黄瓜的一个中短果突变体基因的初步定位

doi:10.16420/j.issn.0513-353x.2021-0194

摘要/Abstract

摘要：

中短果（medium short-fruit）突变体msf黄瓜为种植‘长春密刺’黄瓜自交系的过程中发现的1株自然突变材料,表现为果实长度以及果把长度相对野生型偏短,还表现出植株偏矮,侧枝发育异常,以及主茎脆弱等表型。通过石蜡切片和扫描电镜观察发现突变体果实长度的差异是由于细胞变小引起的。遗传分析发现F₂分离群体中野生型表型长果与突变体表型中短果的分离比符合孟德尔3:1遗传定律,表明msf突变性状为单基因控制的隐性性状。BSA-seq（Bulked Segregant Analysis Coupled with Whole-Genome Sequencing）分析将候选基因定位于黄瓜基因组1号染色体26.7 ~ 30.9 Mb区域内。利用437株中短果和‘hazerd’（欧洲温室型短果）杂交的F₂群体将基因定位在28.4 ~ 29.8 Mb之间的1.4 Mb区间内。结合dCAPS标记验证将CsaV3_1G044310作为候选基因,该基因编码拟南芥Ⅱ形肌醇多磷酸5-磷酸酶（Ⅱ型5PTase）同源蛋白。通过基因定量表达和内源激素生长素和赤霉素的测定进一步表明该突变体中Ⅱ型肌醇多磷酸5-磷酸酶表达缺陷可能引起激素水平的变化从而影响植株的生长发育。

关键词: 黄瓜, 果实长度, 突变体, 基因定位, Ⅱ型肌醇多磷酸5-磷酸酶;

Abstract:

We isolated a natural mutant,msf（medium short-fruit）,from the cucumber inbred line CCMC,a North China type with long fruits. The mutant showed the phenotype of shorter fruit length and fruit neck length than the wild type and also showed short plant,abnormal development of lateral branches and fragile main stem. Through paraffin section and scanning electron microscope observation,it was found that the difference of mutant fruit length was caused by the change of cell size. Genetic analysis showed that the segregation ratio of wild-type phenotype long fruit and mutant phenotype medium short fruit in F₂ segregation population conformed to Mendel’s 3︰1 genetic law. This indicated that msf mutation was a recessive trait controlled by a single gene. BSA-seq analysis showed that the candidate gene was located in a 26.7-30.9 Mb region of chromosome 1 of cucumber genome. The msf gene was mapped to a 28.4-29.8 Mb region using a F₂ population containing 437 individuals from a cross between msf and ‘hazerd’（a European greenhouse-type inbred line with short fruit）. CsaV3_1G044310 was identified as the candidate gene by genome-wide screening combined with dCAPS marker verification,which encodes a homologous protein of Arabidopsis type Ⅱ inositol polyphosphate 5-phosphatase（type Ⅱ 5 Ptase）. The quantitative expression of gene and the determination of endogenous hormones auxin and gibberellin the mutant may cause the change of hormone levels and affect the growth and development of plant.

Key words: cucumber, fruit length, mutant, gene mapping, type Ⅱ inositol polyphosphate 5-phosphatase;

中图分类号:

S642.2

程凤, 宋蒙飞, 曹蕾, 张孟茹, 杨志歌, 陈劲枫, 娄群峰. 黄瓜的一个中短果突变体基因的初步定位[J]. 园艺学报, 2021, 48(7): 1359-1370.

CHENG Feng, SONG Mengfei, CAO Lei, ZHANG Mengru, YANG Zhige, CHEN Jinfeng, LOU Qunfeng. Genetic Mapping for a Medium Short-fruit Mutant of Cucumber[J]. Acta Horticulturae Sinica, 2021, 48(7): 1359-1370.

