Scientia Agricultura Sinica

Major Characteristics, Often-Raised Queries and Potential Usefulness of the Restricted Two-Stage Multi-Locus Genome-Wide Association Analysis

JunYi GAI,JianBo HE

Scientia Agricultura Sinica. 2020, 53(9): 1699-1703. doi:10.3864/j.issn.0578-1752.2020.09.001

Abstract ( 695 )

HTML ( 61 )

PDF (361KB) ( 573 )

Save

References | Related Articles | Metrics

Restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) is a novel GWAS procedure which provides a way to identify the QTL system with various multiple alleles in natural and bi- or multi-parental derived populations. The major purposes and its two major characteristics of the RTM-GWAS procedure were presented, including the establishment of the SNPLDB markers with multiple alleles fitting the property of the natural and bi- or multi-parental derived populations and the establishment of multi-locus model GWAS procedure with the total genetic contribution controlled within heritability value. Generally, the readers and editors do not doubt about the methods and principles, the multiple allele markers and the multi-locus model, but have questions and queries on the large amount of detected QTLs many more than those from single locus MLM-GWAS procedure and on the general significance level without correction used in RTM-GWAS. These doubts were carefully and seriously explained and relieved. Furthermore, the potential usefulness of the RTM-GWAS procedure in genetic and evolutionary studies were summarized, including usefulness in relatively thorough identification of the QTL-allele system in populations and major gene finding and cloning, usefulness in relatively thorough identification of the QTL-allele system in bi-and multi-parental derived populations, usefulness in studies on population genetic differentiation and evolution and usefulness in breeding by genetic design.

Restricted Two-Stage Multi-Locus Genome-Wide Association Analysis and Its Applications to Genetic and Breeding Studies

JianBo HE,FangDong LIU,WuBin WANG,GuangNan XING,RongZhan GUAN,JunYi GAI

Scientia Agricultura Sinica. 2020, 53(9): 1704-1716. doi:10.3864/j.issn.0578-1752.2020.09.002

Abstract ( 449 )

HTML ( 36 )

PDF (1843KB) ( 385 )

Save

Figures and Tables | References | Related Articles | Metrics

Genome-wide association studies (GWAS) take genome-wide high-density molecular markers to identify associations between genotype and phenotype, which have been widely used for genetic dissection of quantitative traits in plants and animals. However, previous GWAS methods focused on finding a handful of major loci and were not able to detect multi-allelic genetic variation in natural populations based on bi-allelic SNP marker, which caused limitations in extending application of GWAS. The restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) firstly groups multiple adjacent and tightly linked SNPs based on linkage disequilibrium to form multi-allelic SNPLDB markers with multiple haplotypes as alleles. Secondly, population structure bias is estimated using the genetic similarity coefficient matrix calculated from SNPLDB marker, and the eigenvectors of the similarity matrix are extracted and incorporated as model covariates to correct for population structure bias and to reduce false positives. Finally, RTM-GWAS utilizes two-stage association analysis to detect genome-wide QTLs and their multiple alleles efficiently based on the SNPLDB marker and multi-locus multi-allele model, and builds the final multi-QTL genetic model with the total QTL genetic contribution restricted to trait heritability. RTM-GWAS can also detect QTL-by-environment interaction effect using plot-based phenotype data, and can detect not only the main effect QTL, but also QTL with only interaction effect with environment. RTM-GWAS solves the issue that multiple alleles are not estimable in previous GWAS, and also improves the detection power and reduces the false positive rate by fitting multiple QTLs simultaneously in a multi-locus model. It provides a potential solution for a relatively thorough detection of genome-wide QTLs and their multiple alleles, and the allele effect and relative frequency can also be estimated. From RTM-GWAS results, a QTL-allele matrix can be constructed as a compact form of the population genetic constitution, and can be further used for gene discovery. QTL-allele matrix also provides a new tool for studies on the dynamic change of QTLs and their multiple alleles (genes and their multiple alleles), such as population genetic differentiation and population-specific and new alleles. According to QTL-allele matrix, the progeny genotype of cross between parental lines can be simulated by using computer simulation, and then the phenotype can be predicted to assist optimal cross design and molecular design breeding. In addition, RTM-GWAS is more efficient in QTL detection for bi-parental recombinant inbred line population and multi-parental nested association mapping population because the population structure bias can be well-controlled. The present paper presents the principles and procedures of the RTM-GWAS method at first, and then provides some potential applications of RTM-GWAS in plant genetic and breeding studies.

Genome-Wide QTL-Allele Dissection of 100-Seed Weight in the Northeast China Soybean Germplasm Population

XiaoShuai HAO,MengMeng FU,ZaiDong LIU,JianBo HE,YanPing WANG,HaiXiang REN,DeLiang WANG,XingYong YANG,YanXi CHENG,WeiGuang DU,JunYi GAI

Scientia Agricultura Sinica. 2020, 53(9): 1717-1729. doi:10.3864/j.issn.0578-1752.2020.09.003

Abstract ( 469 )

HTML ( 23 )

PDF (1974KB) ( 338 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】A genome-wide association study in the Northeast China soybean germplasm population was conducted for a relatively thorough detection of the QTL-allele constitution of 100-seed weight, which may provide a theoretical basis for soybean breeding for seed size improvement. 【Method】In the present study, a total of 290 soybean accessions that were frequently used for soybean breeding and production in the Northeast China were tested in 2013 and 2014 for 100-seed weight at four locations, including Keshan, Mudanjiang, Jiamusi and Changchun, which are all in the second sub-ecoregion of the Northeast China. RAD-seq (restriction site-associated DNA sequencing) was used for SNP genotyping, and 82 966 high-quality SNPs were obtained after filtering and imputation. According to the RTM-GWAS (restricted two-stage multi-locus genome-wide association analysis) method, firstly a total of 15 546 multi-allelic SNPLDBs were constructed, and then a multi-locus model was used for genome-wide association study of 100-seed weight. The genes near (within 50kb) the detected SNPLDBs were analyzed, and candidate genes for 100-seed weight were identified and annotated according to Chi-square test of independence between the SNPs within genes and the detected SNPLDBs. Finally, genetic differentiation among maturity groups were investigated based on the detected QTL-allele system of 100-seed weight. 【Result】The 100-seed weight of the present population ranged from 18.3 to 20.7 g, and the trait heritability was 92.3%. A total of 76 SNPLDBs were detected to be associated with 100-seed weight, among which there were 15 SNPLDBs with non-significant main effect and the 61 SNPLDBs with significant main effect explained 65.40% phenotypic variation. There were 68 SNPLDBs that had significant interaction effect with environment and explained 17.46% phenotypic variation. In addition, 34 out of 76 detected SNPLDBs overlapped 30 previously reported QTLs and 42 SNPLDBs were novel loci. A total of 137 candidate genes for 100-seed weight were annotated in the detected SNPLDB regions, and functional annotation showed that these genes were not only involved in regulation of 100-seed weight, but also involved in primary metabolism, translation, protein modification, material transport, stress response and signal transduction, etc. Although there was no obvious difference in the 100-seed weight among different maturity groups, genetic differentiation analysis showed varying changes of allele emergence and exclusion in QTL-allele structure of 100-seed weight among maturity groups. 【Conclusion】The RTM-GWAS method used in the present study provided a relatively thorough detection of genome-wide QTLs and their multiple alleles for 100-seed weight in the Northeast China soybean germplasm population. The 100-seed weight of the Northeast China soybean germplasm population was controlled by a large number of QTLs with large significant interaction effect with environment, and there was also abundant multiple allelic variation in these QTLs. The QTL-allele matrix established from RTM-GWAS provided an efficient tool for population genetics and evolution study.

