Scientia Agricultura Sinica

Identification of miRNAs and tRFs in Response to Salt Stress in Rice Roots

ShuJun MENG, XueHai ZHANG, QiYue WANG, Wen ZHANG, Li HUANG, Dong DING, JiHua TANG

Scientia Agricultura Sinica. 2020, 53(4): 669-682. doi:10.3864/j.issn.0578-1752.2020.04.001

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【Objective】 Rice (Oryza sativa L.) is the most important food crop in China. But as a salt sensitive plant, to study the expression pattern of salt stress responding genes in rice has theoretical and practical significance. The dissection of the molecular mechanism and regulatory network in response to salt stress will contribute to salt-tolerant rice breeding. 【Method】 The rice variety Nipponbare seeds were used as experimental materials. Samples were treated with 150 mmol·L ^-1 NaCl for 21 days in 1/2MS medium. Small RNAs were extracted from treated and non-treaded root samples for sequencing. Differentially expressed miRNAs and tRFs (tRNA-derived RNA fragments) were identified using the cut-off of log₂ fold change (log₂FC) >1 or ＜-1, and their predicted target genes were analyzed. The sequencing results and target genes were further verified by real-time quantitative PCRs.【Result】 RPM > 500 for at least one set of data and log₂ FC > 1 or ＜-1 were used as the cut-off threshold, and 31 differently expressed miRNAs were obtained. Among them, the expression of 8 miRNA were up-regulated and 23 miRNA were down-regulated under salt treatment. The differently expressed miRNAs belonged to 12 miRNA families. 8 of these miRNA families have been reported as salt responding ones in Arabidopsis, maize and wheat, including osa-miR397, osa-miR396, osa-miR156, osa-miR167, osa-miR1432, which were down-regulated miRNAs, and osa-miR159, osa-miR168, osa-miR164, which were up-regulated ones under salt treatment, The other 4 miRNA families, namely osa-miR1882, osa-miR1876, osa-miR1423 and osa-miR5077, have not yet been reported in relation to salt stress. 162 target genes of these 31 differentially expressed miRNAs were predicted. Under salt stress, the expression of 34-38nt tRFs were significantly enhanced in rice root, indicating these tRFs were not produced randomly, but induced by salt stress. RPM > 50 and log₂ FC > 1 or ＜-1 were used as the threshold, 3 tRFs were detected as 5' terminal tRFs and 3 tRFs were detected as 3' terminal tRFs. These tRFs are produced from 6 tRNAs. It is suggested that these tRFs function potentially response to salt stress in rice root. 【Conclusion】 In this study, 12 miRNA families responding to salt stress were detected in rice root. Most of their target genes are transcription factors. It is suggested that these miRNAs are involved in the salt response through post-transcriptional regulation of their target genes. In addition, 8 of them were conserved salt responsive miRNAs among plant species. Furthermore, the salt responsive tRFs of rice was examined in transcriptome level. 6 tRFs induced by salt stress were identified.

Construction of Genetic Map and Mapping QTL for Flowering Time in A Summer Planting Soybean Recombinant Inbred Line Population

YongCe CAO, ShuGuang LI, XinCao ZHANG, JieJie KONG, TuanJie ZHAO

Scientia Agricultura Sinica. 2020, 53(4): 683-694. doi:10.3864/j.issn.0578-1752.2020.04.002

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【Background】 Flowering time (FT) is an important agronomic trait, which determines the planting range of cultivars and has a significant influence on the yield and quality of soybean. The Chinese Jiang-Huai valley is an important soybean producing area. However, little is known about the genetic basis of flowering time in these genotypes. 【Objective】 The objectives of this study were to map mapping quantitative trait loci (QTLs) and identify stable and reliable loci that can be used for molecular marker-assisted selection (MAS) and map-based gene cloning, and then dissect the genetic basis of flowering time in summer planting soybean. 【Method】 A recombinant inbred lines (RIL) population containing 91 lines (F_2:8) developed by crossing KF35 with NN1138-2 was planted in six environments to investigate phenotypic data. Restriction-site associated DNA sequencing (RAD-seq) technology was used to genotype all lines and their parents. And then bin markers were obtained by window sliding method based on the SNP markers and used to construct the genetic map. The mixed-model based composite interval mapping (MCIM) method in the software of QTL Network 2.2 and the composite interval mapping (CIM) method in the software of Windows QTL Cartographer V2.5_011 were used to reveal the effects of the QTLs of FT. 【Result】 A total of 36778 high-quality SNP markers were obtained in the whole soybean genome, and further divided into 1733 bin markers. A high-density genetic map with 1733 bin markers was constructed that spanned 2362.4 cM of the soybean genome with an average marker distance of 1.4 cM. Nine additive QTLs, two pairs of epistatic QTLs and one environmental interaction QTL were detected by MCIM method. The cumulative contribution of additive, epistatic and environmental interaction effects were 63.9%, 4.6% and 2.1%, respectively. Ten QTLs were detected by CIM method, and four of them, qFT-8-1, qFT-11-1, qFT-15-1 and qFT-16-1 could be detected in three or more environments. Altogether, 12 QTLs controlling FT were mapped using MCIM and CIM methods. Six of them, qFT-8-1, qFT-11-1, qFT-15-1, qFT-16-1, qFT-16-2, qFT-20-1 and qFT-20-2 could be detected by two methods. Six novel QTLs, qFT-5-1, qFT-8-1, qFT-8-2, qFT-13-1, qFT-15-1 and qFT-20-2 were detected in this study. 【Conclusion】 The genetic composition of FT in summer planting soybean is relatively complex. However, the additive effect was dominant, epistatic interaction and environmental interaction had little effect on FT. Four QTLs, qFT-8-1, qFT-11-1, qFT-15-1 and qFT-16-1 can be detected by two methods and in multiple environments, which are important loci for controlling FT in NJK3N-RIL population.

