甘蓝型油菜光叶性状的遗传分析及基因定位

doi:10.3864/j.issn.0578-1752.2024.04.003

中国农业科学 ›› 2024, Vol. 57 ›› Issue (4): 650-662.doi: 10.3864/j.issn.0578-1752.2024.04.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

甘蓝型油菜光叶性状的遗传分析及基因定位

关志林¹^,²(), 靳丰蔚¹, 刘婷婷¹, 王毅¹, 谭莹莹², 杨春慧², 李蕊彤², 王博³, 刘克德², 董云¹()

¹ 甘肃省农业科学院作物研究所，兰州 730070
² 华中农业大学作物遗传改良全国重点实验室，武汉 430070
³ 武汉基诺赛克科技有限公司，武汉 430070

收稿日期:2023-07-14 接受日期:2023-08-30 出版日期:2024-02-16 发布日期:2024-02-20
通信作者:
董云，E-mail：dongyungs@163.com。
联系方式: 关志林，E-mail：zhilinggg@163.com。
基金资助:
甘肃省科技计划重大项目(21ZD4NA022); 国家自然科学基金(31960436); 甘肃省农业科学院院列项目(2022GAAS54); 甘肃省农业科学院院列项目(2021GAAS13); 甘肃省农业科学院院列项目(2021GAAS43); 甘肃省农业科学院院列项目(2023GAAS43)

Genetic Analysis and Gene Mapping of Glossy Leaf in Brassica napus

GUAN ZhiLin¹^,²(), JIN FengWei¹, LIU TingTing¹, WANG Yi¹, TAN YingYing², YANG ChunHui², LI RuiTong², WANG Bo³, LIU KeDe², DONG Yun¹()

¹ Crop Research Institute of Gansu Academy of Agricultural Sciences, Lanzhou 730070
² National Key Laboratory of Crop Genetic and Improvement, Huazhong Agricultural University, Wuhan 430070
³ Wuhan Genoseq Technology Co., Ltd., Wuhan 430070

Received:2023-07-14 Accepted:2023-08-30 Published:2024-02-16 Online:2024-02-20

摘要/Abstract

摘要：

【目的】表皮蜡质是覆盖植物叶片和茎秆等部位的疏水层，在植物抵御逆境方面发挥着重要作用，同时会影响光合作用和生长发育;甘蓝型油菜作为世界上主要的油料作物之一，研究表皮蜡质突变体的遗传机制，为实现油菜高产稳产提供参考。【方法】对油菜光叶突变体M8和普通蜡质叶中双11（ZS11）和C20的叶片表征进行记载，使用便携式植物光合作用测量系统测定叶片光合速率;利用M8和ZS11、C20分别杂交获得2个F₁、自交构建2个F₂分离群体，用于分析油菜光叶性状的遗传规律;选取M8和ZS11杂交的F₂群体中光叶和蜡质叶表型单株分别进行混池，通过集团分离分析法结合靶向测序技术进行基因克隆，结合比较基因组和转录组数据进行候选基因预测，并通过RT-PCR验证候选基因。【结果】甘蓝型油菜光叶表型叶片气孔导度更大、光合效率更高;遗传分析表明M8光叶性状受1对基因控制，光叶相对蜡质叶为隐性。图位克隆将光叶控制基因定位至A08染色体0.134-0.699 Mb物理区间内。进一步分析发现，相比于ZS11，光叶突变体M8中A08染色体0.22-0.58 Mb区间存在大片段缺失，ZS11在该区段中的BnaA08G0006900ZS（BnaA08.SAGL1）可能为光叶的候选基因，该基因编码一个Kelch-F-box蛋白，在ZS11叶片中表达量高而M8中未检测到，该基因缺失可能导致了M8光叶表型。【结论】甘蓝型油菜光叶新突变体M8相比野生型蜡质叶片光合速率更高，该光叶表型受1对隐性基因控制;图位克隆鉴定到光叶性状调控基因为BnaA08.SAGL1，基因缺失产生了光叶表型。

关键词: 甘蓝型油菜, 光叶, 蜡质, 图位克隆, BnaA08.SAGL1

Abstract:

【Objective】Epicuticular waxes is a hydrophobic layer covering plant leaves and stems, playing an important role in stress resistance and affecting photosynthesis, growth and development. Rapeseed (Brassica napus L.) is one of the main oil crops in the world, but the mechanism of epicuticular waxes formation on its leaves is still unclear. This study aims to reveal the genetic mechanism of epicuticular wax on leaves through a mutant, which will help to achieve high and stable yield of rapeseed.【Method】In this study, leaf of glossy leaf mutant M8 and common waxy leaf inbred lines Zhongshuang 11 (ZS11) and the C20 were characterized, and its photosynthetic rate were measured by portable plant photosynthesis measurement system. Two F₁ hybrids and F₂ segregating populations were constructed by crossing the M8 with ZS11 and C20, respectively, and were used to analyze the heredity of leaf characters in rapeseed. The F₂ population hybridized by M8 and ZS11 was constructed to analyze the genetic regulation of the glossy leaf trait in rapeseed by bulked segregation analysis (BSA) combined with Target-sequencing (Target-seq). Combined with comparative genome and transcriptome database, candidate genes were predicted and then verified by RT-PCR.【Result】The leaf stomatal conductance and photosynthetic efficiency were higher in the glossy leaf mutant M8, compared with ZS11 in rapeseed. The genetic analysis results showed that the glossy leaf was controlled by one pair of recessive genes and glossy leaf was recessive compared with waxy leaf. It was finally fine mapped in the physical region of 0.134-0.699 Mb on chromosome A08 by map based cloning method. Further analysis revealed that there was a large segmental deletion in the 0.22-0.58 Mb region of chromosome A08 in the M8 glossy leaf mutant compared to ZS11, and BnaA08G0006900ZS (BnaA08.SAGL1) in this region of the genome was identified as the candidate gene for the glossy leaf trait. This gene encodes a Kelch-F-box protein, and its deletion may lead to the observed glossy leaf trait in the M8 mutant, as it was highly expressed in ZS11 but none in M8. 【Conclusion】Compared with wild-type waxy leaves, the photosynthetic rate of the new glossy leaf mutant M8 was higher, and the glossy leaf phenotype was controlled by a recessive gene. In the picture, the glossy leaf phenotype regulatory gene was identified as BnaA08.SAGL1, and deletion of the gene resulted in glossy leaf in rapeseed.

Key words: Brassica napus L., glossy leaf, epicuticular wax, map-based gene cloning, BnaA08.SAGL1

关志林, 靳丰蔚, 刘婷婷, 王毅, 谭莹莹, 杨春慧, 李蕊彤, 王博, 刘克德, 董云. 甘蓝型油菜光叶性状的遗传分析及基因定位[J]. 中国农业科学, 2024, 57(4): 650-662.

GUAN ZhiLin, JIN FengWei, LIU TingTing, WANG Yi, TAN YingYing, YANG ChunHui, LI RuiTong, WANG Bo, LIU KeDe, DONG Yun. Genetic Analysis and Gene Mapping of Glossy Leaf in Brassica napus[J]. Scientia Agricultura Sinica, 2024, 57(4): 650-662.

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杂交组合 Combination	父本 Male parent	母本 Female parent	世代 Generation	光叶 Glossy leaf	蜡质叶 Waxy leaf	期望分离 Expect separation	卡方值 Chi-square value (χ² _0.05=3.84)
组合1 Group 1	M8	ZS11	F₁	0	15
组合1 Group 1	M8	ZS11	F₂	95	348	1﹕3	2.80
组合2 Group 2	M8	C20	F₁	0	13
组合2 Group 2	M8	C20	F₂	109	275	1﹕3	2.17

