黄瓜—酸黄瓜异附加系的创制与鉴定

doi:10.16420/j.issn.0513-353x.2021-0187

摘要/Abstract

摘要：

异附加系是重要的种间材料,可用于种间外源遗传物质的渐渗及基因定位等相关研究,创制多种类型的异附加系对于栽培黄瓜（Cucumis sativus）的品种改良及相关研究均具有重要作用。本试验中以来源于酸黄瓜（Cucumis hystrix）和栽培黄瓜‘北京截头’杂交的种间异源四倍体为母本,以‘北京截头’为轮回父本,连续回交获得22株植株。利用基因组荧光原位杂交（GISH）技术确定22株BC₂群体中附加有外源染色体的植株,然后利用oligo-FISH技术和12个酸黄瓜染色体单拷贝序列标记准确识别外源染色体。共筛选出4种异附加系材料,包括3种单体异附加系和1种双单体异附加系。其中3种单体异附加系分别附加酸黄瓜6号、10号和12号染色体,双单体异附加系同时附加了酸黄瓜6号和10号染色体。

关键词: 黄瓜, 酸黄瓜, 异附加系, oligo-FISH, 单拷贝序列标记

Abstract:

Alien addition lines are important interspecific materials,which can be used for introgression of interspecific genetic material and gene mapping. Creating a variety of alien addition lines plays an important role in cultivar improvement and related research of cucumber. In this experiment,based on the allotetraploid Cucumis hytivus from the cross of C. sativus and C. hystrix,two backcrosses with diploid cultivar C. sativus were carried out to obtain 22 plants of BC₂. The plant containing alien chromosome in BC₂ population was identified by GISH technique. Further,oligo-FISH technology and 12 C. hystrix chromosome specific single-copy sequence markers were used to accurately identify the alien chromosomes. Finally,a total of four different alien additional lines were screened,including three monosomic alien addition lines（MAALs）which contained C. hystrix chromosomes H06,H10,and H12,respectively,and one double alien monosomic addition line containing H06 and H10.

Key words: Cucumis sativus, Cucumis hystrix, alien addition line, oligo-FISH, single-copy sequence marker

中图分类号:

S642.2

胡伟, 刘昱希, 赵勤政, 陈劲枫, 娄群峰. 黄瓜—酸黄瓜异附加系的创制与鉴定[J]. 园艺学报, 2021, 48(7): 1349-1358.

HU Wei, LIU Yuxi, ZHAO Qinzheng, CHEN Jinfeng, LOU Qunfeng. Creation and Identification of Cucumis sativus-C. hystrix Alien Addition Lines[J]. Acta Horticulturae Sinica, 2021, 48(7): 1349-1358.

图/表 9

图1 材料的创建过程

Fig. 1 The process of creating materials

表1 黄瓜oligo探针与酸黄瓜染色体对应关系

Table 1 Corresponding relationship between cucumber oligo probe and Cucumis hystrix chromosome

黄瓜oligo探针 Cucumber oligo probe	酸黄瓜染色体 Cucumis hystrix chromosomes	物理位置/Mb Physical positions
Oligo C7-a	H01	0 ~ 19
Oligo C1-a	H02	0 ~ 4/12.5 ~ 20/24 ~ 33
Oligo C2-a	H03	12.4 ~ 22.6
Oligo C3-a	H04	25.2 ~ 40.8
Oligo C2-b	H05	0 ~ 12.4
Oligo C3-b	H06	0 ~ 25.2
Oligo C4-a	H07	0 ~ 10.5/19.5 ~ 23.3
Oligo C4-b	H08	10.5 ~ 19.5
Oligo C5-a	H09	0 ~ 4/11 ~ 25
Oligo C5-b	H10	4 ~ 16.5/20 ~ 31.9
Oligo C6-a	H11	0 ~ 17.8
Oligo C1-b	H12	4 ~ 12.5/20 ~ 24

表2 酸黄瓜染色体单拷贝序列标记引物

Table 2 The chromosomes single-copy sequence marker primers in Cucumis hystrix

单拷贝序列标记Single-copy sequences marker	前引物（5′-3′）Primer forward	后引物（5′-3′）Primer reverse
ss H01	AGCCTTTTTCCTACACCATAA	ATTCCCTAGATCCCTTGACTG
ss H02	GTCTTTTACTCTTGGTTTTCG	TAATCGTTCCACCCATCACTA
ss H03	CAATAGAATCCATATCTTTGA	GTCATTCGTTTACTATCACAT
ss H04	GGGATTAAGCTTGTTTAGGTT	ATTTCCTTGGGATCACTACTT
ss H05	GAATAGGGAAAAGTTGCCGTT	TGATGCTGTCAAGTCGAGTCA
ss H06	CGACAAGTTTGACGTTGATAT	CTAGTTTGAACTGCGTGTATTA
ss H07	TATACCCAACCAACCGATTCC	ACTTTGAGGGCTTGTGTCTCC
ss H08	ATCGTAGAAAGGAAAGGAGAC	GATCAATTGTTGGGAGTGACT
ss H09	AGAAAATTTGGATGAAAACTAGC	ATAGAGAAAACGAACACGACT
ss H10	TACATTCCAATAGGGTGCCGT	GCCGTACTCACAGATTAAAAAA
ss H11	ACTACACTAGCGAGCAAGGGC	ACAGTAATGGTTTGGAAACGG
ss H12	GGACCCAACCTTACACCCTTA	TTCGAGTTTCAATTCCTCACC