Detection Power of RTM-GWAS Applied to 100-Seed Weight QTL Identification in a Recombinant Inbred Lines Population of Soybean

LiYuan PAN,JianBo HE,JinMing ZHAO,WuBin WANG,GuangNan XING,DeYue YU,XiaoYan ZHANG,ChunYan LI,ShouYi CHEN,JunYi GAI

Scientia Agricultura Sinica. 2020, 53(9): 1730-1742. doi:10.3864/j.issn.0578-1752.2020.09.004

Abstract ( 630 )

HTML ( 26 )

PDF (1349KB) ( 359 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】To thoroughly dissect the QTL system conferring 100-seed weight in a recombinant inbred lines population, the restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) method was compared with other mapping methods for method optimization, which will provides basis for further exploration of candidate gene system and molecular marker-assisted design breeding. 【Method】A recombinant inbred line population consisting of 427 lines derived from a cross between Kefeng-1 and NN1138-2 was tested for its 100-seed weight under three environments. A total of 3 683 SNPLDBs (SNP linkage disequilibrium blocks) composed of 39 353 SNPs were applied to QTL mapping using three different mapping procedures, including the composite interval mapping (CIM) method, the mixed linear model (MLM-GWAS) method and the RTM-GWAS method, and the best mapping procedure was selected for the analysis of the 100-seed weight genetic system in NJRIKY population through comparing their detection power, including the detected number of QTLs and total phenotypic variation explained. 【Result】The 100-seed weight of Kefeng-1 and NN1138-2 were 9.0 g and 17.9 g, respectively, showing significant difference. The genotypic coefficient of variation and heritability of the trait were 12.4% and 85.4%, respectively. These results indicated that the population was suitable for genetic analysis of 100-seed weight trait. The RTM-GWAS procedure performed the best with the largest number of QTLs (57) explaining the most phenotypic variation (PVE=70.78%), while a total of 14 and 6 QTLs contributing 56.47% and 18.47% phenotypic variation were detected using CIM and MLM-GWAS, respectively. The 57 QTLs detected from the RTM-GWAS distributed on 19 chromosomes, of which 41 QTLs overlapped with 81 QTLs identified from 30 bi-parental populations in the literature. Furthermore, the PVE of 57 QTLs ranged from 0.03% to 7.57%, of which 16 QTLs were novel ones, including one large contribution major QTL Sw-09-2 (PVE>3%). Furthermore, 20 QTLs had significant interaction effect with environment. A total of 36 candidate genes were annotated from 37 QTLs through χ² test between SNPLDB markers and SNPs harboring in the predicted genes, of which 4 candidate genes were from the large contribution QTLs and other 32 candidate genes were from the small contribution QTLs. These candidate genes were included in different biological processes, of which 13 candidate genes were grouped in seed development directly, and the remaining candidate genes were grouped into different functions, such as transport, transcriptional regulators, etc., indicating that these genes from different biological pathways regulate the expression of 100-seed weight trait in NJRIKY together. 【Conclusion】Among the three different mapping procedures, RTM-GWAS procedure is the most powerful one which can provide a relatively thorough detection of 100-seed weight QTLs in NJRIKY population, therefore, it is more suitable for QTL mapping in bi-parental population such as RIL population. The candidate genes with various functions jointly regulated the complex expression of 100-seed weight trait.

Genetic Dissection of Protein Content in a Nested Association Mapping Population of Soybean

ShuGuang LI,YongCe CAO,JianBo HE,WuBin WANG,GuangNan XING,JiaYin YANG,TuanJie ZHAO,JunYi GAI

Scientia Agricultura Sinica. 2020, 53(9): 1743-1755. doi:10.3864/j.issn.0578-1752.2020.09.005

Abstract ( 389 )

HTML ( 21 )

PDF (1701KB) ( 420 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Soybean is an important cash crop, a major source of plant protein and oil for human diet. As a major objective of soybean breeding, protein content is a complex trait controlled by multiple genes with varying genetic effects interacting with environment. A genome-wide association study (GWAS) was conducted to dissect the genetic architecture of protein content in a soybean nested association mapping (NAM) population, and the detected genetic constitution can be further used for molecular design in soybean breeding for high protein content. 【Method】Four soybean recombinant inbred line (RIL) populations (Linhe×M8206, Zhengyang×M8206, M8206×Tongshan and M8206×WSB) with a common parent (M8206) as a NAM population were constructed, genotyped with RAD-seq (restriction-site-associated DNA sequencing) and tested under multiple locations in 2012 - 2014. Protein content was measured at full maturity (R8) stage. The restricted two-stage multi-locus GWAS (RTM-GWAS) procedure was used to dissect the genetic architecture of seed protein content of the population. 【Result】The protein content varied widely in the population with trait heritability estimated as high as 85.00%. The analysis of variance for protein content showed significant differences across genotypes, environments and genotype-by-environment interactions. A total of 90 QTLs were detected to be associated with protein content, with 20 loci being novel ones. The phenotypic variation explained by each QTL ranged from 0.06% to 3.99%, with a sum of 45.60%. The number of alleles at each locus ranged from 2 to 5, and the allele effects ranged from -2.434% to 2.845%, while most of them were between -1.000% and 1.000%. From the detected QTLs, 73 candidate genes were annotated. Among these candidate genes, Glyma18g03540 involved in cysteine biosynthetic process, and Glyma20g24830 involved in glycine and aromatic amino acid family metabolic process. The two genes may be selected for further functional study. Based on the QTL-allele matrix of protein content, the predicted transgressive potential of cross progeny was as high as 56.5%. 【Conclusion】A total of 90 QTLs for protein content were detected with 20 loci being novel, from which 73 candidate genes were annotated, indicating that protein content is a complex trait conferred by multiple genes or a gene network.