Discovery of Microsatellite Markers from RNA-seq Data in Cultivated Peanut (Arachis hypogaea)

ZhiJun XU, Sheng ZHAO, Lei XU, XiaoWen HU, DongSheng AN, Yang LIU

Scientia Agricultura Sinica. 2020, 53(4): 695-706. doi:10.3864/j.issn.0578-1752.2020.04.003

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【Objective】 This study aimed to identify SSRs in peanut RNA-seq data,clarify their distribution and structural characteristics,and develop gene-associated SSR markers. The study may lay the foundation for the excavation of important functional genes of peanut, the study of isometric variation and molecular markers assisted breeding. 【Method】From 22 different cultivated peanut tissue types and ontogenies that represent its full development, the reported RNA-Seq data, were used to analyze the distribution and characteristics of SSR using MISA software. Gene-associated SSR primers were designed by Primer 3.0 and its quality were detected by e-PCR 2.3.9. 38 pairs of primers were randomly synthesized for polymorphism testing. 【Result】A total of 19 143 SSRs were identified from 52 280 transcripts, distributed in 14 084 transcripts, with a frequency of 26.94%. The dominant SSR repeat unit types were mononucleotide and trinucleotide in mononucleotide to pentanucleotide, accounting for 39.24% and 38.40% of the total SSR locus. Dominant motif types of each repeat unit were A/T, AG/CT, AAG/CTT, AAAG/CTTT, AACAC/GTGTT, accounting for 97.62%, 72.01%, 30.96%, 24.59%, and 16.67% in the corresponding repeat units, respectively. The repetition of repeat units was 5-47 times, and the length distribution range of single SSR site was 10-47bp, mainly concentrated at 10-14 bp. The length range of compound SSR locus was 21-249 bp, mainly concentrated at 31-40 bp. Among all the SSR,13 477 SSR could be used to develop SSR markers, of which 5 020 transcript sequences were annotated to specific genes, containing 5 859 SSR markers locus. These SSRs were unevenly distributed on the 20 chromosomes of A and B genomes, and chromosomes B03 had the most SSR locus of 484. Using electronic PCR, 4 468, 4 929 and 10 188 effective loci were amplified in the genome of A. duranensis, A. ipaensis, A. hypogaea, with 3 968 (67.74%), 4 232 (72.25%) and 5 174 (88.33%) effective markers, respectively. In the genome of A. hypogaea, SSR primers amplified mainly with 2 loci, while 1 477 pairs of SSR primers were single-locus markers. And the physical map of amplified SSR loci was drawn according to the loci position in cultivated peanut genome. Among the randomly synthesized primers, 35 pairs (92.1%) of SSR primers amplified stable and clear bands in two peanut varieties, among which 11 pairs (28.9%) of SSR primers amplified different band. 【Conclusion】 In this study, 13 477 potential primer design SSRs were identified, 5 859 gene-associated SSR markers were developed and detected,with high amplification efficiency in cultivated peanut genome,and the physical map of gene-associated SSR were constructed.

Yield Gap of Ratoon Rice and Their Influence Factors in China

YuXian CAO, JianQiang ZHU, Jun HOU

Scientia Agricultura Sinica. 2020, 53(4): 707-719. doi:10.3864/j.issn.0578-1752.2020.04.004

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【Objective】The yield gap and the influence factors of ratoon rice was investigated for revealing yield potential in this study, so as to provide a scientific basis for developing high-yielding and high-efficiency ratoon rice management practices.【Method】The publications were collected by searching in China National Knowledge Infrastructure and Web of Science by using the keywords of “ratoon rice yield, variety, fertilizer application, density, stubble height, planting methods and harvesting methods” and “ratoon rice, variety, fertilizer and China”, respectively. A total of 119 published literatures were collected. This study summarized the yield potential and yield gap for ratoon rice in China. Meta-analysis method was applied to quantify the effect of variety, fertilization, density, stubble height, planting and harvesting methods on yield of main and ratoon crops of ratoon rice. This paper made a preliminary illustration about the factors that affect yield gap and ways to close the yield gap.【Result】The ratoon rice yield potential was 11.65 t·hm ^-2 for main crop, 6.90 t·hm ^-2 for ratoon crop and 17.10 t·hm ^-2 in total. The sample average yield of main crop, ratoon crop, and total crop for ratoon rice was 71%, 53% and 68% of the corresponding yield potential, respectively. Indica and hybrid variety could increase the ratoon rice yield by 24%-19% and 18%-8% than japonica and inbred variety, respectively; Optimized N, P₂O₅ and K₂O application rate was 168, 123 and 124 kg·hm ^-2 for main crop, and 145, 50 and 200 kg·hm ^-2for ratoon crop. Optimized density for ratoon rice was 22.4-29.1×10 ⁴ hills/hm ^-2. Optimized stubble height was 40-50 cm. Artificial transplanting could increase ratoon crop yield and thus maximize the total yield; Manual harvesting yield of ratoon crop was 12% higher than mechanized harvesting. But it’s no significant difference between artificial transplanting/harvesting and mechanized planting/harvesting.【Conclusion】 There was still a huge room to increase the yield of ratoon rice, and the yield gap of main crop, ratoon crop and total was 3.38, 3.27 and 5.41 t·hm ^-2, respectively. Furthermore, suitable variety, fertilizer application rate, density, stubble height, planting and harvesting method were important to close yield gap. For ratoon rice, the main varieties were indica and hybrid variety. Compared to average yield from all the samples, the yield of main crop and ratoon crop could increase by 9% and 22% respectively after optimized fertilization. Suitable density could increase the yield by 8% for main crop, 17% for ratoon crop; For ratoon rice, the optimized stubble height was 40-50 cm. Mechanized planting/harvesting as simplified pattern with low labor input, could more suitable for Chinese modern agriculture.

