032 Large White litters. The effects of parity and classified litter size on CVB, CVT, TNB, NBA, NBD, GL, M₀, M₃, TLW₀, and TLW₃ were also estimated. The heritabilities of these reproductive traits ranged from 0.06 to 0.17, with the lowest heritability for CVB and the highest heritability for TLW0. Phenotypic and genetic correlations between these reproductive traits were low to highly positive and negative (ranging from −0.03 to 0.93, and −0.53 to 0.93, respectively). The genetic correlations between TNB and CVB, and between M₀ and CVB were 0.32 and 0.29, respectively. In addition, CVB was significantly influenced by parity and litter size class (P<0.05). All the results suggest that piglet uniformity should be maintained in pig production practices and pig breeding programs.

   Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3 (LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 cDNA. The complete cDNA contains 4 843 bp, including a 2 736-bp open reading frame (ORF), a 37-bp 5´-untranslated region (UTR) and a 2 070-bp 3´-UTR. The complete LEMD3 gene is 126 241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms (SNPs): L964C>A in the complete coding region, L4625A>G in the 3´ UTR, and L-394T>C in the promoter region. Genome-wide association study (GWAS) revealed that all of SNPs were shown significant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log₁₀(P-value) decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strategies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA fragments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNA was sufficient to confer sour orange with CTV resistance/tolerance.

Although quantitative trait loci (QTLs) for number of thoracic-lumbar vertebrae have been identified on Sus scrofa chromosomes (SSCs) 1 and 7, the influence of these QTLs on the thoracic and lumbar vertebrae is not clear. The aim of this study was to identify single nucleotide polymorphisms (SNPs) associated with total number of thoracic-lumbar vertebrae and for each trait (number of thoracic and lumbar vertebrae) separately. A total of 581 individuals from an F2 Large White×Minzhu population were genotyped using an SNP60K chip. Performing a genome-wide association study (GWAS) for total number of thoracic-lumbar vertebrae, 38 significant SNPs were identified in two QTL regions located on SSC1 and SSC7. Performing a GWAS for number of thoracic vertebrae only, 72 significant SNPs were located on SSC7. While performing a GWAS for number of lumbar vertebrae only, 17 significant SNPs were identified on SSC1. Gene mining suggested that the gene encoding orphan nuclear receptor, germ cell nuclear factor (NR6A1) on SSC1 was a strong candidate affecting the number of lumbar vertebrae in pigs. Additionally, genes encoding vertnin (VRTN), prospero homeobox 2 (PROX2), Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS), and transforming growth factor beta 3 (TGFB3) may be important candidates affecting the number of thoracic vertebrae in pigs. QTLs on SSC1 and SSC7 independently influenced the numbers of thoracic and lumbar vertebrae. These results shed light on the complex genetic background of vertebrae development in pigs.

Porcine carcass traits and organ weights have important economic roles in the swine industry. A total of 576 animals from a Large White×Minzhu intercross population were genotyped using the Illumina PorcineSNP60K Beadchip and were phenotyped for 10 traits, specifically, backfat thickness (6-7 libs), carcass length, carcass weight, foot weight, head weight, heart weight, leaf fat weight, liver weight, lung weight and slaughter body weight. The genome-wide association study (GWAS) was assessed by Genome Wide Rapid Association using the mixed model and regression-genomic control approach. A total of 31 single nucleotide polymorphisms (SNPs) (with the most significant SNP being MARC0033464, P value=6.80×10-13) were located in a 9.76-Mb (31.24-41.00 Mb) region on SSC7 and were found to be significantly associated with one or more carcass traits and organ weights. High percentage of phenotypic variance explanation was observed for each trait ranging from 31.21 to 67.42%. Linkage analysis revealed one haplotype block of 495 kb, in which the most significant SNP being MARC0033464 was contained, on SSC7 at complete linkage disequilibrium. Annotation of the pig reference genome suggested 6 genes (GRM4, HMGA1, NUDT3, RPS10, SPDEF and PACSIN1) in this candidate linkage disequilibrium (LD) interval. Functional analysis indicated that the HMGA1 gene presents the prime biological candidate for carcass traits and organ weights in pig, with potential application in breeding programs.

The erythropoietin receptor (EPOR) has shown to play an important role in fetal survival by promoting the maturation of red blood cells in many studies of uterine capacity and litter size in swine. In this study, we screened the porcine EPOR gene for mutations and identified five single nucleotide polymorphisms (SNPs): g.705G>T in intron 1, g.2 373C>T in intron 4, and g.2 882C>T, g.3 035A>G, and g.3 132A>T in intron 6. We then genotyped 247 Beijing Black (BB) sows and compared the polymorphism data with the litter sizes of 1 375 parities among the sows. At first parity, there was no association of g.2 882C>T and g.3 132A>T with litter sizes. However, the CT sows in g.2 882C>T had 2.13 higher total number born (TNB) (P<0.01) and 1.81 higher number born alive (NBA) (P<0.01) than the CC sows and the heterozygous sows in g.3 132A>T had the highest litter size when compared to the two homozygotes for the later parities (P<0.05). In the g.3 035A>G SNP, for the later parities, the TNB of the sows with the GG genotype was 3.81 higher (P<0.01) and the NBA was 2.75 higher (P<0.01) than that with the AA genotype but no difference at first parity. The G allele of the EPOR g.705G>T SNP was associated with a greater litter size at both the first parity (P<0.05) and later parities (P<0.01). Furthermore, we determined the allele frequencies for this SNP among five Chinese indigenous pig breeds (Erhualian, Laiwu Black, Meishan, Min, and Rongchang) and three western commercial pig breeds (Duroc, Landrace, and Large White). The G allele of the EPOR g.705G>T SNP was significantly more common in the more prolific Chinese breeds. These results indicated that the EPOR could be an important candidate gene for litter size and g.705G>T can serve as a useful genetic marker for improving litter size in both first and later parities in swine.

The pathogenesis-related protein PR10 is essential for plant growth, development, and stress responses.  In this study, PR10 genes in cultivated peanut (Arachis hypogaea L.) were systematically identified, after which their phylogenetic relationships, conserved motifs, gene structures, and syntenic relationships were analyzed.  A total of 54 AhPR10 genes were identified.  They were then divided into eight groups according to their phylogenetic relationships, which were supported by the characterization of gene structures and conserved motifs.  Analyses of chromosomal distribution and synteny revealed that segmental duplications were critical for the expansion of the AhPR10 gene family.  In addition, the identified AhPR10 genes had constitutive and inducible expression patterns.  Notably, AhPR10-7, AhPR10-33, and AhPR10-41 may have crucial functions affecting the resistance of peanut to A. flavus.  In vitro fungistatic experiments indicated that recombinant AhPR10-33 can effectively inhibit A. flavus mycelial growth.  The study results provide useful insights for future research on AhPR10 functions that protect peanut from the detrimental effects of A. flavus.