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Identification of Cashmere Dermal Papilla Cells Based on Single- Cell RNA Sequencing Technology ZHANG WeiDong,ZHENG YuJie,GE Wei,ZHANG YueLang,LI Fang,WANG Xin Scientia Agricultura Sinica    2022, 55 (12): 2436-2446.   DOI: 10.3864/j.issn.0578-1752.2022.12.014 Abstract （368）   HTML （39）    PDF （3368KB）（105）       Knowledge map    Save 【Objective】 Based on single-cell RNA sequencing, this article aims to explore the marker genes of cashmere dermal papilla cells, and to optimize the methods to identify dermal papilla cells in vitro, thereby laying a cell model for future pertinent research in cashmere hair follicle development. 【Methods】 The single-cell transcriptional data from the skin tissues of Shanbei white cashmere embryonic stage (E60, E90 and E120) were analyzed with Seurat package. After quality control, filter and normalization of raw data, the dimension reduction analysis and cell cluster identification were performed by uniform manifold approximation and projection (UMAP). Moreover, depending on cluster-specific expressed gene expression, the principal cell lineage information was identified. The type-specific marker genes of the dermal papilla were obtained after gene expression analysis. The immunofluorescence staining was used to validate the expression position of marker protein to identify the dermal papilla specific protein in goat skin. Whole hair follicles were isolated mechanically under stereoscope, and combined with enzyme detach, cashmere dermal papilla region was isolated and cultured in vitro until cell separation. The dermal papilla cells were purified by different-speed adherence methods. When the cells were highly pure, the expression of candidate marker protein was verified by immunofluorescence assay. 【Result】 In current study, the key transcription information of goat hair follicle cells was analyzed at single cell level. Information of 17 subsets of cells in cashmere goat skin structure was obtained successfully including dermal cell lineage, epidermal cell lineage, dermal papilla cell, hair stem cell and inner root sheath cell, as well as other functional cell groups such as pericyte cell, macrophage and muscle cell. 427 specific markers of dermal papilla cells including SOX2, FGF7, APOD, BMP3, HHIP, HEY2 and SPON1 were screened. By comparison, the expression of these marker genes in cashmere dermal papilla cells was much higher than that in other cell types, which could be confirmed as the specific genes of hair papilla cells. Immunofluorescence result further proved that SOX2, FGF7 and APOD were specifically expressed in the dermal papilla region, and could be used to trace the dermal papilla cells in vivo. In addition, in current study, the single cashmere goat secondary hair follicle was separated successfully, and the adherent culture of dermal papilla was realized. A large number of cells were observed migrating from the hair papilla area. Immunofluorescence assay showed that SOX2, FGF7 and APOD were all expressed in goat dermal papilla cells, and about 76% of cells were SOX2 positive, while more than 98% of cells were FGF7 and APOD positive. Combined with the immunofluorescence results, SOX2, FGF7 and APOD genes factually could be used to identify the cultured goat dermal papilla cells in vitro. 【Conclusion】 In this study, single cell RNA sequencing technology was used to describe the main transcriptome information of cells in cashmere goat skin, and the specific marker genes of dermal papilla cells were sifted out successfully. And it proved that single-cell sequencing based method was simple and efficient to identify marker genes further identified by immunofluorescence. The discovering of SOX2, FGF7 and APOD not only provided the markers for the localization of hair papilla cells in vivo, but also provided the possibility for the identification of dermal papilla cells with multiple markers, which laid the foundation for further study of the gene functions in regulating hair follicle development. Table and Figures | Reference | Related Articles | Metrics

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DUS Traits Variation Analysis and Application of Standard Varieties of Lolium multiflorum Introduced from Japan FENG JunJie,ZHAO WenDa,ZHANG XinQuan,LIU YingJie,YUAN Shuai,DONG ZhiXiao,XIONG Yi,XIONG YanLi,LING Yao,MA Xiao Scientia Agricultura Sinica    2022, 55 (12): 2447-2460.   DOI: 10.3864/j.issn.0578-1752.2022.12.015 Abstract （320）   HTML （24）    PDF （2382KB）（87）       Knowledge map    Save 【Objective】This study aimed to determine the consistency and specificity of DUS test traits of Lolium multiflorum (annual ryegrass) standard varieties introduced from Japan, and to optimize the DUS test system of L. multiflorum in China, thus identify new varieties (lines) more quickly and accurately. 