Spatial-Temporal Variations of Photo-Temperature Potential Productivity and Yield Gap of Highland Barley and Its Response to Climate Change in the Cold Regions of the Tibetan Plateau

KaiYuan GONG, Liang HE, DingRong WU, ChangHe LÜ, Jun LI, WenBin ZHOU, Jun DU, Qiang YU

Scientia Agricultura Sinica. 2020, 53(4): 720-733. doi:10.3864/j.issn.0578-1752.2020.04.005

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【Objective】The climate change of highland barley during the growth season and effect on photo-temperature potential productivity as well as yield gap over Tibetan Plateau from 1977 to 2017 were investigated.【Method】The DSSAT-CERES-barley was validated against statistical and field observational data, and then applied to simulate the potential yield of the highland barley on Tibetan Plateau. Then yield gaps were calculated by using observed yields and simulations. Finally, we analyzed the impact of climate change on highland barley production and yield gaps by using statistical methods.【Result】(1) Temperature and precipitation during highland barley growth period significantly increased on Tibetan Plateau over the past 40 years, whereas solar radiation decreased and it decreased significantly at Lizhi station; (2) The growth period of highland barley has significantly decreased if using the same variety at a fixed sowing date. The decrease of growth period in high-altitude was mainly caused by the increasing of the average maximum temperature, however, at low-altitude, which were mainly caused by the increase of the effective accumulated temperature during the whole growing period due to rising of mean temperature; (3) The potential barley yield was limited by the altitude and more sensitive to solar radiation at the high altitude stations. It was large and stable at the high-altitude stations with an altitude of 3 500 m. The average potential yield of Shannan station approached to 12 000 kg·hm ^-2 while only 6 000 kg·hm ^-2at low altitude stations around 3 000 m; (4) The yield gaps of highland barley in Tibetan Plateau in the past 30 years has decreased from 58.2% to 34.5% due to the increase of actual production. And the decreasing rate of yield gaps decelerated in recent decade. The yield gaps in Lasa and Shigatse were the least during 2007-2017, which were less than 25%.【Conclusion】The potential yields of highland barley on Tibetan Plateau were different greatly in different stations on Tibetan Plateau. The potential yield of the high-altitude areas was significantly larger than that of the low-altitude areas in study region. Climate change in the past 40 years had caused the higher variation of potential yield at low-altitude, while relatively stable potential yield at high-altitude. The yield gaps in Tibetan Plateau gradually decreased over the past 30 years because of the increase of actual yield, which was caused by the improvement of varieties and cultivation management. However, the yield gaps except Lasa and Shigatse were still large. Therefore, there was great potential to increase crop production in the future.

Effects of Different Mulching Patterns on Growth of Potato and Characteristics of Soil Water and Temperature in Dry Farmland

XueKe PU, ChunHua WU, YouMing MIAN, FangFang MIAO, XianQing HOU, Rong LI

Scientia Agricultura Sinica. 2020, 53(4): 734-747. doi:10.3864/j.issn.0578-1752.2020.04.006

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【Objective】 The effects of different mulching methods on soil water, temperature and potato growth in dry farmland of northwest China were studied. 【Method】 A field experiment was conducted in 2015 and 2016 for two consecutive years, and three mulching patterns were set up: full mulching of furrows and ridges with plastic film (DD), half film mulching of ridge and furrow (DB) and dual-mulching of ridge and furrow (DJ), the conventional flat field without mulching was used as the control check (CK). 【Result】 The soil water content with mulching treatments at the 0-200 cm soil layer during the whole growth period was higher than that under CK, especially the soil water retention effect under DJ treatment after 70-90 d sowing was the best. Different mulching treatments could reduce the soil water coefficient of variation at 0-200 cm soil layer, and the DJ treatment had the strongest vertical stability of soil water. The DD treatment had significant thermal insulation effect in 0-15 cm soil layer after 30-50 d sowing, while the DJ treatment had double effects of cooling and increasing temperature at 0-25 cm layer during the whole growth period, compared with CK. All the mulching treatments could reduce the variation coefficient of soil temperature at 0-25 cm soil layer, and the variation coefficient of soil temperature under DJ treatment was the smallest, while the vertical stability of soil temperature was the best. Mulching could increase the potato emergence rate, and affect emergence days. The average emergence days under DD treatment was 4.5 d earlier than that under CK, while DJ treatment delayed 3.5 d. The cumulative dynamics of biomass during the growth period of potato were consistent with the logistic growth equation. The theoretical value and growth rate of biomass under DJ treatment were the largest, and the rapid growth duration was the longest. 【Conclusion】 Through the principal component analysis, it was found that besides some factors of potato biomass, number of potatoes per plant, and potato per hectare, the soil water had a higher effect on potato growth than soil temperature, which was an important factor limiting potato high yield. DJ could improve soil moisture status and maintain relatively stable soil. Water temperature environment was beneficial to potato growth and tuber yield.

Evaluation of Resistance to Stripe Rust and Genetic Diversity and Detection of Resistance Genes in 103 Wheat Cultivars (Lines)

MoRan XU,RuiMing LIN,FengTao WANG,Jing FENG,ShiChang XU

Scientia Agricultura Sinica. 2020, 53(4): 748-760. doi:10.3864/j.issn.0578-1752.2020.04.007

