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A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus and atypical porcine pestivirus

SONG Xiang-peng, XIA Ying-ju, XU Lu, ZHAO Jun-jie, WANG Zhen, ZHAO Qi-zu, LIU Ye-bing, ZHANG Qian-yi, WANG Qin

2023, 22 (2): 559-567. DOI: 10.1016/j.jia.2022.08.115

Abstract （211） PDF in ScienceDirect

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation. African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety. Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China. These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry. They are difficult to be distinguished only by clinical diagnosis. Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential. In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5´ UTR), African swine fever virus (ASFV) (B646L), and APPV (5´ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay. The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV). The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL^–1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%. Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively. The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the compliance results were the same (100%). A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively. This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

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A high-quality genome of Actinidia eriantha provides new insight into ascorbic acid regulation

LIAO Guang-lian, HUANG Chun-hui, JIA Dong-feng, ZHONG Min, TAO Jun-jie, QU Xue-yan, XU Xiao-biao

2023, 22 (11): 3244-3255. DOI: 10.1016/j.jia.2023.07.018

Abstract （283） PDF in ScienceDirect

Actinidia eriantha is one of the species of kiwifruit with a particularly high ascorbic acid (AsA) content. However, the molecular mechanism driving AsA richness in fruit remains unclear. In order to reveal the molecular mechanism of AsA richness in A. eriantha, this study constructed a regulatory network related to AsA metabolism by combining genomics, metabolomics and transcriptomics. We assembled a high-quality genome of A. eriantha ‘Ganlv 1’ with only five remaining gaps. The assembly is comprised of 29 pseudochromosomes with a total size of 615.95 Mb, and contig N50 of 20.35 Mb. Among them, 24 of the pseudochromosomes were obtained directly from telomere-to-telomere. The LTR assembly index score and consensus quality value were 21.34 and 39.90%, respectively. Subsequently, 61 metabolites and 2 092 genes were found to be differentially accumulated/expressed during fruit development by metabolome and transcriptome assays, respectively. AsA metabolism and the cyclic regeneration pathway were found to have high expression levels throughout fruit growth and development, suggesting its crucial role in the regulation of AsA. Furthermore, the AsA contents are highly associated with ascorbate peroxidase genes. The genome obtained in this study provides genomic resources for the genetic and breeding research of A. eriantha, and the constructed regulatory network can provide a public data platform for future research on kiwifruit.

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Dek219 encodes the DICER-LIKE1 protein that affects chromatin accessibility and kernel development in maize

XIE Si-di, TIAN Ran, ZHANG Jun-jie, LIU Han-mei, LI Yang-ping, HU Yu-feng, YU Guo-wu, HUANG Yu-bi, LIU Ying-hong

2023, 22 (10): 2961-2980. DOI: 10.1016/j.jia.2023.02.024

Abstract （369） PDF in ScienceDirect

Chromatin accessibility plays a vital role in gene transcriptional regulation. However, the regulatory mechanism of chromatin accessibility, as well as its role in regulating crucial gene expression and kernel development in maize (Zea mays) are poorly understood. In this study, we isolated a maize kernel mutant designated as defective kernel219 (dek219), which displays opaque endosperm and embryo abortion. Dek219 encodes the DICER-LIKE1 (DCL1) protein, an essential enzyme in miRNA biogenesis. Loss of function of Dek219 results in significant reductions in the expression levels of most miRNAs and histone genes. Further research showed that the Heat shock transcription factor17 (Hsf17)-Zm00001d016571 module may be one of the factors affecting the expression of histone genes. Assay results for transposase-accessible chromatin sequencing (ATAC-seq) indicated that the chromatin accessibility of dek219 is altered compared with that of wild type (WT), which may regulate the expression of crucial genes in kernel development. By analyzing differentially expressed genes (DEGs) and differentially accessible chromatin regions (ACRs) between WT and dek219, we identified 119 candidate genes that are regulated by chromatin accessibility, including some reported to be crucial genes for kernel development. Taken together, these results suggest that Dek219 affects chromatin accessibility and the expression of crucial genes that are required for maize kernel development

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Dissecting the genetic basis of maize deep-sowing tolerance by combining association mapping and gene expression analysis

