Ogura CMS青花菜育性恢复材料创制及其遗传背景研究

doi:10.16420/j.issn.0513-353x.2021-0100

园艺学报 ›› 2022, Vol. 49 ›› Issue (3): 533-547.doi: 10.16420/j.issn.0513-353x.2021-0100

Ogura CMS青花菜育性恢复材料创制及其遗传背景研究

黄静静¹, 刘伟², 刘玉梅¹, 韩风庆¹, 方智远¹, 杨丽梅¹, 庄木¹, 张扬勇¹, 吕红豪¹, 王勇¹, 季家磊¹, 李占省¹^,^*()

¹中国农业科学院蔬菜花卉研究所,农业部园艺作物生物学与种质创制重点实验室,北京 100081
²中蔬生物科技（寿光）有限公司,山东寿光 262700

收稿日期:2021-03-08 修回日期:2021-11-08 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 李占省 E-mail:lizhansheng@caas.cn
基金资助:
国家重点研发计划项目(2017YFD0101805);国家现代农业产业技术体系建设专项资金项目(CARS-23-A05);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS)

Creation of Fertility Restorer Materials for Ogura CMS Broccoli and the Study of Its Genetic Background

HUANG Jingjing¹, LIU Wei², LIU Yumei¹, HAN Fengqing¹, FANG Zhiyuan¹, YANG Limei¹, ZHUANG Mu¹, ZHANG Yangyong¹, LÜ Honghao¹, WANG Yong¹, JI Jialei¹, LI Zhansheng¹^,^*()

¹Key Laboratory of Horticultural Crop Biology and Germplasm Creation,Ministry of Agriculture,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China
²China Vegetable Biotechnology Shouguang Co.,Ltd,Shouguang,Shandong 262700,China

Received:2021-03-08 Revised:2021-11-08 Online:2022-03-25 Published:2022-03-25
Contact: LI Zhansheng E-mail:lizhansheng@caas.cn

摘要/Abstract

摘要：

利用含有育性恢复基因Rfo的芥蓝中间材料（14Y12）,通过杂交转育途径将该基因导入6份优良Ogura CMS青花菜中,并从农艺性状、细胞水平和遗传多样性方面揭示其变化特征及遗传背景。分子鉴定结果表明,Ogura CMS青花菜Rfo杂交后代（F₁代）中,14个株系含有Rfo基因,Rfo基因传递效率约为40%。在农艺性状方面,Rfo转育后代农艺性状偏向青花菜,且遗传分离明显。在细胞水平方面,Rfo转育后代花粉活力（70% ~ 85%）存在显著差异（P < 0.05）;减数分裂后期有染色体不均等分裂和染色体丢失的异常现象;部分Rfo转育后代的花粉管能够正常生长,而一些后代花粉管发育受阻或不正常;倍性鉴定表明,Rfo转育后代中二倍体12份,占总体比例54.55%,介于三倍体和四倍体的杂合倍性10份,占总体比例45.45%。遗传分析结果表明,在遗传相似系数0.50处,芥蓝14Y12、青花菜17B1253及Rfo转育后代可分为两大亚群：A亚群有10份,遗传背景偏向芥蓝;B亚群有22份,全部为青花菜。Rfo转育后代与芥蓝遗传相似系数为0.24 ~ 0.88,其中19B809-8单株与芥蓝遗传背景最远,遗传相似系数为0.24,可作为Ogura类型青花菜育性恢复的亲本材料。

关键词: 青花菜, Ogura CMS, 育性恢复, 遗传背景

Abstract:

The Chinese kale material（14Y12）containing the Ogura CMS fertility restorer gene Rfo was used to introduce the Rfo restorer gene into six excellent Ogura CMS broccoli through cross breeding,and revealed its change characteristics and genetic background from the aspects of agronomic traits,cell level and genetic diversity. The results showed that the agronomic traits of Ogura CMS broccoli hybrids（F₁ generation）were biased towards broccoli and genetic segregation was obvious. At the cellular level,there was a significant difference in the pollen viability of Rfo-transformed offsprings（P < 0.05）,and the range was between 70% and 85%. There were abnormal phenomena of unequal division of chromosomes and loss of chromosomes in the late stage of meiosis. The pollen tubes of some Rfo-transformed offspring could be formed normally,while the pollen tube development of some Rfo-transformed offspring was blocked or abnormal. The ploidy identification showed that there were 12 diploids in Rfo-transformed offspring,accounting for 54.55% of the total,and 10 heterozygous ploidy between triploid and tetraploid,accounting for 45.45% of the total. The results of genetic analysis showed that at the genetic similarity coefficient of 0.50. Chinese kale 14Y12,broccoli 17B1253 and their Rfo-transformed offspring could be divided into two categories：A subgroup had 10 copies,with a genetic background biased toward Chinese kale;B subgroup had 22 copies,all of which are broccoli. The genetic similarity coefficient between Rfo-transformed offspring and Chinese kale was from 0.24 to 0.88,among which 19B809-8 had the farthest genetic background to Chinese kale,and the genetic similarity coefficient was 0.24,which could be used as the parent material for the fertility restoration of Ogura broccoli.

Key words: broccoli, Ogura CMS, fertility restorer, genetic background

中图分类号:

S635.9

黄静静, 刘伟, 刘玉梅, 韩风庆, 方智远, 杨丽梅, 庄木, 张扬勇, 吕红豪, 王勇, 季家磊, 李占省. Ogura CMS青花菜育性恢复材料创制及其遗传背景研究[J]. 园艺学报, 2022, 49(3): 533-547.

HUANG Jingjing, LIU Wei, LIU Yumei, HAN Fengqing, FANG Zhiyuan, YANG Limei, ZHUANG Mu, ZHANG Yangyong, LÜ Honghao, WANG Yong, JI Jialei, LI Zhansheng. Creation of Fertility Restorer Materials for Ogura CMS Broccoli and the Study of Its Genetic Background[J]. Acta Horticulturae Sinica, 2022, 49(3): 533-547.

图/表 12

参考文献 36

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引物名 Primer name	引物序列（5′-3′） Primer sequence	扩增长度大小/bp Amplification length	退火温度/℃ Melting temperature
BoRfo-2F	CAAGTGTTCTTCTTCTCCTGC	250	57
BoRfo-2R	CCATCTTCCATCATCCGA	250	57
BoRfo-6F	AGAGTTGCGAAGCAGGTT	300	55
BoRfo-6R	CCATAAGTAATCTGGGTAGGCT	300	55
BnRfo-AS2F	CATGCTTCGATCTCGTCCTTTA	400	57
BnRfo-AS2R	TACGACATTGGGCCTACATGTC	400	57
Bo138F	CCATGGACAATAATCTTAGT	250	55
Bo138R	TAATGTGCATGAAGTTGAAG	250	55

引物名 Primer name	引物序列（5′-3′） Primer sequence	扩增长度大小/bp Amplification length	退火温度/℃ Melting temperature
BoRfo-2F	CAAGTGTTCTTCTTCTCCTGC	250	57
BoRfo-2R	CCATCTTCCATCATCCGA	250	57
BoRfo-6F	AGAGTTGCGAAGCAGGTT	300	55
BoRfo-6R	CCATAAGTAATCTGGGTAGGCT	300	55
BnRfo-AS2F	CATGCTTCGATCTCGTCCTTTA	400	57
BnRfo-AS2R	TACGACATTGGGCCTACATGTC	400	57
Bo138F	CCATGGACAATAATCTTAGT	250	55
Bo138R	TAATGTGCATGAAGTTGAAG	250	55