A Comparative Study on Linkage and Association QTL Mapping for Seed Isoflavone Contents in a Recombinant Inbred Line Population of Soybean

ZaiDong LIU,Shan MENG,JianBo HE,GuangNan XING,WuBin WANG,TuanJie ZHAO,JunYi GAI

Scientia Agricultura Sinica. 2020, 53(9): 1756-1772. doi:10.3864/j.issn.0578-1752.2020.09.006

Abstract ( 417 )

HTML ( 20 )

PDF (2930KB) ( 380 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Isoflavones are a group of phenolic secondary metabolites which are relatively abundant in soybean and some other legumes, and are important for food and healthcare industry. A total of 12 kinds of components are isolated from soybean seed, and can be grouped into three categories: daidzin group, genistin group and glycitin group. To understand the complex genetic constitutions of isoflavone content in soybean, the additive and epistatic quantitative trait loci (QTLs) conferring the total isoflavone content and its component contents were detected in the present study. 【Method】The NJRSXG recombinant inbred line (RIL) population derived from Xianjin 2 and Gantai-2-2 were used in this study. Four isoflavone content traits, i.e. the total seed isoflavone content (SIFC), the total daidzin group content (TDC), the total genistin group content (TGC) and the total glycitin group content (TGLC) were tested in five environments. The mixed model composite interval mapping (MCIM) and restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) were used for QTL detection. 【Result】There was a large difference in isoflavone content between the two parental lines of NJRSXG population, and transgressive segregations were observed in NJRSXG population while the transgressive trend in low isoflavone content direction were stronger than that in high isoflavone content direction. A total of 19 additive QTLs and 16 pairs of epistatic QTLs for the four isoflavone traits on 15 chromosomes were detected by MCIM. Three novel additive QTLs, i.e. qSifc-14-1, qTdc-14-2 and qTgc-14-1, were detected in the same important marker interval GNE186b-Sat020 on chromosome 14, and explained the highest phenotypic variation. A total of 51, 66, 42 and 36 significantly associated markers were detected by RTM-GWAS for SIFC, TDC, TGC and TGLC, respectively. The phenotypic variation explained by these markers was ranged from 39.7% to 52.5%, covering 11 additive QTLs and 11 epistatic QTLs detected by MCIM. Furthermore, a total of 93 and 100 candidate genes were annotated in the additive and epistatic QTL regions, respectively. Gene enrichment analysis indicated that three genes located in the important marker interval GNE186b-Satt020 on chromosome 14, i.e. Glyma14g33227, Glyma14g33244 and Glyma14g33715, were related to isoflavone metabolism. 【Conclusion】A relatively thorough detection of isoflavone content QTLs was achieved by using linkage and association mapping. Compared with the linkage mapping method MCIM, the association mapping method RTM-GWAS can detect more QTLs with larger total contribution to phenotypic variation, but cannot detect epistatic QTLs as in MCIM. The QTLs detected from the two methods can be used for complementary verification from each other. A large number of QTLs/genes are involved in the seed isoflavone contents of soybean.

Spatio-Temporal Variations of Winter Wheat Planting Frequency and Their Analysis of Influencing Factors in Henan Province

FangJie LI,JianQiang REN,ShangRong WU,ZhongXin CHEN,NingDan ZHANG

Scientia Agricultura Sinica. 2020, 53(9): 1773-1794. doi:10.3864/j.issn.0578-1752.2020.09.007

Abstract ( 483 )

HTML ( 85 )

PDF (12543KB) ( 480 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Quantitative analysis was made on spatio-temporal variations of winter wheat planting frequency (WWPF) and its main influencing factors in different stages (2001-2005, 2006-2010 and 2011-2015) in Henan province, in order to further clarify the characteristics of crop planting frequency change and the sequence of main influencing factors. 【Method】In this study, winter wheat in Henan province was taken as the research crop. The spatial information of winter wheat planting frequency in different periods in the study area were obtained by extracting the spatial crop distribution information for 15 consecutive years (2001-2015) using mid-low resolution MODIS EVI time series remote sensing data and CART decision tree algorithm. On this basis, the spatio-temporal variations of winter wheat planting frequency in different stages were analyzed, and the influencing factors of the winter wheat planting frequency variations in different stages were investigated by correlation analysis, principal component analysis and linear regression analysis. Finally, the sequence of the main influence factors was determined. 【Result】It was verified that the average overall precision of winter wheat extraction was 90.39%, and the Kappa coefficient was between 0.82 and 0.92, which could meet the requirements of spatial distribution accuracy of crops required for regional winter wheat planting frequency change research. By analyzing the spatial and temporal variation information of the winter wheat planting frequency in different periods in Henan province, most of the main winter wheat producing regions in Henan province had higher winter wheat planting frequency (WWPF>80%), while the planting frequency of winter wheat was generally lower in southwestern Henan and in mountainous areas of southern Henan due to complex topography and poor natural conditions (WWPF≤40%). In addition, during the three periods, the area of winter wheat planted at high frequency increased gradually in the main winter wheat producing areas of Henan province, the area proportion of WWPF>80% was 42.68%, 59.94% and 63.07%, respectively. And the area planted at low frequency showed a decreasing trend, the area proportion of WWPF≤40% was 28.53%, 17.99% and 16.63%, respectively, which were of great significance to stabilize the planted area of grain in the main winter wheat producing areas of China. According to the analysis results of influencing factors of winter wheat planting frequency, there was a significant correlation between the WWPF in Henan province and the ratio of effective irrigation area, soil quality comprehensive index, climate suitability at sowing stage, slope and elevation, etc. Besides the negative correlation between slope/elevation and WWPF, other factors were all positively correlated with WWPF. The ranking results of the above indicators affecting on the WWPF were as follows: soil comprehensive quality index>climatic suitability at sowing stage>effective irrigated area ratio>slope (elevation), that was to say, the soil quality>sowing date climatic condition>irrigation condition>topographic condition. 【Conclusion】Based on quantitative analysis of spatio-temporal variations of winter wheat planting frequency and its influencing factors in Henan province, the spatio-temporal distribution characteristics and variation law of winter wheat planting frequency in Henan province were clarified, and the influencing factors and importance ranking of winter wheat planting frequency in Henan province were gotten, which could provide some technical methods and ideas for the analysis of crop planting area change, and provide some basic theoretical support for the construction of regional agricultural land use decision-making model.