【Method】In this study, seven standard varieties of annual ryegrass introduced from Japan were planted in Chengdu Plain, China. Fifteen individuals with the similar growth status were selected from each standard variety. Cluster analysis of the standard varieties was conducted based on their DUS test data to identify the DUS trait expression of the standard varieties in Chengdu Plain. In order to screen out the characters with the high intra-population consistency and obvious inter-population specificity, the coefficient of variation and variance values of 18 tested DUS characters were calculated, and the nested variance analysis of the studied characters was also conducted. Furthermore, the probability classification was conducted based on the selected characters, which was used to clustering analysis and identification of the tested national approved varieties and new lines, and to evaluate the correlation coefficients of test traits. 【Result】According to the values of 18 DUS test traits of standard varieties, the standard varieties could be clearly clustered into 7 groups. The DUS traits were fully expressed and could be clearly distinguished from each other, which were suitable for subsequent correlation analysis. The results of intraspecific consistency analysis showed that, six traits, including leaf color degree, growth habit after vernalization, plant width at booting stage, flag leaf width at booting stage, flag leaf length-width ratio at booting stage, and spikelet length, possessed the poor intra-population consistency for multiple varieties. When conducting the inter-population specificity analysis of varieties, however, the poor specificity among the tested varieties was found in plant height at booting stage, ratio of flag leaf length to width at booting stage and spikelet density among cultivars, among which the variance component between populations of flag leaf length-width ratio at booting stage was 54.83%, and the variance component between individuals within populations was 45.17%. The selected ten quantitative traits were conducted the K-S test and χ2 test analysis, however, only the basal spikelet length conformed to χ2 test. Afterwards, the ten quantitative traits were divided into five grades. On the basis of these traits grading criterion, the identification of national approved varieties and new lines of L. multiflorum. The results showed that the tested varieties and new lines of annual ryegrass could be clustered into eight groups according to its classification results and corresponding to varieties origin, which revealed the reliability of traits grading criterion built in this study on variety identification. The heat map drawn based on the trait values of the tested varieties showed that the leaf length of ‘Tetragold’ was significantly different from that of other tested varieties in the vegetative growth period, which was manifested as shorter leaf length; the variety ‘Chuannong No. 2’ showed higher plant height after vernalization; the internode length under spike of ‘Diamond T×changjiang No. 2’ was significantly shorter than that of other varieties; the poor intra-population consistency of ‘Double Barrel’, ‘Chuannong No. 1’ and ‘Chuannong No. 2’ showed good intra-population consistency. The low correction and strong independence were existed within the selected ten traits, indicating that these traits were very well suited for DUS test. 【Conclusion】The character classification system for DUS test based on Japanese standard varieties could be applied to the identification and distinguish of L. multiflorum varieties released in China. This study could provide the important technical and theoretical reference for the optimization of DUS test method system of annual ryegrass varieties. Table and Figures | Reference | Related Articles | Metrics

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Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang Scientia Agricultura Sinica    2022, 55 (12): 2461-2471.   DOI: 10.3864/j.issn.0578-1752.2022.12.016 Abstract （459）   HTML （35）    PDF （1275KB）（151）       Knowledge map    Save 【Background】As an important native resource insect, the Chinese honeybee (Apis cerana cerana) plays important ecological roles in pollinating the plants that bloom at low temperatures in early winter in China, and the pollination behavior of A. c. cerana is closely related to its olfactory system. According to the analysis of antenna transcriptome data collected from foragers treated at high and low temperatures, it was found that the Niemann-Pick type C2 protein (NPC2) gene family related to insect olfaction was up-regulated expression at low temperatures.【Objective】Therefore, this study aims on the A. c. cerana NPC2 family genes, including cloning and analysis of their structural characteristics and expression profiles. Moreover, the NPC2 family gene expression under high and low temperatures was also studied. It will provide an evidence of the AcNPC2 gene family in the low-temperature adaptation involved in the chemosensory and olfactory function of A. c. cerana.【Method】Based on the results of high and low temperatures transcriptome sequencing of A. c. cerana, the ORF sequence of AcNPC2 genes was cloned by RT-PCR, and phylogenetic tree analysis and three-dimensional structure prediction were performed. Then, the spatio-temporal expression profile of AcNPC2 genes in different developmental stages and different tissues, as well as the amount of expression at high and low temperatures of A. c. cerana were analyzed by qRT-PCR.