图2 异源三倍体的植株形态和果实及体细胞染色体GISH分析

Fig. 2 Cucumis allotriploid plant morphology and fruit and chromosome GISH analysis

图3 ‘北京截头’黄瓜（a）及其染色体（b）和利用hy-gDNA探针（绿色）分别筛选出的携带1条（c）和2条（d）酸黄瓜染色体的异附加系材料

Fig. 3 ‘Beijing Jietou’cucumber（a）and its chromosomes（b）and alien addition lines carrying one（c）and two（d）C. hystrix chromosome

图4 黄瓜—酸黄瓜单体异附加系CC-1、CC-4和CC-5中染色体的FISH鉴定 Hy-gDNA：酸黄瓜基因组DNA探针;oligo：黄瓜染色体探针。

Fig. 4 FISH identification of chromosomes in Cucumis sativus-chystrix monosomic alien addition lines CC-1,CC-4 and CC-5 Hy-gDNA：C. hystrix genome DNA probe;oligo：Cucumber chromosome probe.

图5 黄瓜—酸黄瓜双单体异附加系CC-9中染色体的FISH鉴定 a：CC-9的植株形态;b ~ d：1条外源染色体被鉴定为酸黄瓜6号染色体（H06）;e ~ g：1条外源染色体被鉴定为酸黄瓜10号染色体（H10）。Hy-gDNA：酸黄瓜基因组DNA探针;oligo：黄瓜染色体探针。

Fig. 5 FISH identification of chromosomes in Cucumis sativus-chystrix double monosomic alien addition lines CC-9 a：Plant morphology of CC-9;b-d：One alien chromosome identified as H06;e-g：One alien chromosome identified as H10. Hy-gDNA：C. hystrix genome DNA probe;oligo：Cucumber chromosome probe.

图6 酸黄瓜染色体单拷贝序列标记引物ssH01 ~ ssH12的扩增结果 HH：酸黄瓜;HCC：异源三倍体;CC：‘北京截头’黄瓜。

Fig. 6 Amplification results of single-copy sequence marker primers ss H01-ss H12 for Cucumis hystrix chromosome HH：C. hystrix;HCC：Allotriploid;CC：C. sativus‘Beijing Jietou’.

图7 酸黄瓜染色体特异的单拷贝序列标记引物在黄瓜—酸黄瓜异附加系中的扩增结果 HH：酸黄瓜;HCC：异源三倍体;CC：‘北京截头’黄瓜;ss H06、ss H10和ss H12是酸黄瓜6号、10号和12号染色体单拷贝序列。

Fig. 7 Amplification results of C. hystrix chromosome specific single-copy sequence marker primers in Cucumis sativus-hystrix MAALs HH：C. hystrix;HCC：allotriploid;CC：C. sativus;ss H06,ss H10 and ss H12 were C. hystrix H06,H10 and H12 chromosome single-copy sequence markers.