LAI Estimation Based on Multi-Spectral Remote Sensing of UAV and Its Application in Saline Soil Improvement

FengZhi SHI,RuiYan WANG,YuHuan LI,Hong YAN,XiaoXin ZHANG

Scientia Agricultura Sinica. 2020, 53(9): 1795-1805. doi:10.3864/j.issn.0578-1752.2020.09.008

Abstract ( 388 )

HTML ( 15 )

PDF (2247KB) ( 302 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Vegetation yield can comprehensively and intuitively reflect the improvement effect of saline soil. Leaf area index (LAI) of winter wheat during its vigorous growth period is a commonly used measure of vegetation yield. In this study, unmanned aerial vehicle (UAV) multispectral remote sensing was used to obtain the LAI distribution data of winter wheat during its vigorous growth period to objectively and accurately evaluate the improvement effect of saline soil, so as to provide scientific guidance for people to screen saline soil improvement technology and products. 【Method】Taking the experimental area of coastal saline soil improvement in Bohai granary in Wudi county as the research area, based on UAV multispectral remote sensing data, linear regression analysis, partial least squares, random forest, and support vector machine were used to construct LAI inversion model of winter wheat. The factor analysis method was used to evaluate the improvement effect of the sampled land in saline soil area, and the LAI evaluation model of saline soil improvement effect was established to evaluate the improvement effect of saline soil in the entire experimental area. 【Result】The results showed that, for the winter wheat LAI remote sensing estimation, it was not that the higher the resolution, the better, but the smoothed 5×5 mean spectral data corresponded best to the LAI of a ridge of wheat. Among the LAI remote sensing estimation models, the models were built by using SVM with the highest accuracy. The prediction result of the improvement effect LAI evaluation model showed that the prediction accuracy of the improvement effect of saline soil by LAI was higher, and the best improvement land numbers were 26, 27, 28, 29, 30, and 31, and the optimal improvement method was cited. The best improvement method was comprehensive improvement measures of diversion irrigation and adding organic fertilizer. 【Conclusion】UAV remote sensing could accurately invert the LAI of winter wheat at the jointing stage in saline soil area. The evaluation of the improvement effect of saline soil based on the results of LAI inversion could locate the optimal improvement effect from many experimental plots. Compared with the traditional method, this method had the advantages of low cost and high accuracy. The research results had a broad prospect and could provide important technical support for the improvement of saline soil.

The Resistance Prediction of Wheat Hybrids Based on the Sensibility of Their Parents to Stripe Rust

TianYu ZHOU,JiangLing LI,Lan YANG,RenWu RUAN,YuHeng YANG,ZhongAn LI

Scientia Agricultura Sinica. 2020, 53(9): 1806-1819. doi:10.3864/j.issn.0578-1752.2020.09.009

Abstract ( 595 )

HTML ( 18 )

PDF (2671KB) ( 353 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】The objective of this study is to predict the resistance of wheat hybrids based on the resistance evaluation of their parental lines to stripe rust, and to enhance the predictability of disease resistance breeding in hybrid wheat. 【Method】A sensitive wheat variety, Mingxian 169, was used as negative control. A total of 13 restorer lines (male parents), 21 sterile lines (female parents), and their F₁ hybrids were inoculated with a suspension of a mixture of fresh urediniospores (equal amounts of stripe rust pathogen physiological races CYR23, CYR31, CYR33, and CYR34) in the field. Stripe rust resistance genes Yr5, Yr9, Yr10, Yr15, Yr17, Yr18 and Yr26 were identified by PCR using their gene or linked molecular markers. In addition, semi-quantitative PCR was used to quantify the different stripe rust races in the adult plants of parent lines and a part of F₁ hybrids. 【Result】Yr5, Yr10 and Yr15 were not identified in all materials, Yr26 was mostly existed in Sichuan wheat lines, Yr9 and Yr17 were mostly existed in the northern lines, Yr18 was not identified in restorer lines. The parental resistance genes were polymerized in F₁ hybrids, which is consistent with the genetic law, indicating that the molecular marker can be used in wheat assistant breeding. The restorer lines from Sichuan and their F₁ hybrids showed high resistance against stripe rust, speculating that there are homozygous dominant resistance genes against stripe rust. Meanwhile, these wheat lines can be used for stripe rust resistance breeding in China. The infection types (ITs) of F₁ hybrids tended to the average of parental infection types. The results of binary regression analysis showed a significant correlation between parent lines and F₁ hybrids in ITs (R²=0.812). Although Yr5 and Yr15 resistance genes were absent in all tested wheat lines, the restorers from Sichuan Province and their F₁ hybrids showed higher resistance, speculating that those lines might carry unknown resistance genes against CYR34. The semi-quantitative PCR results indicated that CYR23 was not detected in all wheat lines. Only the restorer line 15CA50, sterile lines 17L6078 and 15L7128 were infected with a small amount of CYR31. The tested restorer lines Chuan 13 pin 6, MR1101, Chuanmai 98 and their F₁ hybrids were not infected by CYR33 or CYR34. Meanwhile, the parents with complementary resistance to different races were found to increase the resistance level of F₁ hybrids effectively. 【Conclusion】The F₁ hybrids resistance to strip rust can be predicted according to the average of the ITs of parental lines, the higher levels of disease resistance of parents, the better resistance of F₁ hybrids. Wheat varieties or lines with complementary resistance to different races of stripe rust should be screened as parents to improve the resistance level of F₁ hybrids. The results revealed the rule of disease resistance between parents and their F₁ hybrids, as well as providing a practical strategy for hybrid wheat disease resistance breeding.

Verification of SNPs Associated with Citrus Bacterial Canker Resistance and Induced Expression of SNP-Related Calcium-Dependent Protein Kinase Gene

Yun PENG,TianGang LEI,XiuPing ZOU,JingYun ZHANG,QingWen ZHANG,JiaHuan YAO,YongRui HE,Qiang LI,ShanChun CHEN

Scientia Agricultura Sinica. 2020, 53(9): 1820-1829. doi:10.3864/j.issn.0578-1752.2020.09.010

Abstract ( 332 )

HTML ( 19 )