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【Objective】The objective of this study is to understand the resistance level to stripe rust and genetic diversity of wheat cultivars (lines), master the utilization of stripe rust resistance genes, and to lay a foundation for wheat breeding and rational utilization of new cultivars with excellent resistance to stripe rust.【Method】A total of 103 wheat cultivars (lines) were identified by physiological races CYR32, CYR33 and CYR34 singly at seedling stage and CYR32 at adult stage. SSR molecular marker technique was used to evaluate the genetic diversity and the molecular markers of the important known resistance genes of wheat stripe rust Yr5, Yr9, Yr10, Yr15, Yr18 and Yr26 were used to detect the stripe rust resistance genes in the tested wheat cultivars (lines).【Result】The identification of seedling resistance showed that 20 out of 103 cultivars (lines) were resistant to physiological race CYR32, accounting for 19.42% of the tested materials; 34 cultivars (lines) showed resistance to CYR33, accounting for 33.01% of the tested materials; 36 cultivars (lines) showed resistance to CYR34, accounting for 34.95% of the tested materials; only six cultivars including Zheng 6 fu, Ningmai 3, Laolanmai, Jing 411, Jingzuo 278 and Yangmai 158 showed resistance to all the three physiological races CYR32, CYR33 and CYR34. The identification by CYR32 at adult stage showed that 55 cultivars (lines) such as Zhengzhou 021 had adult plant resistance, accounting for 53.40% of the tested materials. Genetic diversity analysis showed that the variation of genetic similarity coefficient of 103 cultivars (lines) ranged from 0.50 to 0.93, with an average of 0.66. Cluster analysis found that 103 cultivars (lines) were divided into three categories. The first category included four cultivars, namely Huapei 112-2, Jubilejina Ⅱ, Luzhan 1 and Elkhart. The second category included 43 cultivars (lines), among which the cultivars (lines) from the same region or with the same pedigree source were clustered in the same category, indicating that there was a certain relationship between the genetic relationship and the source. In the third category, the cultivars (lines) with the same pedigree were all clustered in one sub-category, indicating that the same or similar materials were mostly used in the breeding process of wheat cultivars in the same region and it resulted in the similar genetic relationship among wheat cultivars. The detection of known resistance genes suggested that Yr9, Yr10, Yr18 and Yr26 existed in 15, 8, 19 and 1 cultivars, respectively. Yr5 and Yr15 were not detected in the cultivars (lines).【Conclusion】The resistance level of the cultivars (lines) was low at seedling stage. Six cultivars, Zheng 6 fu, Ningmai 3, Laolanmai, Jing 411, Jingzuo 278 and Yangmai 158, may contain unknown all stage resistance genes, which are suitable for the control of wheat stripe rust in cultivar rotation planting. The resistance at adult stage was better and the frequency of Yr18 detection was higher. Therefore, wheat breeding should make full use of high-quality known resistant resources, explore new resistant materials, and cultivate durable resistant cultivars of multi-gene combination.

Identification of the Resistance to Sclerotinia Stem Rot in HIGS-SsCCS Transgenic Arabidopsis thaliana

YaRu CHAI,YiJuan DING,SiYu ZHOU,WenJing YANG,BaoQin YAN,JunHu YUAN,Wei QIAN

Scientia Agricultura Sinica. 2020, 53(4): 761-770. doi:10.3864/j.issn.0578-1752.2020.04.008

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【Objective】Sclerotinia stem rot is a kind of fungal disease caused by Sclerotinia sclerotiorum. The host range of S. sclerotiorum is wide, which seriously endangers the quality of many crops. The objective of this study is to enhance the resistance to stem rot by silencing the virulence genes of S. sclerotiorum in host via the host-induced gene silencing (HIGS) technology, and to provide new ideas for breeding of sclerotinia stem rot resistance.【Method】The gene encoding a copper chaperone for copper/zinc superoxide dismutase of S. sclerotiorum (SsCCS) was selected as the target gene, the sequences were analyzed by bioinformatics tools, and the phylogenetic tree was constructed using MEGA6.0 software. The specific interference fragment was selected for amplification after comparing the genome of Arabidopsis thaliana and S. sclerotiorum, respectively. HIGS vector containing the RNAi structure of SsCCS was transferred into wild type A. thaliana Col-0 mediated by Agrobacterium, and the stable HIGS-CCS transgenic A. thaliana lines were screened by DNA identification and labeling. The leaves of HIGS-CCS transgenic plants grown for 4-5 weeks were selected to analyze the resistance to sclerotinia stem rot according to the lesion area at 24 h after inoculated with S. sclerotiorum. The relative expression level of SsCCS during infecting was analyzed by qRT-PCR. The accumulation of H₂O₂ during the period of interaction between transgenic plants and S. sclerotiorum was detected by DAB staining at 6, 12 and 24 hpi.【Result】Bioinformatics analysis showed that the length of genome sequence of SsCCS (SS1G_00102) is 1 010 bp, while the length of its coding sequence (CDS) is 759 bp, encoding a protein with 253 amino acids, the molecular weight is 2 7176.96 Da, the isoelectric point (PI) is 5.04. SsCCS has 87% amino acid homology to BcCCS (EDN25358) while far to AtCCS (AT1G12520.1). By aligning with the genome of S. sclerotiorum and A. thaliana, a 314 bp specific interference fragment was selected and constructed the HIGS vector successfully and transformed into A. thaliana. The lesion of T₁ and T₂ generation transgenic lines was smaller than that of wild type A. thaliana 24 h after inoculated with strain 1980. From the T₂ generation, three stably expressed T₃ generation HIGS-CCS transgenic A. thaliana lines (HIGS-CCS-5, HIGS-CCS-8, HIGS-CCS-13) were obtained. Compared with wild type A. thaliana, the lesion area on the HIGS-CCS transgenic plants was reduced by 46% to 61% 24 h after inoculated with strain 1980. The expression of SsCCS was significantly reduced by 98% in the HIGS-CCS transgenic plants compared to that in wild type A. thaliana at 6 h after inoculated with S. sclerotiorum strain 1980. Furthermore, the accumulation of H₂O₂was decreased in transgenic plants as revealed by DAB staining, indicating the ROS production was reduced in transgenic plants.【Conclusion】The resistance to stem rot can be significantly enhanced by silencing the SsCCS of S. sclerotiorum in A. thaliana via the HIGS. This study provides a reference for the resistance improvement to sclerotinia stem rot of host crops, such as rapeseed.

Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana

MingYue GONG,XiaoTian DUAN,TingTing YU,Jie WANG,LiLi SHEN,Ying LI,MingHong LIU,YongLiang LI,HongKun LÜ,SongBai ZHANG,JinGuang YANG

Scientia Agricultura Sinica. 2020, 53(4): 771-781. doi:10.3864/j.issn.0578-1752.2020.04.009

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【Objective】Potato virus Y (PVY) is an important virus that harms major crops such as tobacco, potato, tomato, pepper and so on, and brings great losses to agricultural production. The objective of this study is to clone NbHsc70-2, analyze the bioinformatics of NbHsc70-2 protein, research the effect of NbHsc70-2 protein on PVY infection in Nicotiana benthamiana, and to provide a theoretical basis for further analyzing the infection mechanism of PVY.【Method】The gene coding sequence (CDS) of Hsc70-2 protein from N. benthamiana was cloned in pEarleyGate100 vector. Multi-sequence alignment and phylogenetic analysis were performed using MEGA 6.0 software. Fluorescence quantitative RT-PCR (qRT-PCR) analysis of NbHsc70-2 expression level was performed in different tissues of N. benthamiana. Bioinformatics analysis using online software BaCeILo and SignalP 4.0.was performed for construction of NbHsc70-2-RFP fusion protein and determination of the subcellular localization of the identified NbHsc70-2 protein. PVY-GFP was used to analyze the effect of PVY infection on NbHsc70-2 in N. benthamiana leaves and localization of this protein. Furthermore, NbHsc70-2 VIGS silencing system and transient expression vector were constructed, and the effect of the protein on PVY expression after silencing/overexpression was analyzed by comparing the results with qRT-PCR assay.【Result】NbHsc70-2 encodes a total of 649 amino acids. The phylogenetic analysis indicated that NbHsc70-2 belongs to the heat shock protein (HSP) family and has the highest similarity with the NaHsc70-2. The C-terminal has the highly conserved motif structure of the HSP family. qRT-PCR analysis showed that NbHsc70-2 had the highest expression level in leaves and low expression in roots and stems. Moreover, BaCeILo prediction and laser confocal microscopy depicted that NbHsc70-2 was localized in the cytoplasm, and after PVY-GFP infection to N. benthamiana, NbHsc70-2-RFP was partially transferred to the nucleus and colocalized with virus in the cytoplasm and nucleus. The induction of NbHsc70-2 gene silencing vector pTRV::NbHsc70-2 into N. benthamiana resulted in stunted growth of infected plants compared to the control at 7 dpi. Whilst, inoculation of silent plants with PVY-GFP didn’t reflect green fluorescence, whereas PVY-GFP inoculation resulted in the green fluorescence production from the leaves of plants kept as control, the number of fluorescent spots in the silent group was about 28% of that in control group. qRT-PCR analysis showed that further inoculation of PVY resulted in accumulation of PVY CP at 1, 3, and 5 d after silencing NbHsc70-2. Moreover, the level of PVY CP decreased after silencing NbHsc70-2, and the difference between 3 and 5 dpi was significant compared to the control, the gene expression level was 14% and 0.004% of that in the control, respectively. Contrarily, high level of PVY CP was observed when N. benthamiana plants were inoculated with NbHsc70-2 overexpression vector along with PVY. The detailed analysis showed that the accumulation of PVY CP was significantly increased at 48 h and 72 h, and the gene expression level was 2.31 and 2.56 times of that in the control group, respectively.【Conclusion】PVY infection causes increased expression of NbHsc70-2. NbHsc70-2 is an important component of PVY infection to N. benthamiana, silencing NbHsc70-2 significantly inhibited PVY expression, and overexpression of NbHsc70-2 significantly increased PVY expression, i.e., the expression level of NbHsc70-2 was positively correlated with PVY replication. NbHsc70-2 protein promoted PVY infection in tobacco.

Nitrogen Fertilizer and Its Combination with Straw Affect Soil Labile Carbon and Nitrogen Fractions in Paddy Fields

ShiChao WANG,ZhiHao YAN,JinYu WANG,ShengChang HUAI,HongLiang WU,TingTing XING,HongLing YE,ChangAi LU

Scientia Agricultura Sinica. 2020, 53(4): 782-794. doi:10.3864/j.issn.0578-1752.2020.04.010

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【Objective】Soil microbial biomass carbon & nitrogen (N) (SMBC & SMBN) and water soluble organic carbon & nitrogen (DOC & DON) are the active pools in soil, which are essential indexes for assessing soil carbon and nitrogen turnover and nutrient availability for providing scientific information and guidelines and nitrogen management under straw returning in rice-wheat rotation system. The objective of the study was to investigate the effects of straw incorporation, nitrogen application rate and basal/topdressing ratios of nitrogen fertilizers on the SMBC, SMBN, DOC, DON, ROC and AN at paddy field, so as to explicit the optimal nitrogen rate and basal/topdressing ratios of nitrogen fertilizers under the straw incorporation condition. 【Method】A 4-year field experiment with straw incorporation matched with nitrogen fertilizer was conducted in rice cropping system from 2012 to 2015 in Jingmen city, Hubei Province. Three field experiments including different nitrogen application, straw incorporation combined with nitrogen fertilization and different ratios of base N to dressing N. Field experiments included: (1) different nitrogen rates, including No N fertilizer (N0), Optimal N fertilizer (165 kg N·hm ^-2, N165), the farmer common N rate (195 kg N·hm ^-2, N195); (2) different straw incorporation combined with nitrogen fertilization: straw removal (CK), straw incorporation (wheat straw incorporation before rice transplanting, S), straw incorporation matched with farmer common N rate (SN), straw incorporation matched with optimal N rate (SF), SF plus straw decomposing microbial inoculants (SM); (3) three basal/topdressing ratios of nitrogen fertilizers in the shooting and heading stage: 7﹕3﹕0 (R1), 5﹕3﹕2 (R2); 10﹕0﹕0 (R3). 【Result】The results showed that the SMBC content significantly increased at rice jointing stage and the DOC content obviously decreased after the maturity under the SN treatment. The DON content increased under the SF treatment at rice jointing stage. DON and ROC contents decreased under the SM treatments at the maturity and jointing stage. The AN content increased in the SF treatment at rice heading and grain filling stage. Appropriate N application rate (165 kg N·hm ^-2) was favorable to increasing the DON and AN in soil. However, the DON and AN contents decreased under high application rate of N fertilizer (195 kg N·hm ^-2). The higher proportion of topdressing fertilizer increased the SMBN and ROC contents at rice jointing stage, but had no impacts on the SMBC and DOC contents. 【Conclusion】Nitrogen rate and basal/topdressing ratios were main factor for determining active soil carbon and nitrogen contents under straw incorporation in Paddy field. Appropriate nitrogen fertilizer could increase SMBC, AN, and DON contents, and the higher topdressing fertilizer could also increase active soil carbon and nitrogen contents during rice growth stage.