图/表 10

图1 黄瓜‘长春密刺’野生型（WT）和中短果突变体（msf）果实表型特征

Fig. 1 Phenotypic characteristics of wild type and msf mutant

图2 黄瓜‘长春密刺’野生型（WT）和中短果突变体（msf）果实长度和横径

Fig. 2 Fruit length and width of‘Changchun Mici’wild type and msf mutant

图3 黄瓜‘长春密刺’野生型（WT）和突变体（msf）开花后不同时期果实细胞大小的石蜡切片观察 “**”表示与WT在0.01概率水平上的显着差异。比例尺为20 μm。

Fig. 3 Observation of paraffin sections of fruit cell size in wild type（WT）and mutant（msf）at different stages after flowering “**”indicates significant difference from WT at the 0.01 probability level. Scale bars represent 20 μm.

图4 黄瓜‘长春密刺’野生型（WT）和突变体（msf）花后16 d果实表皮和果肉细胞的扫描电镜观察

Fig. 4 Scanning electron microscope observation of epidermis cells and pulp cells in wild type（WT）and mutant（msf）fruit at 16 d after flowering

表1 中短果突变体和野生型黄瓜的表型数据统计

Table 1 Phenotypic data for short fruit mutant and wild-type cucumber

群体	总株数	长果株数	中短果株数	期望比	χ²	P
Population	Total	Long fruit	Medium Short fruits	Expected ratio	χ²	P
P1（WT）	20	20	0
P2（msf）	20	0	20
F₁	40	40	0
F₂	447	339	108	3︰1	0.17	0.68

图5 msf基因定位 A：两个子代混池∆（All-index）在7条染色体上的分布;（每1个点代表1个SNP位点,不同染色体间用不同颜色区分;黑色的线是SNP-index均值线,蓝色的线为95%阈值线,紫色的线为99%阈值线）;B：两个子代混池∆（All-index）在1号染色体上的分布;（每1个点代表1个SNP位点,红色的线是SNP-index均值线,绿色的线为95%阈值线,紫色的线为99%阈值线）C：利用437株msf和‘hazerd’构建的F2群体在1号染色体定位msf基因。

Fig. 5 Mapping of the msf gene A：Distribution of ∆（All-index）in two pools on seven chromosomes.（Each point represents a SNP site,and different chromosomes are distinguished by different colors;the black line is the SNP-index mean line,the blue line is the 95% threshold line,and the purple line is the 99% threshold line）;B：Distribution of ∆（All-index）in two pools on chromosome 1.（Each point represents a SNP site,the red line is the SNP-index mean line,the green line is the 95% threshold line, and the purple line is the 99% threshold line）;C：The msf gene was mapped to a region on chromosome 1 in an F2 population containing 437 individuals from a cross between msf and‘hazerd’.

表2 黄瓜1号染色体目标区域内的候选位点

Table 2 Candidate loci in the target region on chromosome 1

位置/bp Position	参考基因组 REF	碱基Base		SNP指数 SNP-index		类型 Category	氨基酸变化 Amino acid change
		野生型	变异	野生池	突变池
		WT	ALT	WT-pool	msf-pool
28 730 253	T	T	A	0.28	0.96	基因间Intergenic
28 790 458	A	A	T	0.28	0.91	上游 Upstream
29 026 627	G	G	C	0.25	1	基因间Intergenic
29 045 423	G	G	A	0.23	0.93	基因间Intergenic
29 057 944	C	C	T	0.21	0.96	基因间Intergenic
29 114 093	T	T	G	0.29	0.97	基因间 Intergenic
29 380 231	G	G	A	0.26	0.92	基因间 Intergenic
29 440 371	C	C	G	0.25	0.9	内含子Intronic
29 501 452	G	G	A	0.19	1	内含子Intronic
29 567 774	G	G	C	0.27	1	外显子Exonic	谷氨酰胺到谷氨酸 Q to E
29 647 321	G	G	A	0.26	0.9	基因间Intergenic
29 765 691	G	G	A	0.25	0.96	内含子Intronic
29 825 348	A	A	C	0.24	0.95	基因间Intergenic

图6 黄瓜‘长春密刺’野生型（WT）和突变体（msf）不同组织中候选基因CsaV3_1G044310的相对表达量 ** P < 0.01.