PDF (1163KB) ( 251 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】In the previous study, single nucleotide polymorphisms (SNPs) of citrus varieties were screened based on transcriptome, and 14 SNPs were defined to be associated with citrus bacterial canker (CBC) resistance via association analysis. In this study, it is aimed to verify the correlation between these SNP loci and CBC resistance in order to obtain significantly related SNPs and to find the inducible expression profiles of corresponding genes by plant hormones and Xanthomonas citri subsp. citri (Xcc) infection. 【Method】The sensitive and resistant varieties and their F₁ populations were used for CBC resistance identification via in vitro acupuncture inoculation and SNP-based genotyping was conducted via high resolution melting (HRM) technology. The phenotypes and genotypes were then associated by software DPS, and inducible expression profiles of SNP (HP031) related calcium-dependent protein kinase gene (CDPK) were analyzed by quantitative real-time PCR (qRT-PCR). 【Result】The lesion areas of F₁ populations ranged from 0.75 to 3.29 mm², and the disease index ranged from 30.2 to 100, while the resistant variety Jindan had a disease index of 11.1, and disease index of the susceptible variety Bingtangcheng was 100. For the offspring of hybrids with a low disease index, at least one of their parents is a disease-tolerant variety, and most of the female parents have strong disease-tolerance. According to the disease index, the populations could be grouped into immune (1 variety), highly resistant (17), moderately resistant (31), moderately sensitive (38), and highly sensitive (56). The SNP typing results showed that all the 14 SNP loci were polymorphic among 143 test materials, which could be divided into 3 different genotypes, 2 homozygous types and 1 heterozygous type. The results of simple correlation analysis showed that the genotypes of multiple SNP loci were significantly correlated with the lesion area and disease index. The results of canonical correlation analysis showed that the correlation between the 5 SNP loci and the lesion area was high, the absolute values of the correlation coefficients were all >0.2, and their numbers were HP31, HP42, HP85, HP87, and HP170. The genotypes of these 5 SNP loci could be used to predict the tolerance to CBC. SNP HP31 located in the coding region of CsCDPK (CAP ID: Cs4g10370). The expression of CsCDPK was analyzed at 6, 12, 24, 48 and 72 hpi (hours post inoculation), it was found that the expression of CsCDPK all increased first and then decreased, and reached the highest relative expression at 48 hpi in Jindan (highly resistant), Xinshengxi No. 3 (moderately resistant), and Bingtangcheng (highly sensitive) varieties. At 12 hpi, the relative expression level of CsCDPK in Jindan was 3 times of that in the control, but there was no significant difference in Xinshengxi No. 3 and Bingtangcheng. Besides, CsCDPK was also differently induced by salicylic acid (SA), methyl jasmonate (MeJA), and abscisic acid (ABA) in CBC resistant and sensitive varieties. 【Conclusion】Five SNPs associated with CBC resistance were verified, which can be used for marker-assistant selection. SNP HP31 related gene CsCDPK can be induced by Xcc and phytohormones, which may play an important role in the signal transduction process of citrus response to Xcc.

Mapping of Soil Organic Matter and Its Driving Factors Study Based on MGWRK

Lei QIAO,WuPing ZHANG,MingJing HUANG,GuoFang WANG,Jian REN

Scientia Agricultura Sinica. 2020, 53(9): 1830-1844. doi:10.3864/j.issn.0578-1752.2020.09.011

Abstract ( 421 )

HTML ( 22 )

PDF (3337KB) ( 298 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Spatial prediction is an important approach to obtain location-specific values of soil organic matter (SOM), which is an important figure of soil fertility and farmland management properly. This study was performed to compare different digital soil spatial mapping methods of SOM to get better prediction accuracy and to reveal the spatial non-stationarity characteristics of environmental covariates and the spatial scale of different environmental covariates at the same time. 【Method】In this study, the digital soil spatial mapping method was used, which was a combination of Multiscale Geographically Weighted Regression Model with simple Kriging of the residuals (MGWGK) for mapping SOM in seven towns from Jinzhong Basin. The performance of MGWRK with those of Ordinary Kriging (OK), Regression Kriging (RK), and Geographically Weighted Regression Kriging (GWRK) were compared to explore the relationship between influence factors and SOM on the influence degree and space scale. 【Result】Based on the stepwise regression method, 13 indexes were selected as environmental covariables in the modeling, including aspect, slope, height, annual average precipitation, annual average temperature, gross primary productivity (GPP), evapotranspiration (ET), topographic wetness index (TWI), plan curvature, stream power index (SPI), terrain position index (TPI), terrain ruggedness index (TRI), and the annual NDVI. In the multiple linear regression (MLR), the formula had statistical significance. In the Radius index, the performance of each model was in order from good to bad: RK, OK, GWRK, MGWRK. In the mapping performance, MGWRK was close to GWRK, and both of which were better than OK method and RK method. The SOM in the study area, showed a spatial pattern of higher in middle than east and west side, among which the SOM was high in the east of the Fenhe river and the Changyuan river. The influence of aspect, annual average precipitation, annual average temperature, height, TPI and the annual NDVI on SOM in the eastern of the study area was stronger than that in the western. Whiles slope, GPP, ET, plan curvature, SPI and TRI showed opposite influence in spatial. The influence of TWI on SOM was stronger in the northern than the southern. 【Conclusion】The spatial prediction accuracy of MGWRK was 69% of RK, 71.74% of OK, and 71.17% of GWRK. MGWRK performed well in the spatial non-stationary features and the spatial visualization, which provided a reference for prediction of SOM and description spatial non-stationarity characteristics.

Effects of Soil Erosion on Physical and Mechanical Properties of Cultivated Layer of Purple Soil Slope Farmland

Na JIANG,DongMei SHI,GuangYi JIANG,Ge SONG,ChengJing SI,Qing YE

Scientia Agricultura Sinica. 2020, 53(9): 1845-1859. doi:10.3864/j.issn.0578-1752.2020.09.012

Abstract ( 320 )

HTML ( 15 )

PDF (868KB) ( 318 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Purple soil slope farmland is an important cultivated land resource for agricultural production in southern hilly area. In order to study the effects of soil erosion on the physical properties and mechanical properties degradation of purple soil slope arable land, based on the classification of soil degradation, the change characteristics of physical and mechanical properties and soil degradation index under different erosion degrees were quantitatively analyzed. 【Method】With non-eroded plots as control group, the soil permeability, soil mechanical properties and soil degradation index of cultivated-layer were compared and analyzed under 5 cm (S_-5), 10 cm (S_-10), 15 cm (S_-15) and 20 cm (S_-20) erosion conditions by shovel soil erosion simulation test method, and the degradation degree of physical and mechanical properties of sloping farmland was quantitatively analyzed. 【Result】Soil permeability of cultivated-layer under different erosion degrees was CK>S_-5>S_-10>S_-15>S_-20. The initial infiltration rate, stable infiltration rate, average infiltration rate and saturated water conductivity of soil decreased with the increase of erosion degree. Soil permeability index of each layer under different erosion degrees was 0-20 cm soil layer>20-40 cm soil layer. Soil mechanical properties of different erosion degrees were CK-5-10-15-20. Soil shear strength and soil compacted degree increased with erosion degree. Soil mechanical indexes of all layers under different erosion degrees were 0-20 cm soil layer<20-40 cm soil layer. The contribution rate of soil shear strength to the first axis was the largest, and soil shear strength was the main factor affecting the change of soil physical properties and mechanical properties under different erosion degrees. Soil physical properties and mechanical properties were ranked as stable infiltration rate>soil compaction>saturated water conductivity>average infiltration rate>initial infiltration rate>shear strength. Soil degradation index of under different erosion degrees was S_-5 (-8.71%)>S_-10 (-10.95%)>S_-20 (-12.17%)>S_-15 (-15.37%). S_-15 had the greatest influence on the topsoil physical properties, and the S_-15 soil degradation index was the smallest, with the degree of soil degradation being severe. 【Conclusion】Soil compaction was serious in slope farmland of purple soil. According to soil infiltration and mechanical properties, the soil degradation grade with different erosion degree could be classified into four grades: undegraded, mild degradation, moderate degradation and severe degradation. The results could provide the technical parameters for the identification and restoration control of the quality degradation process of sloping farmland.