Present Situation and Analysis of Soil Acidification in Chinese Tea Garden

Peng YAN,WenYan HAN,Xin LI,LiPing ZHANG,Lan ZHANG

Scientia Agricultura Sinica. 2020, 53(4): 795-801. doi:10.3864/j.issn.0578-1752.2020.04.011

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【Objective】Tea is an important economic crop in China. Because of its high economic value, its planting area has been expanding rapidly in the past years. However, the soil acidification is more and more serious. Understanding the soil acidification of tea gardens in the main tea planting provinces in China will be useful for guiding soil management and tea development in future.【Method】To achieve our objective, 99 papers published in Chinese magazine Network and web of science were collected and the data about soil pH and its distribution in there interval of ＜4.5,4.5-5.5 and ＞5.5 were analyzed by weighted average method. We further compared the soil pH between forest and tea garden to understand the effect of tea cultivation on soil acidification. And depending on the data from National Soil Testing and Formula Fertilizing Project, the soil pH with different crops were also analyzed in Songyang and Anxi countries, which were two representative tea planting countries.【Result】The averaged soil pH of the whole county was 4.73, while it was various among different provinces. The averaged soil pH of Shandong and Henan were above 5.5, which were 5.76 and 5.54, respectively. While the averaged soil pH of Jiangxi was as low as 3.86. As for the percentage of averaged soil pH distribution in the interval of ＜4.5, 4.5-5.5 and ＞5.5, only 41% soil pH were in the interval of 4.5-5.5, which was suitable for tea growth. 52% sites were in the interval of pH＜4.5, which was too low for tea growth. Depending on the data from 22 literatures, the soil pH of tea garden was 4.28, which was significantly lower than that 5.17 of forest. In Anxi, the country with long history of tea plantation, the soil pH of tea garden was 4.2, which was lower than that 5.2 and 6.2 of rice and fruit or vegetable soil. In Songyang, the country started to growth tea in the last two decades, the soil pH of tea garden was 5.1, this was also lower than that 5.2 and 5.4 of rice and fruit or vegetable soil. 【Conclusion】 Soil acidification was serious in our tea garden, the averaged soil pH was 4.73, and 52% of the tea garden with soil pH below 4.5, which was the critical value for tea growth. It is important to pay more attention on developing various technologies to ameliorate soil acidity in tea garden, and achieve its sustainable development in China.

Fine Mapping and Candidate Gene Analysis of a Short Internodes Gene Cmdm1 in Melon (Cucumis melo L.)

Jian MA,CongCong LI,JianShe WANG

Scientia Agricultura Sinica. 2020, 53(4): 802-810. doi:10.3864/j.issn.0578-1752.2020.04.012

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【Objective】In this study, the Z8 was a mutant with short internode in melon, and its gene fine mapping and candidate gene analysis were conducted to provide the theoretical guidance for the improvement of melon architecture. 【Method】Agronomic traits, such as internode number, main stem length, main stem internodes length and lateral branch length were measured in the Z8 and wild-type B15. The Z8 was crossed with B15, and the F₂ and BC₁F₁ generations were used for genetic analysis. The recessive individuals in F₂ were used to locate the gene by the map-based cloning strategy, and the candidate gene in the mapping region was determined by gene sequencing. 【Result】The Z8 mutant was dwarf because the internodes were significantly shorter compared with the wild-type B15. In addition, the flower buds of Z8 mutant were clustered at the top, which showed significantly compact inflorescences. Genetic test indicated that the mutant phenotype segregated as a single recessive gene locus named Cmdm1, which had been mapped to a 56 kb interval on the short arm of chromosome 7, flanked by markers c7-112 and s2 and co-segregated with marker dm-1. The MELO3C016916 gene was homologous to Arabidopsis ERECTA gene, which was one of the four annotated open reading frames (ORFs) in the 56 kb region. Sequence analysis of MELO3C016916 gene in the Z8 mutant indicated that a single nucleotide substitution (T ¹⁹⁹⁵→G ¹⁹⁹⁵) in its 25 ^th exon caused premature termination of the encoded protein, thus placing MELO3C016916 as a candidate gene for Cmdm1.【Conclusion】The short internode trait of Z8 was controlled by a single recessive gene named Cmdm1, which was finally mapped to a 56 kb interval between markers c7-112 and s2 on chromosome 7, and the MELO3C016916 gene was presumed to be the candidate gene.

Influence of Three Enzymes on Oxidation of Ascorbic Acid in Postharvest 'Hayward' and 'Huate' Kiwifruit

Qi FENG,ChaoZheng LI,GuiTian GAO,You WU,Yan XIAO,WuQi ZHAO,YuShan LEI

Scientia Agricultura Sinica. 2020, 53(4): 811-822. doi:10.3864/j.issn.0578-1752.2020.04.013