YANG Yue, MA Yu-ting, LIU Yang-yang, Demar LYLE, LI Dong-dong, WANG Ping-xi, XU Jia-liang, ZHEN Si-han, LU Jia-wen, PENG Yun-ling, CUI Yu, FU Jun-jie, DU Wan-li, ZHANG Hong-wei, WANG Jian-hua

2022, 21 (5): 1266-1277. DOI: 10.1016/S2095-3119(21)63649-3

Abstract （150） PDF in ScienceDirect

Deep-sowing is an important method for avoiding drought stress in crop species, including maize. Identifying candidate genes is the groundwork for investigating the molecular mechanism underlying maize deep-sowing tolerance. This study evaluated four traits (mesocotyl length at 10 and 20 cm planting depths and seedling emergence rate on days 6 and 12) related to deep-sowing tolerance using a large maize population containing 386 inbred lines genotyped with 0.5 million high-quality single nucleotide polymorphisms (SNPs). The genome-wide association study detected that 273 SNPs were in linkage disequilibrium (LD) with the genetic basis of maize deep-sowing tolerance. The RNA-sequencing analysis identified 1 944 and 2 098 differentially expressed genes (DEGs) in two comparisons, which shared 281 DEGs. By comparing the genomic locations of the 273 SNPs with those of the 281 DEGs, we identified seven candidate genes, of which GRMZM2G119769 encoded a sucrose non-fermenting 1 kinase interactor-like protein. GRMZM2G119769 was selected as the candidate gene because its homologs in other plants were related to organ length, auxin, or light response. Candidate gene association mapping revealed that natural variations in GRMZM2G119769 were related to phenotypic variations in maize mesocotyl length. Gene expression of GRMZM2G119769 was higher in deep-sowing tolerant inbred lines. These results suggest that GRMZM2G119769 is the most likely candidate gene. This study provides information on the deep-sowing tolerance of maize germplasms and identifies candidate genes, which would be useful for further research on maize deep-sowing tolerance.

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Recent advances in plant immunity with cell death: A review

YIN Jun-jie, XIONG Jun, XU Li-ting, CHEN Xue-wei, LI Wei-tao

2022, 21 (3): 610-620. DOI: 10.1016/S2095-3119(21)63728-0

Abstract （181） PDF in ScienceDirect

Cell death is an important physiological phenomenon in life. It can be programmed or unprogrammed. Unprogrammed cell death is usually induced by abiotic or biotic stress. Recent studies have shown that many proteins regulate both cell death and immunity in plants. Here, we provide a review on the advances in plant immunity with cell death, especially the molecular regulation and underlying mechanisms of those proteins involved in both cell death and plant immunity. In addition, we discuss potential approaches toward improving plant immunity without compromising plant growth.

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Evidence of silk growth hampering in maize at high planting density using phenotypic and transcriptional analysis

ZHANG Min, XING Li-juan, REN Xiao-tian, ZOU Jun-jie, SONG Fu-peng, WANG Lei, XU Miao-yun

2022, 21 (11): 3148-3157. DOI: 10.1016/j.jia.2022.08.083

Abstract （350） PDF in ScienceDirect

Increasing the planting density is an effective way to increase the yield of maize (Zea mays L.), although it can also aggravate ovary apical abortion-induced bald tips of the ears, which might, in turn, reduce the yield. While the mechanism underlying the regulation of drought-related abortion in maize is well established, high planting density-related abortion in maize remains poorly understood. Therefore, the present study was designed to investigate the mechanism underlying the ovary apical abortion response to high density. This was achieved by evaluating the effects of four different plant densities (60 000 plants ha^–1 (60 k), 90 k, 120 k, and 150 k) on plant traits related to plant architecture, the plant ear, flowering time, and silk development in two inbred lines (Zheng58 and PH4CV) and two hybrid lines (Zhengdan958 and Xianyu335). The phenotypes of both inbred and hybrid plants were observed under different planting density treatments, and the high planting density was found to increase the phenotypic performance values of the evaluated traits. The anthesis–silking interval (ASI) was extended, and the amount of the silk extruded from husks was reduced upon increasing the planting density. Delayed silk emergence resulted in asynchronous flowering and ear bald tips. Observations of the silk cells revealed that the silk cells became smaller as planting density increased. The changes in transcript abundances in the silks involved the genes associated with expansive growth rather than carbon metabolism. These findings further our understanding of silk growth regulation under high planting density and provide a theoretical basis for further research on improving high planting density breeding in maize.