Effects of Different Root Exudates on Soil N₂O Emissions and Isotopic Signature

Shan ZHUANG,Wei LIN,JunJun DING,Qian ZHENG,XinYue KOU,QiaoZhen LI,YuZhong LI

Scientia Agricultura Sinica. 2020, 53(9): 1860-1873. doi:10.3864/j.issn.0578-1752.2020.09.013

Abstract ( 358 )

HTML ( 24 )

PDF (594KB) ( 312 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】The aim of this study was to investigate the effects of the main plant root exudates (organic acids, amino acids, sugars) on N₂O emission and its microbial processes, so as to provide a support for selecting suitable plants to control soil N₂O emissions. 【Method】Three main components of root exudates, including oxalic acid, serine and glucose, were added in the soil, and two levels of concentration were set for each component: low concentration (150 μg C·d ^-1) and high concentration (300 μg C·d ^-1). There were totally 7 treatments with a control treatment treated with distilled water, and all treatments were incubated in 120 mL glass bottles in incubator. The gas samples were sampled 7 times within 24 hours after 2-hour incubation each time. After sampling, the N₂O emission rate, daily cumulative emission and isotope signatures (δ ¹⁵N ^bulk, δ ¹⁸O and SP (intramolecular ¹⁵N site preference, δ ¹⁵N ^α-δ ¹⁵N ^β)) were measured, then the optimal sampling time was determined according to their daily variation rules. 【Result】The N₂O emission rate of soils increased gradually after adding three components of root exudates, which were higher than the control treatment. The cumulative emission of N₂O in the high concentration treatment was: Glucose treatments ((3.2±1.3) mg·kg ^-1·d ^-1)>Serine treatments ((2.6±0.5) mg·kg ^-1·d ^-1)>Oxalic acid treatments((1.4±0.2) mg·kg ^-1·d ^-1), low concentration treatment: Oxalic acid ((2.7±1.3) mg·kg ^-1·d ^-1)>Serine ((1.8±0.4) mg·kg ^-1·d ^-1)>Glucose ((1.6±0.8) mg·kg ^-1·d ^-1); the values of δ ¹⁸O of N₂O were not different among different root exudate treatments and were stable at 24.1‰-25.6‰, but significantly higher than the control treatment ((20.1±1.5) ‰); the δ ¹⁵N ^bulk value of N₂O was related to the component of root exudates added, which was (-20.06±2.22) ‰ of oxalic acid treatment, (-22.33±1.10) ‰ of serine treatment, (-13.86±1.11) ‰ treated of glucose treatment, and (-23.14±3.72) ‰ of the control treatment. The SP value of N₂O of each treatment ranged from 13.13‰ to 15.03‰, and the higher the root exudate concentration, the lower the SP value; after a comprehensive analysis of the correction coefficients of four indexes (N₂O emission rate, the value of δ ¹⁵N ^bulk, δ ¹⁸O and SP) at each sampling time and their daily mean values of 7 treatments, the correction coefficients of all treatments were closest to 1 at the 16th hour after the addition of root exudates. 【Conclusion】In the soil environment with NH+ 4- 300 mg N·kg ^-1, the root exudates promoted N₂O emission and the N₂O emission rate increased gradually during the culture time (24 hours). The promotion effect of glucose in high concentration group was the strongest, while that of oxalic acid in low concentration group was the strongest. Compared with the control treatment, the addition of root exudates significantly increased the δ ¹⁸O value of N₂O; the addition of glucose significantly increased the δ ¹⁵N ^bulk value of N₂O. The higher the concentration of root exudates, the stronger the contribution of denitrification to N₂O was detected.

Genome Wide Identification of CCD Gene Family in Citrus and Effect of CcCCD4a on the Color of Citrus Flesh

YaFei ZHANG,Jie PENG,YanSong ZHU,ShengNan YANG,Xu WANG,WanTong ZHAO,Dong JIANG

Scientia Agricultura Sinica. 2020, 53(9): 1874-1889. doi:10.3864/j.issn.0578-1752.2020.09.014

Abstract ( 640 )

HTML ( 70 )

PDF (3881KB) ( 516 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】To reveal the distribution, structure and evolution of carotenoid cleavage dioxygenas gene family in the citrus genome (CcCCD), this study were performed to develope marker-assisted selection of flesh color in citrus breeding program, bioinformatics predication, expression analysis and genotype of CcCCD4a in flesh color development and different germplasm accessions. 【Method】The CCD gene family of Citrus clementina genome were identified by homologous search according to previously reported CCD in other plant species. Phylogenetic analysis, subcellular localization prediction, relative molecular weight, theoretical isoelectric point (PI), conserved motif prediction, and scaffold location were studied by bioinformatics methods. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study the expression of CcCCD4a in 10 citrus accessions during the flesh color development period. Haplotype analysis was performed by Tassel software after sequencing of CcCCD4a in 30 citrus varieties. 【Result】Fourteen CCD family genes were found in the Citrus clementina genome, and these genes could be divided into five subfamily, namely, CcCCD1, CcCCD4, CcCCD7, CcCCD8 and CcNCED. Its theoretical isoelectric point were 6.05 to 8.53 and these CCD family genes encoded 412-611 amino acids. The subcellular localization prediction indicated that CcCCD genes mainly were located in chloroplast and cytoplasm. Phylogenetic analysis showed that CCD genes in citrus were also found in other plant species. Obviously, CCD8 subfamily had farther genetic distance with other CCD. Scaffold localization analysis showed that 14 CcCCD members were unevenly distributed in all scaffolds except scaffold 5. The phenotyping of flesh color in 10 citrus varieties demonstrated that the hue angle of flesh color was decreased along with fruit maturing. The relative expression of CcCCD4a in different citrus varieties was significantly different. The expression of CcCCD4a in the flesh color of orange red was significantly lower than that in the flesh color of orange or light orange yellow (P<0.05). There was a significant positive correlation between the relative expression of CcCCD4a and the hue angle during fruit ripening. Genotyping of CcCCD4a in 30 citrus varieties revealed that hap-1, hap-4 and hap-5 were dominant haplotype at orange red flesh varieties. 【Conclusion】The whole genome of Citrus clementina contained 14 members of CCD gene family. All these CCD gene family members contained RPE65 conserved domain, but they were located in different cell components and unevenly distributed at different scaffolds. CcCCD4a was involved in the development of citrus flesh color, and there was a significant positive correlation between its relative expression and the hue angle. Therefore, CcCCD4a could be used as potential marker for citrus fruit color breeding. Especially, hap-1, hap-4 and hap-5 had a high correlation with the phenotypes of orange red flesh, which might be helpful for selecting candidate hybrids in early stage of citrus breeding program.

