DNA Fingerprint Construction for 72 Chinese Cabbage (Brassica rapa ssp. pekinensis) Hybrids
LIU Shuan-Tao, ZHANG Zhi-Gang, LI Qiao-Yun, WANG Rong-Hua, WANG Li-Hua, WANG Shu-Bin, ZHAO Zhi-Zhong*
Shandong Branch of National Center for Vegetables Improvement/Vegetable and Flower Institute, Shandong Academy of Agricultural Sciences/Shandong Key Laboratory for Biology of Greenhouse Vegetables/Huang-Huai-Hai Region Scientific Observation and Experimental Station of Vegetables (Shandong), Ministry of Agriculture and Rural Affairs, Ji'nan 250100, China
Abstract：In production practice, the identification of Chinese cabbage (Brassica rapa ssp. pekinensis) hybrids mostly rely on field planting and phenotypic identification, which is inefficient and inaccurate. Under the condition of more and more mature molecular marker research technology, it is necessary to establish a rapid, efficient and accurate identification technology of Chinese cabbage hybrids based on molecular marker technology. Considering this, a total of 86 pairs of insertion/deletion (InDel) primers were used to identify 40 Chinese cabbage hybrids by PCR amplification and polymorphism detection. Powermarker V3.0 software were used to analyze the polymorphic results. MEGA 7.0 software were performed for cluster analysis of hybrids. In order to screen core primers suitable for DNA fingerprint construction of different kind of Chinese cabbage hybrids, the percentage of heterozygous state for each pair of primers were compared in every group. The results showed that, 189 polymorphic loci were obtained, with an average of 2.2 alleles for each primer combination. The heterozygosity index ranged from 0 to 0.648 2 and averaged at 0.304 2. The major allele frequency ranged from 0.391 9 to 1.000 0 and averaged at 0.701 1. The number of alleles detected by each pair of primers ranged from 1 to 4, of which 74 pairs of primers could only detect 2 alleles. Then, cluster analysis revealed that, based on producing area, the 40 hybrids were clearly divided into imported and domestic varieties, among which the domestic varieties could be further divided into folded-, cylindric-, and fluffy-type varieties.The number of loci with heterozygous state ≥50% and their distribution in genome was significantly different in the 4 groups. Finally, 5 pairs of primers with heterozygous state ≥50% and allele number equal to 2 were selected from each group for construction of core primers. Other 32 Chinese cabbage hybrid varieties which were sold in market in recent years were detected with core primers. The detection results of pure loci were assigned a binary numeral 0 or 1 according to the mobility ratio of electrophoresis from small to large, while heterozygous loci were assigned letter H. All primers were ordered according to chromosome number they located from A01 to A10 and primers on the same chromosome were ordered according to their physical position from small to large. DNA fingerprint of 72 hybrids were constructed. The selected core primers will be universal and compatible with DNA fingerprint construction of new hybrid cultivars created in future. The results could also provide reference for gemplasm screening and hybrids identification of Chinese cabbage.
刘栓桃, 张志刚, 李巧云, 王荣花, 王立华, 王树彬, 赵智中. 72个大白菜杂交种DNA指纹构建[J]. 农业生物技术学报, 2021, 29(2): 240-250.
LIU Shuan-Tao, ZHANG Zhi-Gang, LI Qiao-Yun, WANG Rong-Hua, WANG Li-Hua, WANG Shu-Bin, ZHAO Zhi-Zhong. DNA Fingerprint Construction for 72 Chinese Cabbage (Brassica rapa ssp. pekinensis) Hybrids. 农业生物技术学报, 2021, 29(2): 240-250.