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Distribution and accumulation of zinc and nitrogen in wheat grain pearling fractions in response to foliar zinc and soil nitrogen applications

ZHANG Pan-pan, CHEN Yu-lu, WANG Chen-yang, MA Geng, LÜ Jun-jie, LIU Jing-bao, GUO Tian-cai

2021, 20 (12): 3277-3288. DOI: 10.1016/S2095-3119(20)63491-8

Abstract （165） PDF in ScienceDirect

Increasing zinc (Zn) concentration in wheat grain is important to minimize human dietary Zn deficiency. This study aimed to investigate the effect of foliar Zn and soil nitrogen (N) applications on the accumulation and distribution of N and Zn in grain pearling fractions, N remobilization, and the relationships between nutrient concentration in the vegetative tissues and grain or its fractions in two cropping years in the North China Plain. The results showed a progressive decrease in N and Zn concentrations from the outer to the inner parts of grain, with most of the accumulation in the core endosperm. Foliar Zn application significantly increased N concentration in the pericarp, and soil N application increased N concentration in each grain fraction. Both treatments significantly increased core endosperm Zn concentration. Foliar Zn had no effect on grain N and Zn distribution. Soil N application made N concentrated in the aleurone, promoted Zn translocation to the core endosperm and also increased N remobilization and its efficiency from the shoot to the grain, but no improved contribution to grain was found. N concentration in grain and its fractions were positively correlated with N in vegetative organs at anthesis and maturity, while positive correlations were obtained between N concentration in the pericarp and progressive central area of the endosperm and Zn concentration in the core endosperm. Thus, foliar Zn and soil N applications effectively increased yield and N and Zn concentrations in the wheat grain, particularly in the endosperm, and could be promising strategies to address Zn deficiency.

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Optimization of the sex pheromone-based method for trapping field populations of Phthorimaea operculella (Zeller) in South China

YAN Jun-jie, MEI Xiang-dong, FENG Jia-wen, LIN Zhi-xu, Stuart REITZ, MENG Rui-xia, GAO Yu-lin

2021, 20 (10): 2727-2733. DOI: 10.1016/S2095-3119(20)63320-2

Abstract （123） PDF in ScienceDirect

Despite the identification of the potato tuber moth Phthorimaea operculella (Zeller) sex pheromone, no effective application based on this pheromone has yet been developed and evaluated. This study investigated the effect of pheromone lures, trap densities, heights of trap deployment, and pheromone doses in Yunnan, China, for the purpose of increasing the control efficiency of P. operculella and improving the application of pheromone technology in the field. The results showed that lures made of corn oil and red PVC pipes attracted the highest number of moths (11.73±1.90 per trap per day). Sex pheromone loading of 100 μg was optimal for trapping moths, but higher doses of pheromone inhibited attraction. The density of traps did not affect capture rates; therefore, the optimum trap density was 30–40 traps ha^–1. The optimum height of trap deployment was not above the height of the plant canopy. This study provides technical details necessary for the monitoring and control of potato tuber moth using sex pheromones.

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Differentially expressed miRNAs in anthers may contribute to the fertility of a novel Brassica napus genic male sterile line CN12A

Dong Yun, Wang Yi, Jin Feng-wei, Xing Li-juan, Fang Yan, Zhang Zheng-ying, ZOU Jun-jie, Wang Lei, Xu Miao-yun