vvi-miR160s in Mediating VvARF18 Response to Gibberellin Regulation of Grape Seed Development

YunHe BAI,WenRan WANG,TianYu DONG,Le GUAN,ZiWen SU,HaiFeng JIA,JingGui FANG,Chen WANG

Scientia Agricultura Sinica. 2020, 53(9): 1890-1903. doi:10.3864/j.issn.0578-1752.2020.09.015

Abstract ( 387 )

HTML ( 20 )

PDF (6560KB) ( 283 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】This study was performed to investigate the roles and the modes responsive to gibberellin (GA) of the vvi-miR160 family and its target genes in the development of Wink grape seed. 【Method】miR-RACE, RT-qPCR, bioinformatics and RLM-RACE were employed to identify vvi-miR160s and its target gene, and to analyze their modes responsive to GAof spatio-temporal expression and potential functions. 【Result】GA treatment before flowering strongly inhibited the ovule and seed development of Wink grape and induced grape seedless berries with high efficiency, and the seedless rate of berries reached 99.8%. The precursor gene sequence (501 bp) and mature sequence of vvi-miR160s were cloned and identified, which were highly conserved across different plant species. The mature sequences of vvi-miR160s were used as queries to predict the target gene VvARF18. The cleavage sites with 9/17 being their cleavage frequency of vvi-miR160s on VvARF18 were detected between the 10th and 11th sites by RLM-RACE and PPM-RACE, which proved that VvARF18 was the true target gene of vvi-miR160s. VvARF18 encoded 683 amino acids, and a nuclear localization signal existed at positions 398-411, while the protein sub-cellular was localized on the nucleus. The homology of VvARF18 with other in other species was highly conserved. The VvARF18 protein was closely related to tea, tobacco, plum and other species. The number of elements and their order were the same across different species, and the genes structures were similar. The VvARF18 promoter contained four types of cis-elements, which possessed more hormone-related cis-elements. RT-qPCR analysis showed that vvi-miR160c/d/e showed a ‘V’-shaped expression trend with the development of grape berries, and the lowest expression levels were found during the stone-hardening stage. VvARF18 exhibited an opposite expression trend to the former, with the highest expression during stone-hardening stage, indicating that vvi-miR160c/d/e negatively regulates VvARF18, but there was no significant negative correlation between vvi-miR160a/b and VvARF18 expression levels. GA treatment significantly up-regulated the expression of vvi-miR160a/b in the development of grape hardcore seeds, and also conspicuously inhibited the expression of VvARF18 in the corresponding period. The expression levels between vvi-miR160a/b and VvARF18 under GA treatments showed the typical negative correlation, indicating that GA treatment promoted the negative regulation of vvi-miR160a/b on VvARF18; reversely, GA weakened the negative regulation of vvi-miR160c/d/e on VvARF18. 【Conclusion】Among the vvi-miR160 family, vvi-miR160c/d/e may mediated VvARF18 regulation of seed development during specific stages of grape seed development, whereas vvi-miR160a/b may mediated VvARF18, which might be mainly involved in the regulation of GA-induced grape seedless berry development.

Effects of GnIH on Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells via p38MAPK Signaling Pathway

Xin ZHANG,KongLin HUO,XingXing SONG,DuoNi ZHANG,Wen HU,ChuanHuo HU,Xun LI

Scientia Agricultura Sinica. 2020, 53(9): 1904-1912. doi:10.3864/j.issn.0578-1752.2020.09.016

Abstract ( 444 )

HTML ( 34 )

PDF (2774KB) ( 347 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Studies have shown that autophagy and apoptosis restrict each other. As one of the main regulatory pathways of apoptosis, p38MAPK signaling pathway also has the dual effects of promoting and inhibiting autophagy. It has been proved that gonadotropin inhibitory hormone (gonadotropin-inhibitory hormone, GnIH) has effects on autophagy and apoptosis, but the mechanism of action is not clear. This experiment was conducted to study the effects of GnIH on autophagy and apoptosis of porcine ovarian granulosa cells via p38MAPK signaling pathway and its mechanism. 【Method】Oval granulosa cells were extracted from pig ovaries and cultured in vitro. To explore the best time of GnIH on p38MAPK signaling pathway, according to the time gradient of incubation GnIH (0, 10, 30, 60, and 90 min), Western blot was used to detect the protein expression of p38 and p-p38 in pGCs. To verify the effect of GnIH on p38MAPK signaling pathway, the cells were divided into 4 groups (control, GnIH, p38 activating agent (U-46619), and U-46619 +GnIH), Western blot was used to detect the protein expression of p38 and p-p38. To investigate the effects of different concentrations of GnIH on autophagy and apoptosis: the cells were divided into 5 groups ( control, 10 ^-6mol·L ^-1 GnIH, 10 ^-8mol·L ^-1 GnIH, 10 ^-10mol·L ^-1 GnIH, and 10 ^-12mol·L ^-1 GnIH), Western blot was used to detect the protein expression of autophagy and apoptosis. To verify the effects of different concentrations of GnIH on autophagy and apoptosis through p38 signaling pathway: the cells were divided into 6 groups (control, U-46619, U-46619+10 ^-6 mol·L ^-1 GnIH, U-46619+10 ^-8mol·L ^-1 GnIH, U-46619+10 ^-10mol·L ^-1 GnIH, and U-46619+10 ^-12mol·L ^-1 GnIH), Western blot was used to detect the protein expression of autophagy and apoptosis. 【Result】After incubation with GnIH for 10 min, the protein expression of p38 and p-p38 was significantly decreased (P<0.05). The results suggested that the optimum action time of GnIH on p38MAPK signaling pathway was 10 min; U-46619 significantly promoted the phosphorylation of p38 in pGCs (P<0.05), while GnIH significantly inhibited p38 phosphorylation of pGCs (P<0.05). The results suggested that U-46619 activated the p38MAPK signaling pathway, and GnIH inhibited the activation of p38MAPK signaling pathway; When the concentration of GnIH was 10 ^-6 mol·L ^-1, the autophagy and apoptosis of pGCs increased significantly (P<0.05). With the decrease of GnIH concentration, the autophagy level of pGCs increased gradually (P<0.05), while the apoptosis level of pGCs decreased gradually (P<0.05). The results suggest that high concentration of GnIH promote autophagy and apoptosis. With the decrease of GnIH concentration, the autophagy level increased gradually, while the apoptosis level decreased gradually. After adding U-46619, GnIH significantly upregulated the autophagy of pGCs and down-regulated the apoptosis of pGCs (P<0.05), which suggested that different concentrations of GnIH affected the autophagy and apoptosis of pGCs through p38MAPK signaling pathway. 【Conclusion】GnIH might up-regulate the autophagy of pGCs and reduce the apoptosis of pGCs by inhibiting the activation of p38MAPK signaling pathway.