Fig. 6 Relative expression level of CsaV3_1G044310 in different tissues of WT and msf plant

图7 候选基因结构图（A）和蛋白结构域（B）箭头为突变位置。

Fig. 7 Candidate gene structure（A）and protein domain（B） The arrow is the position of mutation.

图8 黄瓜‘长春密刺’野生型（WT）和突变体（msf）不同组织中生长素和赤霉素的含量 ** P < 0.01.

Fig. 8 Endogenous IAA and GA content in different tissues of WT and mutant msf plants

参考文献 22

[1]	Avila L M, Cerrudo D, Swanton C, Lukens L. 2016. Brevis plant1,a putative inositol polyphosphate 5-phosphatase,is required for internode elongation in maize. Journal of Experimental Botany, 67(5):1577-1588. doi: 10.1093/jxb/erv554 pmid: 26767748
[2]	Che G, Zhang X. 2019. Molecular basis of cucumber fruit domestication. Current Opinion in Plant Biology, 47:38-46. doi: 10.1016/j.pbi.2018.08.006 URL
[3]	Chen Y, Hou M, Liu L, Wu S, Shen Y, Ishiyama K, Kobayashi M, McCarty D R, Tan B C. 2014. The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol. Plant Physiology, 166(4):2028-2039. doi: 10.1104/pp.114.247486 URL pmid: 25341533
[4]	Chen Y, Yang Q, Sang S, Wei Z, Wang P. 2017. Rice inositol polyphosphate kinase(OsIPK2)directly interacts with OsIAA11 to regulate lateral root formation. Plant Cell Physiology, 58(11):1891-1900. doi: 10.1093/pcp/pcx125 URL
[5]	Colle M, Weng Y, Kang Y, Ophir R, Sherman A, Grumet R. 2017. Variation in cucumber(Cucumis sativus L.)fruit size and shape results from multiple components acting pre-anthesis and post-pollination. Planta, 246(4):641-658. doi: 10.1007/s00425-017-2721-9 URL
[6]	Daviere J M, Achard P. 2013. Gibberellin signaling in plants. Development, 140:1147-1151. doi: 10.1242/dev.087650 URL
[7]	Gao Z, Zhang H, Cao C, Han J, Li H, Ren Z. 2020. QTL mapping for cucumber fruit size and shape with populations from long and round fruited inbred lines. Horticultural Plant Journal, 6(3):132-144. doi: 10.1016/j.hpj.2020.04.004 URL
[8]	Lin W H, Wang Y, Mueller-Roeber B, Brearley C A, Xu Z H, Xue H W. 2005. At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis. Plant Physiology, 139(4):1677-1691. doi: 10.1104/pp.105.067140 URL
[9]	Liu Xingwang, Zhai Xuling, Zhang Yaqi, Yin Shuai, Feng Zhongxuan, Ren Huazhong. 2020. A review on genetic and molecular biology of fruit morphogenesis in cucumber. Acta Horticulturae Sinica, 47(9):1793-1809. (in Chinese)
	刘兴旺, 翟许玲, 张亚琦, 尹帅, 冯钟萱, 任华中. 2020. 黄瓜果实形态建成的遗传及分子基础研究进展. 园艺学报, 47(9):1793-1809.
[10]	Pan Y, Liang X, Gao M, Liu H, Meng H, Weng Y, Cheng Z. 2017. Round fruit shape in WI7239 cucumber is controlled by two interacting quantitative trait loci with one putatively encoding a tomato SUN homolog. Theoretical and Applied Genetics, 130(3):573-586. doi: 10.1007/s00122-016-2836-6 URL
[11]	Qi J, Liu X, Shen D, Miao H, Xie B, Li X, Zeng P, Wang S, Shang Y, Gu X, Du Y, Li Y, Lin T, Yuan J, Yang X, Chen J, Chen H, Xiong X, Huang K, Fei Z, Mao L, Tian L, Städler T, Renner S S, Kamoun S, Lucas W J, Zhang Z, Huang S. 2013. A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nature Genetics, 45(12):1510-1515. doi: 10.1038/ng.2801 URL