【Result】The full length ORFs of four NPC2 genes of A. c. cerana (AcNPC2a, AcNPC2b, AcNPC2c, and AcNPC2d) were obtained as 447, 480, 459, and 465 bp, respectively, encoding 148, 159, 152, and 154 amino acids. The predicted protein molecular weight is 16.12-18.53 kD, and the isoelectric points are 7.98, 7.57, 6.56, and 6.34, respectively. Phylogenetic tree analysis showed that AcNPC2 sequences were most close to the NPC2 homologous sequence of Apis mellifera ligustica. qRT-PCR results showed that the expression level of AcNPC2a was the highest in the abdomen of the newborns, followed by the abdomen of the nurses and the larval stage. The expression of AcNPC2b was the highest in the thorax of the newborn bees, followed by the head, thorax and metapodium of the foragers. AcNPC2c was notably expressed in high abundance in the antennae of the nurses and the foragers. AcNPC2d had the highest expression in the head of the foragers. After low temperature treatment, the expression levels of all AcNPC2 genes in the forager antennae increased, but there was no significant difference.【Conclusion】AcNPC2 has the conserved structure of NPC2 protein family, and its members show diversity in the spatio-temporal expression profile of A. c. cerana. Among them, AcNPC2c is highly expressed in the antennae, indicating that it is closely related to the olfactory function of A. c. cerana. The expression of the whole AcNPC2 family genes increased in the antennae of the foragers at lower temperature, indicating that these genes might be involved in the low-temperature adaptability of A. c. cerana and pollination behavior in early winter. Table and Figures | Reference | Related Articles | Metrics

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Association Analysis of the ADIPOQ Variation with Sheep Growth Traits LIANG Peng,ZHANG TianWen,MENG Ke,SHAO ShunCheng,ZOU ShiFan,RONG Xuan,QIANG Hao,FENG DengZhen Scientia Agricultura Sinica    2022, 55 (11): 2239-2256.   DOI: 10.3864/j.issn.0578-1752.2022.11.013 Abstract （336）   HTML （40）    PDF （2205KB）（83）       Knowledge map    Save 【Objective】 The aim of the study was to explore the effects of genetic variation of ADIPOQ on growth traits of sheep, and to find the molecular genetic markers related to growth traits in Ningxia high-quality mutton sheep breeding, so as to achieve the purpose of molecular assisted breeding. 【Method】 The mutation sites of ADIPOQ in Dupo sheep, Tan sheep and Small-Tailed Han sheep were obtained by Allegro Targeted Genotyping. At the same time, the ear tissues of 383 different hybrid progenies of three breeds were collected. The SNPs were genotyped by Sequenom Mass ARRAY®SNP. Haploview was used to analyze linkage disequilibrium and construct haplotypes of the polymorphic loci, as well as association analysis between the SNPs in ADIPOQ with growth traits of newborn and 3-month-old sheep.【Result】 A total of 7 SNPs were screened, and 7 SNPs showed polymorphism in the hybrid population. The dominant genotypes of SNP1-SNP7 were CC, GG, GG, CT, AG, GG and AA, and the dominant alleles were C, G, G, C, G, G and A. X2 test showed that all loci were in Hardy Weinberg equilibrium (except SNP7 site deviated from the equilibrium in all individuals). SNP1 (except F2), SNP4, SNP5 and SNP6 were moderately polymorphic (0.25≤PIC<0.50) in all hybrids and all individuals, while SNP2 and SNP3 were moderately polymorphic (0.25≤PIC<0.50) in F2 and SNP7 were moderately polymorphic (0.25≤PIC<0.50) in H1, and were low polymorphic (PIC<0.25) in other populations. The results of linkage imbalance analysis showed that SNP2-SNP3 and SNP5-SNP6 formed two strong linkage, each of which constructed three haplotypes, and formed 4 and 6 genotypes after combination, respectively, among which the dominant genotype was H1H1 and H4H6. 13 genotypes were produced after the haplotypes formed by SNP2-SNP3 and SNP5-SNP6 were recombined, and the dominant genotype was H1H1H4H6. Single SNP correlation analysis revealed that: SNP1 site in F1 population, the primary chest of GG genotype was significantly higher than that of CG genotype (P<0.05); in H2 population, the primary body length of CC genotype was significantly higher than that of GG genotype (P<0.05). SNP2 site in H1 population, the primary body height of CC genotype was significantly lower than that of CG and GG genotypes (P<0.05). SNP3 site in F1 population, the three months body weight of AG genotype was significantly higher than that of GG genotype; in H1 population, the primary body height of AA genotype was significantly lower than AG and GG genotypes (P<0.05). SNP4 site in F1 population, the three months weight of CC genotype was significantly higher than that of CT and TT genotypes (P<0.05); in F2 population, the primary weight and primary chest of CT genotype were significantly higher than that of TT genotype (P<0.05), which of TT genotype was significantly lower than that of CC and CT genotype (P<0.05) in H1 population; in H2 population, the primary body height and primary body length of TT genotype were significantly higher than that of CT genotype (P<0.05). SNP5 site in F2 population, AA genotype had significantly higher primary body height and primary body length than GG genotype (P<0.05). There were significant differences in primary weight, body height, body length, three