标记 Marker	物理位置 Physical position (bp)	参考基因型 Reference (ZS11)	变异类型 Alternative (M8)	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	产物大小 Product size (bp)
P1	133863	C	T	TTACAGCAACGAAGTATGGTGTTATTTCACTT	AGTTTAAGTCTCATCAAACTCTTGATTTCTCC	249
P2	260976	A	G	GATGATCGTGCCAAGGAGTACGTAAG	AACAAAGAAAGAGAATAGTCTAGGTAACTTGC	238
P3	374500	A	T	TACTTCTTCTATACATGGAACGTGACTTATGG	GGCAACCCTATTAAGATCTACTAAGACCA	240
P4	495084	C	T	GTTATAAGTTGTGCTGATGTTTTTAAGTTGCT	GTTCTATGCTTCTTCTTGCCATATCTTTTGAC	249
P5	698791	C	G	GCATATTCACAGGAACATTAAGAAAATGTGTT	TCGGTTAGTTTGATTTGAAATTTGGTTAGTTC	241
P6	791230	A	G	CCCTGAAGGTAAAAGACAGTAAACAGATTTAG	AGCTCTCTTGCTGGTCTTCTATTAATCTT	252
P7	926323	G	C	CTTGACGAACGATGATAGTTTGAAGAGGAT	ATACAAGTTAAAGAAAGAGACTTTGCTCTGTT	265
P8	1105382	A	T	ATAATCGCTACCGATCAAGACGATATAAAGTT	TTTCGTAGTAAACATAACCTTGATATTTTGCG	249
P9	1213167	A	G	AATAAGTCTAATCTCCCGTTACCTTTGAGATG	AATCTTAAGTATGTGTTGGTGTTTTGTTTCAG	240
P10	1321158	A	C	CAGCACATCTTTGTCAAAATCAGATGAGAAAT	GATCATAAGCTATTGATGTAACTACGAGAAGC	266
P11	2025748	T	C	ACTTCTTTTATTCCATTCCCAATCAATTTTGT	GAGAGGTAGAGAACTTGGTCGATGG	230
P12	2315309	T	C	TAGAAGTAATTATCTAGACAACGGAGGAACA	AAACATAACAATTAAGTTATCGAGACATGGGG	238
P13	2400688	G	T	ACAACACAGTTCCCTTATTCAAAAACTTATCT	AAGTACTAATCTTTGTTGAATCTGTGGTCTTC	230
P14	2493038	T	C	GAAAACAAAACTAAAATTGTTTGGAATGTGG	GATAATTCTACCATCATATGAGAATGCACGTC	255
P15	2579076	G	T	ATGTGGTTCTAATTTGCAATGCTACATTTCTG	TTATTCACCCTATAGTTGATTTTGGTTGACAG	230
P16	2725088	A	G	GTTTATTTTATATTAGGCATGGGCATTTCACC	TATACTAGAAGTTTCAGATTTTCGGATACCCT	191

片段 Fragment	物理位置 Physical position (bp)	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	产物大小 Product size (bp)
Fr.1	A08:229280..230322	CTTCCTACGATCACTGCGATTG	CATCGTCTACTGCATAGCTGAGAAT	1042
Fr.2	A08:262560..263824	GCTTCTACCACGTACCCACTTC	TTGGCAACTCGCACGTCT	1264
Fr.3	A08:311145..312544	CATATTCCTCGGTCAACATTGTC	CGAAATTGTATCCACCTGCAT	1399
Fr.4	A08:351515..353298	TTGATTCGGGCTAGCTAAAAGG	TGTTGACACGATTTAGCCTTAACC	1783
Fr.5	A08:395942..396849	TCAGATTCTCACTCTCACCATTG	ATCCAAGTGTGCCATACAGAAC	907
Fr.6	A08:450159..452784	GATTAAATCCTTGGGTTGCAC	GACGGCGTGTGATTCAGAAGC	2625
Fr.7	A08:482496..484254	TATCGTCTAACTCAGCTCCAAGTG	GGTTCTAAGGTCATAGCGTCCAT	1758
Fr.8	A08:585642..586731	GGGCGTGATGAACTCTTTCTAT	GCTCATGCCGCTTCTCAAC	1089
Fr.9	A08:631190..632007	TCATCCGCCTCGCATTGTG	TCATTATTGACAAGTGCATACGTG	817

引物名称 Primer name	引物序列 Primer sequence (5′-3′)
Actin-BnENTH-F	GTTTAGACCCGTTGCTGCTC
Actin-BnENTH-R	TTGTCCATCTCAGCCATTTG
qBnaA08.SAGL1-F	ACCTGATATGCTCAGGAAGAGAC
qBnaA08.SAGL1-R	CATCACTAACCGGACTCCATG
qBnaC03.SAGL1-F	TATGGATTGGATTGTAGGGACG
qBnaC03.SAGL1-R	TTACTGAGAATGCCCTTTGACT
qBnaA02.CER3-F	TGCGAAGAGACTCTTTTGTTTG
qBnaA02.CER3-R	TACATTTTGTTTCCCTGCTTGC
qBnaC02.CER3-F	CGTTTACAACAACATGCTTTGG
qBnaC02.CER3-R	TTCGTGATCAATCTGTTGAACG

甘蓝型油菜光叶性状的遗传分析及基因定位

Genetic Analysis and Gene Mapping of Glossy Leaf in Brassica napus

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