[12]	Qi X, Zhu Y, Li S, Zhou H, Xu X, Xu Q, Chen X. 2020. Identification of genes related to mesocarp development in cucumber. Horticultural Plant Journal, 6(5):293-300. doi: 10.1016/j.hpj.2020.08.001 URL
[13]	Sato-Izawa K, Nakaba S, Tamura K, Yamagishi Y, Nakano Y, Nishikubo N, Kawai S, Kajita S, Ashikari M, Funada R, Katayama Y, Kitano H . 2012. DWARF50(D50),a rice(Oryza sativa L.)gene encoding inositol polyphosphate 5-phosphatase,is required for proper development of intercalary meristem. Plant Cell Environ, 35(11):2031-2044. doi: 10.1111/j.1365-3040.2012.02534.x URL
[14]	Wang Y, Deng D, Ding H, Xu X, Zhang R, Wang S, Bian Y, Yin Z, Chen Y . 2013. Gibberellin biosynthetic deficiency is responsible for maize dominant Dwarf11 (D11) mutant phenotype:physiological and transcriptomic evidence. PLoS ONE, 8(6):e66466. doi: 10.1371/journal.pone.0066466 URL
[15]	Wei Q, Wang Y, Qin X, Zhang Y, Zhang Z, Wang J, Li J, Lou Q, Chen J. 2014. An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment(SLAF)sequencing. BMC Genomics, 15(1):1158. doi: 10.1186/1471-2164-15-1158 URL
[16]	Weng Y, Colle M, Wang Y, Yang L, Rubinstein M, Sherman A, Ophir R, Grumet R. 2015. QTL mapping in multiple populations and development stages reveals dynamic quantitative trait loci for fruit size in cucumbers of different market classes. Theoretical and Applied Genetics, 128(9):1747-1763. doi: 10.1007/s00122-015-2544-7 URL
[17]	Whisstock J C, Wiradjaja F, Waters J E, Gurung R. 2002. The structure and function of catalytic domains within inositol polyphosphate 5-phosphatases. IUBMB Life, 53(1):15-23. pmid: 12018403
[18]	Xin T, Zhang Z, Li S, Zhang S, Li Q, Zhang Z H, Huang S, Yang X. 2019. Genetic regulation of ethylene dosage for cucumber fruit elongation. The Plant Cell, 31(5):1063-1076. doi: 10.1105/tpc.18.00957 URL
[19]	Xu X, Wei C, Liu Q, Qu W, Qi X, Xu Q, Chen X. 2020. The major-effect quantitative trait locus Fnl7.1 encodes a late embryogenesis abundant protein associated with fruit neck length in cucumber. Plant Biotechnology Journal, 18(7):1598-1609. doi: 10.1111/pbi.v18.7 URL
[20]	Zhang Z, Wang B, Wang S, Lin T, Yang L, Zhao Z, Zhang Z, Huang S, Yang X. 2020. Genome-wide Target Mapping Shows Histone Deacetylase Complex1 Regulates Cell Proliferation in Cucumber Fruit. Plant Physiology, 182(1):167-184. doi: 10.1104/pp.19.00532 pmid: 31378719
[21]	Zhao J, Jiang L, Che G, Pan Y, Li Y, Hou Y, Zhao W, Zhong Y, Ding L, Yan S, Sun C, Liu R, Yan L, Wu T, Li X, Weng Y, Zhang X. 2019. A functional allele of CsFUL1 regulates fruit length through repressing CsSUP and inhibiting auxin transport in Cucumber. The Plant Cell, 31(6):1289-1307. doi: 10.1105/tpc.18.00905 URL
[22]	Zhong R, Burk D H, Morrison W H 3rd, Ye Z H. 2004. FRAGILE FIBER3,an Arabidopsis gene encoding a typeⅡinositol polyphosphate 5-phosphatase,is required for secondary wall synthesis and actin organization in fiber cells. The Plant Cell, 16(12):3242-3259. doi: 10.1105/tpc.104.027466 URL