months body height and chest among different genotypes of SNP6 (P<0.05). SNP7 site in H2 population, AA genotype had significantly higher primary body length than GA genotype (P<0.05). When combined with all groups, it was found that: the primary weight and body length of CG genotype at SNP2 were significantly higher than that of GG genotype (P<0.05); the primary weight of TT genotype at SNP4 was significantly lower than CC and CT genotypes (P<0.05); the primary weight of AA genotype at SNP6 was significantly higher than GG and GA genotype (P<0.05); and the primary chest was significantly higher than GG genotypes (P<0.05). There was no significant difference in growth traits among the other five genotypes (P>0.05). The results of haplotype association analysis showed that the primary weight and primary body length of H2H3 genotype were significantly higher than those of other genotypes (P<0.05), while the primary weight of H5H5 genotype was significantly higher than that of H5H6 and H6H6 (P<0.05), the primary body height was significantly higher than that of H5H6 (P<0.05), and the primary chest was significantly higher than that of H6H6 (P<0.05); the three months weight of H4H4 genotype was significantly higher than that of H5H5 genotype (P<0.05), the body weight and body length at three months were significantly higher than those of H4H6 genotype (P<0.05), and the chest of three months was significantly higher than that of H4H5, H5H6 and H5H5 genotype (P<0.05). After SNP2-SNP3 and SNP5-SNP6 haplotypes were recombined, the primary weight, body height, body length and chest of H2H3H4H4 genotype were the highest, which were different from other genotypes, and the body length at three months of H1H1H4H6 genotype were significantly lower than that of H1H2H4H4 and H2H2H4H4 genotypes (P<0.05).【Conclusion】 The results showed that the different SNPs and combined genotypes in ADIPOQ had effect on sheep different growth traits, the seven SNPs in this study could be used as potential molecular markers for growth traits in Ningxia high quality mutton sheep breeding. Table and Figures | Reference | Related Articles | Metrics

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The Response of Leymus chinensis Cloned Offspring to Mowing GUO FengHui,DING Yong,JI Lei,LI XianSong,LI XiLiang,HOU XiangYang Scientia Agricultura Sinica    2022, 55 (11): 2257-2264.   DOI: 10.3864/j.issn.0578-1752.2022.11.014 Abstract （308）   HTML （28）    PDF （473KB）（62）       Knowledge map    Save 【Background】 Grazing could alter the morphological and photosynthetic physiological characters of the Leymus chinensis cloned offspring, but whether grazing influence the adaptability of that to grazing is unclear. 【Objective】 The aim of this study was to investigate whether grazing history enhanced the adaptability of Leymus chinensis cloned offspring to livestock grazing.【Method】 The Leymus chinensis cloned offspring with different grazing histories (enclosed in 1983 VS long-term free grazing) were used to conduct pot experiment in the greenhouse, and their adaptability to simulated livestock grazing (clipping) was compared in terms of individual traits, ramet number, biomass, and biomass allocation. 【Result】 (1) There was a significant interact effect between maternal grazing history and clipping treatment. The individual height and biomass of grazing (GZ) were more resistant to clipping treatment than that of nograzing (NG), while the response of ramet number to clip was not influenced by maternal grazing history. (2) Grazing history altered the responses of aboveground, root, rhizome and total biomass to the clipping treatment. There was significant interaction between grazing history and mowing treatment in terms of the rhizome biomass, while the interaction on other three indicators were not significant. However, the other three indicators of NG had the larger plasticity index and absolute reduction to clipping treatment. Therefore, the maternal grazing experience enhanced the adaptability of L. chinensis cloned offspring to clipping treatment. (3) The cutting biomass of NG was significantly lower than that of GZ, but the cutting degree of NG was significantly higher than that of GZ.(4) The responses of GZ biomass allocation to clipping treatment were not significant, while rhizome biomass allocation of NG significantly decreased under clipping treatment. 【Conclusion】 The grazing disturbance could enhance the adaption of Leymus chinensis cloned offspring to grazing. The maternal grazing history did not enhance the adaptability of L. chinensis cloned offspring to livestock grazing by altering biomass allocation. The response of leaf photosynthetic physiology and grazing avoidance might be the reasons for the enhancement of grazing fitness. In this study, the environmental disturbance factors, such as soil factors, were excluded through control experiments, and the response of L. chinensis cloned offspring traits to grazing was studied from the perspective of the plant itself. Thus, this study provided a new perspective for fully understanding the process of grazing degradation in grassland ecosystem. Table and Figures | Reference | Related Articles | Metrics

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