[1]王春雷,高季平,赵宪坤.利用酶联免疫反应鉴定梨树S基因型[J].江苏农业学报,2015,(05):1124-1128.[doi:doi:10.3969/j.issn.1000-4440.2015.05.028]
　WANG Chun-lei,GAO Ji-ping,ZHAO Xian-kun.S genotyping in pear by PCR-ELISA[J].,2015,(05):1124-1128.[doi:doi:10.3969/j.issn.1000-4440.2015.05.028]
点击复制

利用酶联免疫反应鉴定梨树S基因型()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年05期
页码:
1124-1128
栏目:
园艺
出版日期:
2015-10-31

文章信息/Info

Title:
S genotyping in pear by PCR-ELISA
作者:
王春雷; 高季平; 赵宪坤
（扬州大学园艺与植物保护学院，江苏扬州225009）
Author(s):
WANG Chun-lei1; GAO Ji-ping; ZHAO Xian-kun
(School of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009，China)
关键词:
梨; 自交不亲和性; S基因型鉴定; 酶联免疫反应
Keywords:
pear; self-incompatibility; S genotyping; ELISA
分类号:
S661.2
DOI:
doi:10.3969/j.issn.1000-4440.2015.05.028
文献标志码:
A
摘要:
梨是蔷薇科植物，具有典型的配子体自交不亲和性。获取各品种S基因型是果树生产中一项重要内容。现有S基因型鉴定方法存在一些缺陷。利用通用引物扩增S-RNase高变区，结合单链核苷酸探针杂交、酶联免疫反应，成功鉴定8个品种的7个S基因型。PCR酶联免疫法结果显示爱宕（S2S5），爱甘水（S4S5），二十世纪（S2S4），丰水（S3S5），今村秋（S1S6），新高（S3S9）和幸水（S4S5）7个品种都能与各自对应的S基因型探针杂交结合，颜色反应明显，无明显假阳性反应。秋荣（S4smS5）S4sm-RNase基因在花柱中不表达，只在秋荣花柱中检测到S5-RNase基因信号。与目前已有的方法相比，PCR酶联免疫法操作简单，结果可靠，能够广泛用于梨树S基因型鉴定。
Abstract:
Pear, belonging to the family Rosaceae, possesses gametophytic self-incompatibility. Since SI inhibits self-fertilization, it is essential to obtain information on S genotypes of cultivars for successful planting. A method using a semi-automated sequencer is not convenient. S genotyping by dot-blot analysis requires considerable time and skill, and it is not suitable for identifying S genotypes of a small number of cultivars. A rapid and reliable new method for S genotyping is needed to be developed. To solve those problems, in this study, a PCR-ELISA using a set consensus primers to amplify the fragment covering hypervariable region of S-RNase genes, using allele-specific oligonucleotide probes to discriminate the PCR fragment, and using enzyme linked immunosorbent assays (ELISA) to detect the signal allowed us to identify 7 S haplotypes of 8 cultivars. The signals in Atago（S2S5），Aikansui（S4S5），Nijisseiki（S2S4），Housui（S3S5），Imamuraaki（S1S6），Niitaka（S3S9）, and Kousui（S4S5）were consistent with their respective S genotypes. In‘Akiba’（S4smS5），the S4sm-RNase were not expressed in the style; only S5-RNase signals were detected. Compared with the common methods, the PCR-ELISA method is easily-operated and reliable, which is generally applicable to identify the S genotype of pear. 

参考文献/References:

[1]SONNEVELD T, TOBUTT K R, ROBBINS T P.Allele-specific PCR detection of sweet cherry self-incompatibility (S) alleles S1 to S16 using consensus and allele-specific primers [J].Theor Appl Genet, 2003, 107 (6):1059-1070.
[2]VAUGHAN S P, RUSSELL K, SARGENT D J,et al. Isolation of S-locus F-box alleles in prunusavium and their application in a novel method to determine self-incompatibility genotype [J]. Theor Appl Genet, 2006, 112 (5):856-866.
[3]ORTEGA E, SUTHERLAND B G, DICENTA F,et al. Determination of incompatibility genotypes in almond using first and second intron consensus primers: detection of new S alleles and correction of reported S genotypes[J]. Plant Breeding, 2005, 124 (2):188-196.
[4]KITASHIBA H, ZHANG S, WU J,et al. S genotyping and S screening utilizing SFB gene polymorphism in Japanese plum and sweet cherry by dot-blot analysis [J]. Mol Breeding, 2008, 21 (3):339-349.
[5]INOUYE S, HONDO R. Microplate hybridization of amplified viral DNA segment [J]. J Clin Microbiol, 1990, 28 (6):1469-1472.
[6]KELLER G H, HUANG D P, SHIH J W, et al. Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization [J]. J Clin Microbiol, 1990, 28 (6):1411-1416.
[7]SYVANEN AC, BENGTSTROM M, TENHUNEN J,et al. Quantification of polymerase chain reaction products by affinity-based hybrid collection [J]. Nucleic Acids Res,1988, 16 (23):11327-11338.
[8]TONOSAKI , KUDO J, KITASHIBA H,et al.Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants [J].Mol Breeding, 2013, 31(2): 419-428.
[9]ISHIMIZU T, SHINKAWA T, SAKIYAMA F,et al. Primary structural features of rosaceous S-RNases associated with gametophytic self-incompatibility[J]. Plant Mol Biol, 1998, 37 (6):931-941.
[10]SAPIR G, STERN RA, EISIKOWITCH D,et al. Cloning of four new Japanese plum S-alleles and determination of the compatibility between cultivars by PCR analysis [J]. J Hortic Sci Biotech, 2004, 79 (2):223-227.
[11]TAMURA M, USHIJIMA K, SASSA H,et al.Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis[J]. Theor Appl Genet , 2000, 101(3):344-349.
[12]GUERRA ME, RODRIGO J, LOPEZ-CORRALES M, et al. S-RNase genotyping and incompatibility group assignment by PCR and pollination experiments in Japanese plum[J]. Plant Breeding, 2009, 128(3):304-311.
[13]WANG C L, ZHANG Z P, TONOSAKI K,et al. S genotyping in Japanese plum and sweet cherry by allele-specific hybridization using streptavidin-coated magnetic beads[J]. Plant Cell Reports, 2013, 32(6)：567-576.〖ZK）〗〖FL）〗