[1] 常瑞青. 2020. 鲜食番茄品种遗传多样性分析及指纹图谱构建[J]. 中国蔬菜, (3): 21-27. (Chang R Q. 2020. Genetic diversity analysis and fingerprinting construction of fresh tomato varieties[J]. China Vegetables, (3): 21-27.) [2] 方宣钧, 刘思衡, 江树业. 2000. 品种纯度和真伪的DNA分子标记鉴定及其应用[J]. 农业生物技术学报, 8(02): 106-110. (Fang X J, Liu S H, Jiang S Y.2000. Progress on identification of seed purity and autheticity using DNA molecular markers[J]. Journal of Agricultural Biotechnology, 8(02): 106-110.) [3] 盖树鹏, 王日新, 黄进勇. 2011. 山东省玉米骨干自交系和杂交种的SSR指纹图谱构建[J]. 中国农学通报, 27(24): 40-44. (Gai S P, Wang R X, Huang J Y.2011. Fingerprinting establishment of maize elite inbred lines and hybrids by SSR markers in Shandong province[J]. Chinese Agricultural Science Bulletin, 27(24): 40-44.) [4] 韩晴, 沈雪芳, 陆卫平, 等. 2014. 20个鲜食玉米杂交种DNA指纹数据库构建[J]. 上海农业学报, 30(1): 36-39. (Han Q, Shen X F, Lu W P, et al.2014. Construction of DNA fingerprint database of 20 table core hybrids[J]. Acta Agriculturae Shanghai, 30(1): 36-39.) [5] 匡猛, 杨伟华, 许红霞, 等. 2011. 中国棉花主栽品种DNA指纹图谱构建及SSR标记遗传多样性分析[J]. 中国农业科学, 44(1): 20-27. (Kuang M, Yang W H, Xu H X, et al.2011. Construction of DNa fingerprinting and analysis of genetic diversity with SSR markers for cotton major cultivars in China[J]. Scientia Agricultura Sinica, 44(1): 20-27.) [6] 刘栓桃, 张志刚, 李巧云, 等. 2014. 光敏色素B基因(PHYB)启动子突变及与大白菜开花时间的关联分析[J]. 农业生物技术学报, 22(7): 853-861. (Liu S T, Zhang Z G, Li Q Y, et al.2014. Association analysis between phytochrome B gene (PHYB) promoter mutantions and flowering time in Chinese cabbage (Brassica rapa L. ssp. pekinensis)[J]. Journal of Agricultural Biotechnology, 22(7): 853-861.) [7] 刘栓桃, 张志刚, 王立华, 等. 2020. 牛牌19大白菜杂交种种子纯度快速鉴定方法研究[J]. 中国蔬菜, (03): 28-32. (Liu S T, Zhang Z G, Wang L H, et al. Studies on rapid identification on purity of Chinese cabbage 'Niupai No.19' hybrid seed[J]. China Vegetables, (03): 28-32.) [8] 刘栓桃, 张志刚, 王荣花, 等. 2018. 基于InDels标记的大白菜核心材料的亲缘关系鉴定[J]. 植物遗传资源学报, 19(4): 657-667. (Liu S T, Zhang Z G, Wang R H, et al.2018. Identification of genetic relationships of Chinese cabbage inbred lines using InDels markers[J]. Journal of Plant Genetic Resources, 19(4): 657-667.) [9] 刘栓桃, 张志刚, 王荣花, 等. 2019. 基于InDel标记的大白菜育种材料分子身份证构建[J]. 中国蔬菜, (2): 34-41. (Liu S T, Zhang Z G, Wang R H, et al. 2019. Construction of molecular identity card for Chinese cabbage breeding materials based on InDel markers[J]. China Vegetables, (2): 34-41.) [10] 薛银鸽, 原玉香, 张晓伟, 等. 2014. 利用InDel标记鉴定大白菜杂交种豫新四号种子纯度[J].农业生物技术学报, 22(04): 449-456. (Xu Y G, Yuan Y X, Zhang X W, et al.2014. Purity identification of Chinese cabbage (Brassica rapa L. ssp. pekinensis) hybrid Yuxin No.4 by InDel markers[J]. Journal of Agricultural Biotechnology, 22(04): 449-456.) [11] 肖子发, 朱克永, 刘之熙, 等. 2011. 利用SSR标记构建湖南省审杂交水稻品种DNA指纹图谱[J]. 杂交水稻, 26(03): 51-56. (Xiao Z F, Zhu K Y, Liu Z X, et al.2011. SSR marker based DNA fingerprinting of hybrid rice varieties certified in Hunan province[J]. Hybrid Rice, 26(3): 51-56.) [12] 张全芳, 张荦嘉, 梁水美, 等. 2016. 适用于多重荧光SSR检测的玉米基因组DNA提取方法[J]. 玉米科学, 24(2): 29-34. (Zhang Q F, Zhagn L J, Liang S M, et al.2016. Apply to multiple fluorescent SSR detection of maize genome DNA extraction method[J].Journal of Maize Sciences, 24(2): 29-34.) [13] 张志刚, 赵智中, 李巧云, 等. 2016. 大白菜InDels标记开发及其在剩余杂合体鉴定中的应用[J]. 农业生物技术学报, 24(4): 510-518. (Zhang Z G, Zhao Z Z, Li Q Y, et al.2016. Development of InDels markers and their usage in detection of residual heterozygous lines in Chinese cabbage (Brassica rapa ssp. pekinensis)[J]. Journal of Agricultural Biotechnology, 24(4): 510-518.) [14] 赵久然, 王凤格, 易红梅, 等. 2015. 我国玉米品种标准 DNA 指纹库构建研究及应用进展[J]. 作物杂志, 30(2): 1-6. (Zhao J R, Wang F G, Yi H M, et al.2015. Progress of construction of Chinese maize varieties standard DNA fingerprint database[J]. Crops, 30(2): 1-6.)