参考文献 28

[1]	An D G, Zheng Q, Luo Q L, An D, Zheng Q, Luo Q, Ma P, Zhang H, Li L, Han F, Xu H, Xu Y, Zhang X. 2015. Molecular cytogenetic identification of a new wheat-rye 6R chromosome disomic addition line with powdery mildew resistance. PLoS ONE, 10(8):343-355.
[2]	Bi Y F, Zhao Q Z, Yan W, Li M X, Liu Y X, Cheng C, Zhang L, Yu X, Li J, Qian C T, Wu Y, Chen J F, Lou Q F. 2020. Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis. The Plant Journal, 102(1):178-186. doi: 10.1111/tpj.v102.1 URL
[3]	Chen J F, Kirkbride J H. 2000. A new synthetic species of Cucumis(Cucurbitaceae)from interspecific hybridization and chromosome doubling. Brittonia, 52(4):315-319. doi: 10.2307/2666583 URL
[4]	Chen Jin-feng, Luo Xiang-dong, Qian Chun-tao, Cao Qing-he. 2003. Production and characterizationg of monosomic alien addition lines of cucumber. Acta Horticulturae Sinica, 30(6):725-727. (in Chinese)
	陈劲枫, 罗向东, 钱春桃, 曹清河. 2003. 黄瓜单体异附加系的筛选与观察. 园艺学报, 30(6):725-727.
[5]	Chen Xue-ping, Sheng Er-qiao, Zhang Cheng-he, Li Xiao-feng, Xuan Shu-xin, Sheng Shu-xing. 2010. Transmission of n + 1 gametes and obtaining of two bisomic addition lines of flowering chinese cabbage-chinese kale. Scientia Agricultura Sinica, 43(23):4871-4876. (in Chinese)
	陈雪平, 申二巧, 张成合, 李晓峰, 轩淑欣, 申书兴. 2010. 菜薹—芥蓝单体异附加系n + 1配子传递及两个二体异附加系的获得. 中国农业科学, 43(23):4871-4876.
[6]	Darko E, Janda T, Majláth I, Szopkó D, Dulai S, Molnár I, Türkösi E, Molnár-Láng M. 2015. Salt stress response of wheat-barley addition lines carrying chromosomes from the winter barley“Manas”. Euphytica, 203(3):491-504. doi: 10.1007/s10681-014-1245-7 URL
[7]	Dong Shao-yun, Miao Han, Bo Kai-liang, Zhang Sheng-ping, Gu Xin-fang. 2020. Research progress on wild relatives of cucumber. Journal of Plant Genetic Resources, 21(6):1446-1460. (in Chinese)
	董邵云, 苗晗, 薄凯亮, 张圣平, 顾兴芳. 2020. 黄瓜近缘野生资源的研究进展. 植物遗传资源学报, 21(6):1446-1460.
[8]	Dong T, Feng S, Li S, Yu C, Zhou B. 2018. Ten alien chromosome additions of Gossypium hirsutum-Gossypium bickii developed by integrative uses of GISH and species-specific SSR markers. Molecular Genetics & Genomics Mgg, 293(4):1-11.
[9]	Gu Ai-xia, Zheng Bao-zhi, Wang Yan-hua, Xuan Shu-xin, Luo Shuang-xia, Shen Shu-xing. 2009. Obtaining and studies of Chinese cabbage monosomic alien addition line with chromosome 3 of cabbage. Acta Horticulturae Sinica, 36(1):39-44. (in Chinese)
	顾爱侠, 郑宝智, 王彦华, 轩淑欣, 罗双霞, 申书兴. 2009. 附加甘蓝3号染色体的大白菜单体异附加系的获得与研究. 园艺学报, 36(1):39-44.
[10]	Han Y H, Zhang T, Thammapichai Paradee, Weng Y Q, Jiang J M. 2015. Chromosome-specific painting in Cucumis species using bulked oligonucleotides. Genetics, 200(3):771-779.
[11]	Han Y H, Zhang Z, Liu C, Liu J, Huang S. 2009. Centromere repositioning in cucurbit species:implication of the genomic impact from centromere activation and inactivation. Proceedings of the National Academy of Sciences, 106(35):14937-14941.
[12]	Hechanova S L, Prusty M R, Kim S R, Ballesfin L R, Ramos J, Prahalada G D, K. Jena K. 2018. Monosomic alien addition lines(MAALs)of Oryza rhizomatis in Oryza sativa:production,cytology,alien trait introgression,molecular analysis and breeding application. Theoretical and Applied Genetics, 131(10):2197-2211. doi: 10.1007/s00122-018-3147-x pmid: 30032316
[13]	Jiang J M. 2019. Fluorescence in situ hybridization in plants:recent developments and future applications. Chromosome Research, 27(3):153-165. doi: 10.1007/s10577-019-09607-z URL
[14]	Kong L N, Song X Y, X Jin, H Sun, X Wang. 2018. Development and characterization of a complete set of Triticum aestivum-Roegneria ciliaris disomic addition lines. Theor Appl Genet, 131(8):1793-1806. doi: 10.1007/s00122-018-3114-6 URL
[15]	Li M X, Zhao Q Z, Liu Y X, Qin X D, Hu W, Davoudi M, Chen J F, Lou Q F. 2020. Development of alien addition lines from Cucumis hystrix in Cucumis sativus:cytological and molecular marker analyses. Genome, 63:629-641. doi: 10.1139/gen-2020-0035 URL