引物名 Primer name	引物序列（5′-3′） Primer sequence	扩增片段长度/bp Amplified fragment length
SNP894-F	AAAAATTTCTAACCATATTCTGCATTTGTATTAAGA	250 ~ 500
SNP894-R	GTGATTTTATAGTGGTTATTCCATTTAGGGAG
SNP1743-F	GCCTTGTCCACGTTTTTGATCAGAGTTACA	100 ~ 250
SNP1743-R	ACGGGTGATAGGATCTTAACCTGGAATATGG
SNP1770-F	AAATTCCATGTTCACACGGACCAGAAAA	100 ~ 250
SNP1770 -R	TGAACTTAGAGGATTTCGGAATCACGAGC
SNP1864-F	CACCGGAGAACGTCCCCTGACA	100 ~ 750
SNP1864-R	CTCTTTTGCTTCTTCTTGAAGCTTCCTTGC
LTSSR13-F	TCATCCTCCATACCTCTCCG	250 ~ 750
LTSSR13-R	CCGGTTCAGGTTGAAGAAAA
LTSSR81-F	GTCTCGCCATGTCTCCTCTC	250 ~ 750
LTSSR81-R	TGGCATCACACTAGCTACGC
LTSSR94-F	GCGAAACCAAGAGAGGAGAG	50 ~ 1 000
LTSSR94-R	CAGAGGAAGGGGAGGAAGAG	50 ~ 1 000
LTSSR132-F	CCAGAAGGAACCAGCAAGAG	250 ~ 750
LTSSR132-R	AAATGTCAATGCCACTGCAA	250 ~ 750
BoE134-F	CTCTTATTTCTTGTAGGGCTTTTA	250 ~ 750
BoE134-R	CCGTTGGAGATGACTGACTG	250 ~ 750
LTSSR180-F	TGAATATGATAGCGGAGGGG	100 ~ 500
LTSSR180-R	AACGGGGGAACGTTTAGATT	100 ~ 500
LTSSR188-F	AGACAGCGGAGACGGTAATG	50 ~ 500
LTSSR188-F	CGATAGAGAGATGGGCAACG	50 ~ 500
LTSSR304-F	AAACGAACCACTTTGTTGCC	250 ~ 500
LTSSR304-R	CTTCTGTTTCTTCTTCGCCG	250 ~ 500
LTSSR340-F	GCGACTTCCACTCCTCACTC	100 ~ 500
LTSSR340-R	GAAGACAGCAAAGGACCAGC	100 ~ 500
LTSSR349-F	TTCAACGGCAAACATGTGAT	250 ~ 750
LTSSR349-R	ACCCACCATTGAACAAGAGC	250 ~ 750
BoE450-F	TCTCGCCATGGCTGATAAG	250 ~ 750
BoE450-R	TCGGGGCGTTGATTCTCGTATAT
LTSSR484-F	AAACCAGAGCCAAAAGCAAA	250 ~ 1 000
LTSSR484-R	GTGGTTATGGAAGTGGTGGG
LTSSR544-F	CGGGGGTTTATTAGGGAAAA	100 ~ 500
LTSSR544-R	AGACTGTGGCGCTTTTTGTT
LTSSR584-F	GCCACCGTTGTAAAAGTGCT	250 ~ 750
LTSSR584-R	TCATCATCATCGTCCCTTGA
LTSSR688-F	ATTTTCATGCGTGTGTTCCA	250 ~ 750
LTSSR688-R	ACCACCCCAATCTCGTTACA
LTSSR712-F	GGATTGGTAATGGTTGGTCTG	100 ~ 500
LTSSR712-R	AAGCATCCGTGGTCGTTAAG
LTSSR785-F	TCAAGGCAACTGTGAACCAA	100 ~ 500
LTSSR785-R	GTGATCGCCTTATCCTTTCG
BoSF2313-F	AAGGAGGATCACGAGGAGGT	250 ~ 1 000
BoSF2313-R	CATGGTAGCATCGAAAGCCT
BoSF2421-F	CACTCAGAGGAGGAGGTTGC	250 ~ 750
BoSF2421-F	GCCACGTGTAGGCATGTAGA
BoSF2435-F	CCCACAATTCGGTATTCACC	250 ~ 1 000
BoSF2435-R	GTCTTGCACCACCGAAAGAT
BoSF2587-F	AGCTTCTCGCCCTGACAATA	100 ~ 500
BoSF2587-F	ACCGGCATCAAATCTCAATC
BoSF2733-F	CTCCGGTCATTGATATTGGC	100 ~ 500
BoSF2733-F	GCCTTTTTGGTGCATGTTTT
BoSF2749-F	GCCAAGACTTCACGGTCATT	100 ~ 500
BoSF2749-F	TGTCTGTCTGGTCCCTCTTT
BoSF2860-F	CATGCTTGCCTGAAAAGACA	250 ~ 1 000
BoSF2860-F	CCTTGTACTGCTCCTCTGCC