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【Objective】This study explored the differences in activities of enzymes and their gene expressions related with oxidation of ascorbic acid (AsA) in postharvest Hayward and Huate kiwifruit to provide a theoretical basis for the oxidation mechanism of AsA in postharvest kiwifruit to regulate fruit ripening and senescence, maintain postharvest quality and extend storage life.【Method】 Hayward and Huate kiwifruit were harvested, and total ascorbic acid (T-AsA), dehydroascorbic acid (DHA), AsA/DHA, and AsA content were measured under the storage conditions of 25℃. Enzyme activity of ascorbic acid oxidase (AO), laccase, ascorbate peroxidase (APX) and gene expression related with AsA oxidation were measured. The relationships between AsA content and the enzyme activity of AO, laccase, APX and gene expression were investigated in the two varieties of kiwifruit.【Result】The content of AsA in Hayward was 86.9 mg/100 g FW at the beginning of harvest, and the loss was about 45% at the end of storage. The content of AsA in Huate was higher at the beginning of harvest (610 mg/100 g FW), and it rose to 886 mg/100 g FW, reached its peak value during the storage, and dropped to 778 mg/100 g FW at the end, which was higher than that on the first day after harvest. The DHA content of the two varieties showed a downward trend throughout the whole storage period, but the DHA content of Hayward was lower than that of Huate. The T-AsA content and AsA content change trend was similar. In the later stage of storage, the AsA/DHA ratio of Hayward was much lower than that of Huate. AO activity was significantly negatively correlated with AsA content of two varieties of kiwifruit, and laccase activity was negatively correlated with AsA content. From the 8th day after harvest, the AO activity of Huate was lower than Hayward, and the laccase activity was lower than Hayward during the whole storage period of Huate kiwifruit. The activity of AO and laccase got the lowest value on the 11th day after harvest; laccase activity reached its highest value in Hayward on the 16th day after harvest. APX had little effect on AsA content, and its activity had no significant correlation with AsA content. Among the three genes in the AO gene family, Achn020161 was a key gene for the oxidative decomposition of AsA, while Achn191341 and Achn316521 had no significant correlation with the oxidation of AsA. Among the three genes in the laccase gene family, Achn007661 and Achn191341 had certain effect on the change of AsA content, while Achn163871 had no significant correlation with the oxidation of AsA. Achn123021, Achn082241 and Achn187071 in the APX gene family had certain effect on the change of AsA content, but they were not key genes for oxidative degradation of AsA.【Conclusion】The high ratio of AsA/DHA played an important role in the accumulation of AsA. AO was the key enzyme on the oxidation of AsA, laccase had certain effect, and APX was not a main enzyme in oxidation of AsA. It’s speculated that Achn020161 in the AO gene family was the key gene for oxidizing AsA, and Achn007661 and Achn19134 in the laccase gene family had certain effects on the oxidation of AsA.

Effects of Ozone Fumigation Combined with PE Packaging on Postharvest Storage Quality and Antioxidant Capacity of Flammulina velutipes

Ting WANG,Yu ZHANG,Hong LIU,TianTian HE,Yang BI,JianMin YUN

Scientia Agricultura Sinica. 2020, 53(4): 823-835. doi:10.3864/j.issn.0578-1752.2020.04.014

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【Objective】In order to prolong the shelf life of Flammulina velutipes (F. velutipes), the effects of ozone fumigation combined with PE packaging on postharvest storage quality and antioxidant capacity of F. velutipes after harvest was studied.【Method】Using F. velutipes cultivated in factory as test material, the samples were fumigated with different ozone concentration and time at low temperature (8±1)℃ after pre-cooled 24 h at (4±1)℃, and the best ozone fumigation concentration and treatment time were screened by the sensory score and the indexes of browning index and weight loss rate. And then the storage experiment of F. velutipes was carried out at 4±1℃. To evaluate the fresh-keeping effect of ozone on F. velutipes, during the period, the main physiological quality indexes were determined every 3 d, including mushroom cap diameter, weight loss rate, malondialdehyde (MDA) content, relative conductivity, browning index, total bacteria count, respiratory intensity, active oxygen content ($O^{\bar{·}}_{2}$ and H₂O₂), catalase (CAT), and superoxide dismutase (SOD) activity.【Result】The results showed that the sensory score of F. velutipes treated with 2.711 mg?m ^-3 ozone fumigation for 15 minutes was significantly higher than that of the control group after 21 d storage at 4±1℃. The diameter of mushroom cap, weight loss rate, MDA content, relative conductivity, browning index and aerobic plate count of F. velutipes treated with ozone fumigation were significantly lower than those of the control group (P＜0.05). Respiratory peak of F. velutipes treated with ozone fumigation was delayed and the accumulation of reactive oxygen species (ROS) was significantly lower in the treatment group than in the control group (P＜0.05), while the activities of CAT) and SOD remained at a higher level. During 21 days of storage, in the same day, the sensory quality of F. velutipes treated in the same period was better than that of the control group. Surface depression and yellowing degree of the treated group were slighter and the scores of the treated group were higher. On the 6th d, 12th d, 15th d, 18th d and 21th d, the sensory quality of the treated group was 3.8%, 4.3%, 3.7%, 3.6% and 4.7% higher than that of the control group, respectively. The rate of weightlessness was significantly different after 6 days, the treatment group was lower, and the peak of respiratory jump of fruiting bodies was delayed. The content of MDA increased slowly at the beginning of storage (1-6 d), then accumulated rapidly, and the change trend was consistent with the relative conductivity. The browning index and the aerobic plate count of F. velutipes showed an overall upward trend, and which of the treatment groups were significantly lower than the control. The content of reactive oxygen showed a continuous upward trend, and that of treatment was slower than that of control. The activity of SOD reached its maximum value at 9th d, CAT activity reached its maximum value at 12 d.【Conclusion】Ozone fumigation combined with PE packaging could significantly delay the quality deterioration of F. velutipes, enhance its antioxidant capacity and prolong its shelf life.