2020, 19 (7): 1731-1742. DOI: 10.1016/S2095-3119(19)62780-2

Abstract （95） PDF in ScienceDirect

In Brassica napus L. (rapeseed), complete genic male sterility (GMS) plays an important role in the utilization of heterosis. Although microRNAs (miRNAs) play essential regulatory roles during bud development, knowledge of how GMS is regulated by miRNAs in rapeseed is rather limited. In this study, we obtained a novel recessive GMS system, CN12AB. The sterile line CN12A has defects in tapetal differentiation and degradation. Illumina sequencing was employed to examine the expression of miRNAs in the buds of CN12A and the fertile line CN12B. We identified 85 known miRNAs and 120 novel miRNAs that were expressed during rapeseed anther development. When comparing the expression levels of miRNAs between CN12A and CN12B, 19 and 18 known miRNAs were found to be differentially expressed in 0.5–1.0 mm buds and in 2.5–3.0 mm buds, respectively. Among these, the expression levels of 14 miRNAs were higher and the levels of 23 miRNAs were lower in CN12A compared with CN12B. The predicted target genes of these differentially expressed miRNAs encode protein kinases, F-box domain-containing proteins, MADS-box family proteins, SBP-box gene family members, HD-ZIP proteins, floral homeotic protein APETALA 2 (AP2), and nuclear factor Y, subunit A. These targets have previously been reported to be involved in pollen development and male sterility, suggesting that miRNAs might act as regulators of GMS in rapeseed anthers. Furthermore, RT-qPCR data suggest that one of the differentially expressed miRNAs, bna-miR159, plays a role in tapetal differentiation by regulating the expression of transcription factor BnMYB101 and participates in tapetal degradation and influences callose degradation by manipulating the expression of BnA6. These findings contribute to our understanding of the roles of miRNAs during anther development and the occurrence of GMS in rapeseed.

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Potential of Steinernema carpocapsae (Weiser) as a biological control agent against potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

YAN Jun-jie, Shovon Chandra SARKAR, MENG Rui-xia, Stuart REITZ, GAO Yu-lin

2020, 19 (2): 389-393. DOI: 10.1016/S2095-3119(19)62826-1

Abstract （125） PDF in ScienceDirect

The entomopathogenic nematode, Steinernema carpocapsae, was evaluated for control of the potato tuber moth, Phthorimaea operculella, under laboratory conditions. We evaluated different concentrations of S. carpocapsae for control of 2nd, 3rd, and 4th instar P. operculella. The median lethal concentration (LC₅₀) of S. carpocapsae infective juveniles (IJs) to 2nd, 3rd and 4th instar larvae of P. operculella was 200, 363, 181 IJs mL^–1, respectively. With the extension of treatment time, the cumulative mortality increased for 2nd, 3rd, and 4th instar larvae and pupae of P. operculella. Fourth instars were the most susceptible for all observation periods. Therefore, our results suggest that S. carpocapsae could be an effective biological control agent for P. operculella.

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Effect of dietary supplementation with mulberry (Morus alba L.) leaves on the growth performance, meat quality and antioxidative capacity of finishing pigs

ZENG Zhu, JIANG Jun-jie, YU Jie, MAO Xiang-bing, YU Bing, CHEN Dai-wen

2019, 18 (1): 143-151. DOI: 10.1016/S2095-3119(18)62072-6

Abstract （311） PDF in ScienceDirect

The present study was conducted to evaluate the effect of dietary mulberry (Morus alba L.) leaves powder (MLP) supplementation on meat quality of finishing pigs. A total of 40 Duroc×Landrace×Yorkshire pigs (initial body weight of (40.5±0.63) kg) were randomly allotted into two treatments, fed either with control diet or 15% MLP diet for 85 d. The results showed that MLP diet decreased (P≤0.05) average daily gain (ADG) and increased (P<0.05) feed/gain ratio (F/G) in the finishing and whole period. MLP diet also decreased (P<0.05) carcass weight, dressing percentage, last rib and average backfat depth. However, MLP diet increased (P<0.05) intramuscular fat (IMF) content, decreased (P<0.05) shear force, cooking loss and drip loss. In addition, MLP diet increased (P<0.05) total antioxidative capacity, glutathione peroxidase and tended (P<0.10) to increase total superoxide dismutase in serum. In longissimus thoracis, myosin heavy chain (MyHC) I and IIa mRNA levels were increased (P≤0.05) for MLP diet. In conclusion, 15% MLP supplementation reduced the growth performance and carcass traits, but improved meat quality of finishing pigs possibly through the change of myofiber characteristics, enhancement of antioxidative capacity and increase of IMF.