引物名 Primer name	引物序列（5′-3′） Primer sequence	扩增片段长度/bp Amplified fragment length
SNP894-F	AAAAATTTCTAACCATATTCTGCATTTGTATTAAGA	250 ~ 500
SNP894-R	GTGATTTTATAGTGGTTATTCCATTTAGGGAG
SNP1743-F	GCCTTGTCCACGTTTTTGATCAGAGTTACA	100 ~ 250
SNP1743-R	ACGGGTGATAGGATCTTAACCTGGAATATGG
SNP1770-F	AAATTCCATGTTCACACGGACCAGAAAA	100 ~ 250
SNP1770 -R	TGAACTTAGAGGATTTCGGAATCACGAGC
SNP1864-F	CACCGGAGAACGTCCCCTGACA	100 ~ 750
SNP1864-R	CTCTTTTGCTTCTTCTTGAAGCTTCCTTGC
LTSSR13-F	TCATCCTCCATACCTCTCCG	250 ~ 750
LTSSR13-R	CCGGTTCAGGTTGAAGAAAA
LTSSR81-F	GTCTCGCCATGTCTCCTCTC	250 ~ 750
LTSSR81-R	TGGCATCACACTAGCTACGC
LTSSR94-F	GCGAAACCAAGAGAGGAGAG	50 ~ 1 000
LTSSR94-R	CAGAGGAAGGGGAGGAAGAG	50 ~ 1 000
LTSSR132-F	CCAGAAGGAACCAGCAAGAG	250 ~ 750
LTSSR132-R	AAATGTCAATGCCACTGCAA	250 ~ 750
BoE134-F	CTCTTATTTCTTGTAGGGCTTTTA	250 ~ 750
BoE134-R	CCGTTGGAGATGACTGACTG	250 ~ 750
LTSSR180-F	TGAATATGATAGCGGAGGGG	100 ~ 500
LTSSR180-R	AACGGGGGAACGTTTAGATT	100 ~ 500
LTSSR188-F	AGACAGCGGAGACGGTAATG	50 ~ 500
LTSSR188-F	CGATAGAGAGATGGGCAACG	50 ~ 500
LTSSR304-F	AAACGAACCACTTTGTTGCC	250 ~ 500
LTSSR304-R	CTTCTGTTTCTTCTTCGCCG	250 ~ 500
LTSSR340-F	GCGACTTCCACTCCTCACTC	100 ~ 500
LTSSR340-R	GAAGACAGCAAAGGACCAGC	100 ~ 500
LTSSR349-F	TTCAACGGCAAACATGTGAT	250 ~ 750
LTSSR349-R	ACCCACCATTGAACAAGAGC	250 ~ 750
BoE450-F	TCTCGCCATGGCTGATAAG	250 ~ 750
BoE450-R	TCGGGGCGTTGATTCTCGTATAT
LTSSR484-F	AAACCAGAGCCAAAAGCAAA	250 ~ 1 000
LTSSR484-R	GTGGTTATGGAAGTGGTGGG
LTSSR544-F	CGGGGGTTTATTAGGGAAAA	100 ~ 500
LTSSR544-R	AGACTGTGGCGCTTTTTGTT
LTSSR584-F	GCCACCGTTGTAAAAGTGCT	250 ~ 750
LTSSR584-R	TCATCATCATCGTCCCTTGA
LTSSR688-F	ATTTTCATGCGTGTGTTCCA	250 ~ 750
LTSSR688-R	ACCACCCCAATCTCGTTACA
LTSSR712-F	GGATTGGTAATGGTTGGTCTG	100 ~ 500
LTSSR712-R	AAGCATCCGTGGTCGTTAAG
LTSSR785-F	TCAAGGCAACTGTGAACCAA	100 ~ 500
LTSSR785-R	GTGATCGCCTTATCCTTTCG
BoSF2313-F	AAGGAGGATCACGAGGAGGT	250 ~ 1 000
BoSF2313-R	CATGGTAGCATCGAAAGCCT
BoSF2421-F	CACTCAGAGGAGGAGGTTGC	250 ~ 750
BoSF2421-F	GCCACGTGTAGGCATGTAGA
BoSF2435-F	CCCACAATTCGGTATTCACC	250 ~ 1 000
BoSF2435-R	GTCTTGCACCACCGAAAGAT
BoSF2587-F	AGCTTCTCGCCCTGACAATA	100 ~ 500
BoSF2587-F	ACCGGCATCAAATCTCAATC
BoSF2733-F	CTCCGGTCATTGATATTGGC	100 ~ 500
BoSF2733-F	GCCTTTTTGGTGCATGTTTT
BoSF2749-F	GCCAAGACTTCACGGTCATT	100 ~ 500
BoSF2749-F	TGTCTGTCTGGTCCCTCTTT
BoSF2860-F	CATGCTTGCCTGAAAAGACA	250 ~ 1 000
BoSF2860-F	CCTTGTACTGCTCCTCTGCC