Study of Genotyping and Performance in Late-Feathering Qingyuan Partridge Cocks

Hua LI,GuiJun FANG,GuoHong HUA,ShuWen TAN,ZhengFen ZHANG,YuYu HONG,Hui YU

Scientia Agricultura Sinica. 2020, 53(9): 1913-1920. doi:10.3864/j.issn.0578-1752.2020.09.017

Abstract ( 427 )

HTML ( 24 )

PDF (524KB) ( 255 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】In order to verify the genotyping accuracy of heterozygotic cocks, and to construct the resistant strain without ev21 gene, the relationship among genotypes, four late-feathering sub-phenotypes (named micro-type as L1, inverted type as L2, isometric type as L3, and ungrown type as L4) and the production performance were investigated in the late-feathering Qingyuan partridge cocks. The feasibility was demonstrated by the expression profile difference of two candidate genes, i.e., Prolactin Receptor (PRLR) and Sperm Flagellar Protein 2 (SPEF2), between the early- and late-feathering Qingyuan partridge cocks. 【Method】Genotypes were detected by PCR-RFLP in the late-feathering Qingyuan partridge cocks, and its accuracy was verified by progeny testing using a test cross. The production differences among genotyping groups were compared by variance analysis of percentage data and t-test. The expressions of two candidate genes were quantified by real-time quantitative PCR (RT-PCR) in both the early (R2) feathering and late (L2 and L4) feathering cocks. 【Result】Among the 568 late-feathering cocks, the proportion of the deletant group (ev21 ^- group) was 41.73%, and that of the ev21 ⁺ group was 58.27%, of which the homozygotes and the heterozygote accounted for 8.80% and 49.47%, respectively. By the test cross, the genotyping accuracy was 46.83% both in the homozygote group and the deletant group, indicating that the heterozygote was in fact homozygote. The descendant percentage of the isometric late-feathering type in ev21 ⁺ group was significantly higher than that in ev21 ^- group (P≤0.05). The feather maturity of the late-feathering cocks at 105-day-old in the ev21 ^- group was extremely significantly higher than that in ev21 ⁺group (P≤0.01). At one-day-old chicks, the expression of PRLR gene between R2 versus (L2+L4) showed no any difference (P>0.05),but its expression between R2 versus L4 was significantly down-regulated (0.01<P≤0.05). While the expression of SPEF2 gene in late-feathering chickens was extremely significantly higher than that in early-feathering chickens (P≤0.01). By comparing with the R2 group, SPEF2 significantly differentially up-regulated in the L4 and L2 groups (P≤0.01). 【Conclusion】New genotyping method should be developed for improving the accuracy of the heterozygote in the late feathering cocks. Up to now, a test cross is essential for the chicken production. Construction of the ev21 ^-resistant strain is viable in late-feathering chicken breeding. Furthermore, studies need to be conducted to figure out the difference of isometric subtype ratio and feather maturity between the ev21 ⁺ group and the ev21 ^- group. Besides, SPEF2 and PRLR were candidate genes for subtypes affecting the difference of early-feathering and late-feathering chickens at 1-day-old.

Effect of Recombinant GSTA3 Protein on Expression of the Anti-Apoptotic Gene BAG-3 in Thiram-Induced Tibial Chondrodysplasia

Zhen LI,ShiXiong YANG,Sheng NIU,Ning ZHANG,Xin LI,YangYang ZHANG,YunFei JIA,ZhiXiong TIAN,GuanBao NING,Ding ZHANG,WenXia TIAN

Scientia Agricultura Sinica. 2020, 53(9): 1921-1930. doi:10.3864/j.issn.0578-1752.2020.09.018

Abstract ( 293 )

HTML ( 29 )

PDF (2437KB) ( 233 )

Save

Figures and Tables | References | Related Articles | Metrics

【Objective】Tibial chondrodysplasia (TD) is a common skeletal disease in broilers. The study was conducted to investigate the effect of recombinant GSTA3 protein on the expression of anti-apoptotic gene BAG-3 in the chondrocytes of broilers induced by thiram, so as to provide a new idea and method for the treatment of TD. 【Method】120 one-week-old broiler chicks were randomly divided into six groups (A, B, C, D, E and F), in which Group A, B and C were basic diet groups, and group D, E and F were thiram-containing diet groups. TD was induced by thiram for 2 days. After thiram was added for days 1, 3, 5 and 7, recombinant chicken GSTA3 protein and phosphate buffer were injected into the leg muscles. Group A and group D were injected with PBS (100 μg·kg ^-1); group B and group E were injected with low dose (100 μg·kg ^-1) GSTA3; group C and group F were injected with high dose (200 μg·kg ^-1) GSTA3. The experiment lasted 23 days. Tibia growth plates were collected at the 1st, 2nd, 4th, 6th, 10th and 15th days after thiram treatment. The mRNA level of BAG-3 gene was detected by quantitative PCR, and the protein expression of BAG-3 gene was observed by immunohistochemistry. 【Result】Real-time PCR results showed that the expression level of BAG-3 mRNA in tibia growth plate of broilers in the thiram-containing diet group was significantly increased, compared with that in the basal diet control group during the repair period of TD injury (P<0.05). Compared with the thiram-containing diet group, E and F groups had significant differences on days 2, 4, 10 and 15, and were significantly lower than that under the thiram-containing diet group on days 10 and 15 (P<0.05), indicating a faster recovery compared with group D. Immunohistochemical results showed that BAG-3 protein was not expressed in the proliferation area and the pre-hypertrophic area of the chicken tibial chondrocytes, but only in the cytoplasm of the hypertrophic area. Compared with the control group A, the expression of BAG-3 protein in the thiram-containing diet group was increased. The recombinant GSTA3 induced the protein expression level in the hypertrophic area in the high and low dose of GSTA3 group compared with the thiram-containing diet group D (days 10 and 15). 【Conclusion】In conclusion, GSTA3 recombinant protein could regulate the expression of BAG-3 to participate in the apoptosis pathway and inhibit the apoptosis during the induction of TD in broilers. During the TD-injury repair period, after GSTA3 injection, the expression of anti-apoptotic gene BAG-3 protein was enhanced, which could participate in cell apoptosis to alleviate the TD-injury and make the TD growth plate function of broilers return to normal quickly.

Table of Content