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Selection and evaluation of potential reference genes for gene expression analysis in greenbug (Schizaphis graminum Rondani)

ZHANG Bai-zhong, LIU Jun-jie, YUAN Guo-hui, CHEN Xi-ling, GAO Xi-wu

2018, 17 (09): 2054-2065. DOI: 10.1016/S2095-3119(18)61903-3

Abstract （433） PDF in ScienceDirect

In order to precisely assess gene expression level, a suitable internal reference gene must be chosen to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For greenbug, Schizaphis graminum, a suitable reference gene for assessing the level of transcriptional expression of target genes has yet to be explored. In our study, eight reference genes, elongation fator 1 beta (Ef1β), TATA box binding protein (TBP), alpha-tubulin (α-TUB), 18S ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), actin (ACT), and ribosomal protein L18 (RPL18) were evaluated in S. graminum at different developmental stages, tissues, and insecticide treatments. To further explore whether these genes are suitable to serve as internal control, three software-based approaches (geNorm, BestKeeper, and NormFinder), ?Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized gene expression data of three target genes, heat shock protein gene (HSP70), cytocrome P450 gene (SgraCYP18A1), and glutathione S-transferase (GST). We found that the most suitable reference genes varied considerably under different experimental conditions. For developmental stages, α-TUB and 28S were the optimal reference genes; for different tissues, 18S and ACT were suitable reference genes; for insecticide treatments, 28S and α-TUB were suitable for normalizations of expression data. In addition, 28S and α-TUB were the suitable reference gene as they had the most stable expression among different developmental stages, tissues and insecticide treatments. This should be useful for the selection of the suitable reference genes to obtain reliable RT-qPCR data in the gene expression of S. graminum.

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Identification of novel and differentially expressed microRNAs in ovine ovary and testis tissues using solexa sequencing and bioinformatics

CHANG Wei-hua, ZHANG Yong, CHENG Zhang-rui, ZHAO Xing-xu, WANG Juan-hong, MA You-ji, HU Jun-jie, ZHANG Quan-wei

2015, 14 (8): 1604-1616. DOI: 10.1016/S2095-3119(14)60900-X

Abstract （2131） PDF in ScienceDirect

MicroRNAs (miRNAs) are small, single stranded, non-coding RNA molecules, about 19–25 nucleotides in length, which regulate the development and functions of reproductive system of mammal. To discover novel miRNAs and identify the differential expression of them in ovine ovary and testis tissues, the study constructed two libraries by using next-generation sequencing technologies (Solexa high-throughput sequencing technique). As a result, 9 321 775 and 9 511 538 clean reads were obtained from the ovary and testis separately, which included 130 562 (90 genes of ovary) and 56 272 (85 genes of testis) of known miRNAs and 486 potential novel miRNAs reads. In this study, a total of 65 conserved miRNAs were significantly differentially expressed (P<0.01) between the two samples. Among them, 28 miRNAs were up-regulated and 3 miRNAs were down-regulated on ovary compared with testis. In addition, the known miRNAs with the highest expression level (5 miRNAs) and 30 novel miRNAs with the functions related to reproduction were validated using the real-time quantitative RT-PCR. Moreover, the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differentially expressed miRNAs were involved in ovary and testis physiology, including signal transduction, gonad development, sex differentiation, gematogenesis, fertilization and embryo development. The results will be helpful to facilitate studies on the regulation of miRNAs during ruminant reproduction.

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MicroRNAs Involved in the Pathogenesis of Phytophthora Root Rot of Soybean (Glycine max)

WANG Jing*, LIU Chun-yan*, ZHANG Li-wei, WANG Jia-lin, HU Guo-hua, DING Jun-jie , CHEN Qing-shan

2011, 10 (8): 1159-1167. DOI: 10.1016/S1671-2927(11)60106-5

Abstract （3391） PDF in ScienceDirect

Phytophthora root rot is one of the most prevalent diseases in the world, which can infect the seedlings and plants, withsubstantial negative impact on soybean yield and quality. MicroRNAs (miRNAs) are a class of post-transcriptionalregulators of gene expression during growth and development of organisms. A soybean disease-resistance varietySuinong 10 was inoculated with Phytophthora sojae race No. 1, and the specific miRNA resistant expression profile wasacquired by microarray for the first time. Different expressional miRNAs have been found after comparing the results ofthe treated sample with the control sample. Furthermore, the target genes of different expressional miRNAs were predicted.Two miRNAs, cbr-mir-241 and ath-miR854a, regulated the disease-resistance process directly through their targets, someenzymes. Another two miRNAs, gma-miR169a and ath-miR169h, participated in disease-resistance regulation as transcriptionfactors. Similarly, one miRNA, ptc-miR164f, has been reported to regulate the plant development. All of these studieswould be served as the foundation for exploring the resistance mechanism.

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