[16]	Liu H P, Dai Y, Chi D, Huang S, Li H F, Duan Y M, Cao W G, Gao Y, Fedak G, Chen J M. 2017. Production and molecular cytogenetic characterization of a durum wheat-Thinopyrum elongatum 7E disomic addition line with resistance to fusarium head blight. Cytogenet Genome Res, 153(3):165-173. doi: 10.1159/000486382 URL
[17]	Lou Q F, He Y, Cheng C, Zhang Z, Li J, Huang S, Chen J F. 2013. Integration of high-resolution physical and genetic map reveals differential recombination frequency between chromosomes and the genome assembling quality in cucumber. PLoS ONE, 8(5):e62676. doi: 10.1371/journal.pone.0062676 URL
[18]	Lou Q F, Zhang Y, He Y, Li J, Jia L, Cheng C, W Guan, S Yang, J Chen. 2014. Single-copy gene-based chromosome painting in cucumber and its application for chromosome rearrangement analysis in cucumis. Plant Journal for Cell & Molecular Biology, 78(1):169-179.
[19]	Murray M G, Thompson W F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res, 8(19):4321-4325. pmid: 7433111
[20]	Qin X D, Zhang Z, Lou Q, Xia L, Li J, Zhao X K, Yu X Q, Cheng C Y, Huang S W, Chen J F. 2021. Chromosome-scale genome assembly of Cucumis hystrix-a wild species interspecifically cross-compatible with cultivated cucumber. Horticulture Research,10.1038/s41438-021- 00475-5.
[21]	Shen Shu-xing, Hou Xi-lin, Luo Shuang-xia. 2008. Studies on the gamete formation and transmission rateofa set of primary trisomics in Chinese cabbage. Acta Horticulturae Sinica, 35(9):1285-1290. (in Chinese)
	申书兴, 侯喜林, 罗双霞. 2008. 大白菜初级三体的配子形成及传递率研究. 园艺学报, 35(9):1285-1290.
[22]	Tan C, Cui C, Xiang Y, Ge X H, Li Z Y. 2017. Development of Brassica oleracea-nigramonosomic alien additionlines:genotypic,cytological and morphological analyses. Theor Appl Genet, 130(12):2491-2504. doi: 10.1007/s00122-017-2971-8 URL
[23]	Wang Gui-xiang, Yan Hong, Zeng Xing-ying, Sheng Xiao-guan, Tang Yu, Han Shuo, Zong Mei, Lu Kun, Liu Fan. 2011. New alien additon lines resistance to black rot generated by somatic hybridization between cauliflower and black mustard. Acta Horticulturae Sinica, 38(10):1901-1910. (in Chinese)
	王桂香, 严红, 曾兴莹, 盛小光, 唐宇, 韩硕, 宗梅, 陆坤, 刘凡. 2011. 花椰菜—黑芥体细胞杂交获得抗黑腐病异附加系新材料. 园艺学报, 38(10):1901-1910.
[24]	Wang L, Liu Y, Du W, Jing F, Wang Z, Wu J, Chen X. 2015. Anatomy and cytogenetic identification of a wheat- Psathyrostachys huashanica Keng line with early maturation. PLoS ONE, 10(10):e0131841. doi: 10.1371/journal.pone.0131841 URL
[25]	Yang L M, Dal‐Hoe Koo, Li Y H, Zhang X J, Luan F, Michael J. Havey, Jiang J M, Weng Y Q. 2012. Chromosome rearrangements during domestication of cucumber as revealed by high‐density genetic mapping and draft genome assembly. The Plant Journal, 71(6):895-906. doi: 10.1111/tpj.2012.71.issue-6 URL
[26]	Yin Yue-hui, Xu Qin-xing, Qi Fei, Bao Yin-guang, Wang Hong-gang, Li Xing-feng. 2019. Screening and identification of wheat-Thinopyrum elongatum aline chromosomes lines. Shandong Agricultural Sciences, 51(4):1-6. (in Chinese)
	殷月辉, 徐勤省, 亓斐, 鲍印广, 王洪刚, 李兴锋. 2019. 小麦—长穗偃麦草异染色体系筛选与鉴定. 山东农业科学, 51(4):1-6.
[27]	Zheng Bao-zhi, Sheng Shu-xing, Wang Yan-hua, Chen Xue-ping, Zhang Cheng-he, Xuan Shu-xin, Luo Shuang-xia, Li Xiao-feng. 2008. Production and identification of head cabbage monosomic alien addition lines CO-9-1 in Chinese cabbage. Journal of Plant Genetic Resources, 9(2):239-242. (in Chinese)
	郑宝智, 申书兴, 王彦华, 陈雪平, 张成合, 轩淑欣, 罗双霞, 李晓峰. 2008. 大白菜—甘蓝单体异附加系CO-9-1的选育与鉴定. 植物遗传资源学报, 9(2):239-242.
[28]	Zhuang Fei-yun, Chen Jin-feng. 2003. RAPD analysis of cultivated cucumber,wild Cucumis species,Interspecific hybrid and its progenies from backcrossing. Acta Horticulturae Sinica, 30(1):47-50. (in Chinese)
	庄飞云, 陈劲枫. 2003. 黄瓜栽培种、近缘野生种、种间杂种及其回交后代的RAPD分析. 园艺学报, 30(1):47-50.