Differential Expression of mRNA and lncRNA in Longissimus Dorsi Muscle of Songliao Black Pig and Landrace Pig Based on High-Throughput Sequencing Technique

YaoQun WU,ShaoKang CHEN,XiHui SHENG,XiaoLong QI,XiangGuo WANG,HeMin NI,Yong GUO,ChuDuan WANG,Kai XING

Scientia Agricultura Sinica. 2020, 53(4): 836-847. doi:10.3864/j.issn.0578-1752.2020.04.015

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【Background】Muscle growth and fat deposition are important economic traits in pig production and are also complex quantitative traits. Landrace, originally from Denmark, is one of the world-famous lean-type, with fast growth and high lean meat rate, but its meat quality is poor. Songliao black pig is a black maternal local cultivar cultivated by ternary cross-breeding with the Min pig, the Landrace pig and the Duroc pig, which has the characteristics of high reproduction rate, good meat quality, strong adaptability, roughage resistant and etc. There is a big difference in meat quality between the Songliao Black Pig and Landrace Pig. 【Objective】 In this study, high-throughput sequencing technique was used to analyze the longissimus dorsi transcriptome of Songliao black pig and Landrace. The key mRNA and long non-coding RNA（lncRNA）, affecting muscle growth, meat quality and fat deposition were selected to provide new reference information for the study of pork quality. 【Method】We collected the longissimus dorsi muscle of 6 Songliao black pigs and 6 Landrace to extract RNA. Individuals in each cultivar were sampled in the same RNA mixing pool and sequenced by Illumina HiSeq 2500 high-throughput sequencing technology. Reads were compared, annotated and differentially expressed. Differentially expressed genes and lncRNAs were screened out and their biological functions were enriched. Moreover then, ten genes, including five up-regulated genes and five down-regulated genes, were selected from the differentially expressed RNA and lncRNA, and the relative expression levels of differentially expressed mRNA and lncRNA were detected by real-time PCR. 【Result】After quality control, each sample received 103 M and 150 M reads, of which more than 98% of the reads were of higher quality, meeting the requirements of further analysis. The reads were aligned to the reference genome and a total of 26 774 transcripts were obtained, of which 8 428 were new transcripts. Using edgeR for differential expression analysis, the reference standards are False Discovery Rate(FDR)＜0.05 and fold change (FC)＞2. A total of 4 239 significant differentially expressed genes were obtained, of which 2 023 were up-regulated and 2 216 were down-regulated in Songliao black pig muscle. Meanwhile, 178 significant differentially expressed lncRNAs were detected, of which 84 were up-regulated and 94 were down-regulated in Songliao black pig muscle tissue. The biological function enrichment analysis of differentially expressed genes revealed that it is mainly enriched in cell components and cell development, biological metabolism regulation, and signaling pathways and biological metabolic pathways involved in muscle development and lipid metabolism. The target gene prediction results of the screened lncRNA showed that there was a potential differential relationship between the four differentially expressed lncRNAs and the differentially expressed genes, namely TCONS-00041713, TCONS-00041712, ENSSSCT00000034982, ENSSSCT00000034269. At the same time, the results of real-time PCR analysis of 10 differentially expressed mRNAs and lncRNAs showed that the results of real-time PCR were similar to the expression of transcriptome sequencing of the longissimus dorsi muscle tissue of Songliao black pig and Landrace, indicating that high throughput sequencing results are reliable.【Conclusion】We obtained the key mRNA and lncRNA that affect the muscle growth, meat quality, and fat deposition of pigs. Meanwhile, it was found that there may be a potential regulatory relationship between several pairs of differentially expressed lncRNA and mRNA.The discovery of these differentially expressed mRNAs and lncRNAs and possible potential regulatory relationships may have a certain significance for studying the molecular mechanisms that regulate metabolic activity in muscle tissue and provide new reference information.

Effects of Light Intensity on c-fos, Biological Clock Gene Expression and Melatonin in Cherry Valley Meat Ducks

JiaJie CUI,Qiang XIE,ShuangShuang ZHAI,Tao GONG,YongWen ZHU,Lin YANG,WenCe WANG

Scientia Agricultura Sinica. 2020, 53(4): 848-856. doi:10.3864/j.issn.0578-1752.2020.04.016

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【Objective】 The objective of this study was to investigate the effects of different intensities of light stimulation on c-fos and related clock genes in hypothalamus, diencephalon, pituitary and mesencephalon, as well as melatonin levels in plasma and liver of cherry valley meat duck under continuous darkness. 【Method】 A total of one hundred and forty four 1-day-old cherry valley meat ducks were raised to the age of 21 days. 22-day-old cherry valley meat ducks were randomly divided into 2 groups. Each group had 6 replicates, with 12 ducks per replicate, and kept dark for 7 days, the ducks were given ad libitum access to water and feed. After 7 days lasting darkness, 28-day-old ducks received a LED white light stimulation of different intensities for 1.5 h and then a dark adaptation for 1.5 h. One duck from each replicate was slaughted for sample collection, the expression levels of c-fos and circadian clock genes in the hypothalamus, diencephalon, pituitary and mesencephalon were measured, and plasma and liver melatonin levels were measured. 【Result】 The expression level of c-fos in hypothalamus and mesencephalon of the 10 Lx group was significantly higher than that in the 80 Lx group (P＜0.05). The level of c-fos in the diencephalon was significantly lower than that in the 80 Lx group (P＜0.05), and there was no significant difference of c-fos expression in pituitary in the two groups(P＞0.05). Compared with the 80 Lx group, the expression of clock gene clock, Bmal1, Bmal2 and Per2 in the 10 Lx group in the hypothalamus was significantly increased (P＜0.05), and the expression of Cry1 was little increased in the 10 Lx group (P=0.06) .The expression level of Bmal1 in the diencephalon was significantly higher in the 10 Lx group (P＜0.05), while Bmal2, Clock, Per2 and Cry1 showed no significant difference (P＞0.05) in two groups. The expression of Clock and Per2 in the mesencephalon was significantly higher in the 10 Lx group than that in the 80 Lx group (P＜0.05). The expression of Bmal1, Bmal2 and Cry1 in the two groups had no significant difference (P＞0.05). The expression of clock genes in pituitary was not significant affect by light intensity (P＞0.05). Compared with the 80 Lx, the plasma level of melatonin in 10 Lx group was significantly lower (P＜0.05), while the melatonin levels in the liver were significantly higher. 【Conclusion】 In the continuous darkness, transient high intensity light stimulation could inhibit the expression of c-fos in the hypothalamus and mesencephalon, and increase the expression of c-fos in the diencephalon, while the stimulation could attenuate the amplitude of gene expression in the hypothalamus and diencephalon of the meat duck.

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