相似文献/References:

[1]王冬群,胡寅侠,华晓霞.慈溪市梨农药残留膳食摄入风险评估[J].江苏农业学报,2016,(03):698.[doi:10.3969/j.issn.1000-4440.2016.03.033]
　WANG Dong-qun,HU Yin-xia,HUA Xiao-xia.Dietary intake risk assessment of pesticide residue in pears in Cixi city[J].,2016,(05):698.[doi:10.3969/j.issn.1000-4440.2016.03.033]
[2]王春雷,赵宪坤,高季平,等.芸薹属植物自交不亲和性研究进展[J].江苏农业学报,2015,(06):1442.[doi:doi:10.3969/j.issn.1000-4440.2015.06.039]
　WANG Chun-lei,ZHAO Xian-kun,GAO Ji-ping,et al.Advances in self-incompatibility of Brassica[J].,2015,(05):1442.[doi:doi:10.3969/j.issn.1000-4440.2015.06.039]
[3]许园园,李晓刚,李慧,等.梨CDPK基因家族全基因组序列鉴定分析[J].江苏农业学报,2015,(03):659.[doi:10.3969/j.issn.1000-4440.2015.03.031]
　XU Yuan-yuan,LI Xiao-gang,LI Hui,et al.Identification of calcium-dependent protein kinase(CDPK) gene family in pear（Pyrus bretschneideri Rehd）[J].,2015,(05):659.[doi:10.3969/j.issn.1000-4440.2015.03.031]
[4]谢昶琰,王迪,安祥瑞,等.滴灌减量施肥对梨树体养分及果实产量、品质的影响[J].江苏农业学报,2021,(06):1526.[doi:doi:10.3969/j.issn.1000-4440.2021.05.022]
　XIE Chang-yan,WANG Di,AN Xiang-rui,et al.Effects of drip irrigation and reducing fertilization on tree nutrient, fruit yield and quality of pear[J].,2021,(05):1526.[doi:doi:10.3969/j.issn.1000-4440.2021.05.022]
[5]施露,高庆超,李亚辉,等.梨果实品质的研究进展与潜在技术应用展望[J].江苏农业学报,2022,38(02):567.[doi:doi:10.3969/j.issn.1000-4440.2022.02.033]
　SHI Lu,GAO Qing-chao,LI Ya-hui,et al.Research progress and prospects of potential technology application in pear fruit quality[J].,2022,38(05):567.[doi:doi:10.3969/j.issn.1000-4440.2022.02.033]
[6]倪萌,王娟,王爽,等.克服萝卜自交不亲和性的化学试剂筛选[J].江苏农业学报,2022,38(04):1042.[doi:doi:10.3969/j.issn.1000-4440.2022.04.022]
　NI Meng,WANG Juan,WANG Shuang,et al.Screening of chemical reagents for overcoming the self-incompatibility of radish[J].,2022,38(05):1042.[doi:doi:10.3969/j.issn.1000-4440.2022.04.022]
[7]毕清芮,马新院,薛玉冉,等.基于SSR标记的MCID法鉴定新疆地方梨品种及其遗传多样性分析[J].江苏农业学报,2024,(10):1933.[doi:doi:10.3969/j.issn.1000-4440.2024.10.018]
　BI Qingrui,MA Xinyuan,XUE Yuran,et al.Identification of local pear cultivars in Xinjiang Uygur Autonomous Region by MCID method based on SSR markers and analysis of their genetic diversity[J].,2024,(05):1933.[doi:doi:10.3969/j.issn.1000-4440.2024.10.018]

备注/Memo

备注/Memo:
收稿日期：2015-05-11 基金项目：高等学校全国优秀博士学位论文作者专项基金项目（201467）〖JP〗 作者简介：王春雷，（1981-）男，江苏南京人，博士，教授，研究方向：果树生殖生理。（Tel）18118252060;（E-mail）wangcl@yzu.edu.cn
更新日期/Last Update: 2015-10-31