田间编号 Field number	遗传背景组合 Genetic backcross combination	种子数 Number of seeds	成苗数 Number of viable plants
19B827	18-NH × 18B592-1	7	1
19B829、19B831、19B832	18-YX × 18B592-1	20	18
19B809	18BS-1 × 18B592-1	11	11
19B833	18BS36 × 18B592-1	6	1
19B834	18T2B2 × 18B592-1	6	3
19B835	18B1023 × 18B592-1	1	1

Ogura CMS青花菜育性恢复材料创制及其遗传背景研究

Creation of Fertility Restorer Materials for Ogura CMS Broccoli and the Study of Its Genetic Background

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样品名称 Sample name	株高/cm Plant height	基生叶 Basal leaf			中下部叶 Mid-lower leaf		茎宽/cm Stalk width	侧枝 Lateral branch
样品名称 Sample name	株高/cm Plant height	颜色 Color	性状 Traits	边缘 Margin	叶缘波纹 Edge ripple	叶面 Leaf foliage	茎宽/cm Stalk width	侧枝 Lateral branch
18B592-1	83	深绿 Dark green	柳叶形 Willow leaf shape	全缘 Entire	小 Small	平展 Smooth	2	有 Present
19B711-1	85	绿 green	卵圆 Ovate	波状 Undulant	中 Intermediate	平展 Smooth	2	无 Absent
19B711-2	90	深绿 Dark green	宽卵圆 Broad ovate	波状 Undulant	大 Big	微皱 Slightly wrinkling	3	有 Present
19B809-6	60	深绿 Dark green	宽卵圆 Broad ovate	波状 Undulant	大 Big	平展 Smooth	2	无 Absent
19B831-1	35	灰绿 Grayish green	倒卵圆 Obovate	波状 Undulant	中 Intermediate	平展 Smooth	7	有 Present
19B833-1	50	灰绿 Grayish green	卵圆 Ovate	全缘 Entire	小 Small	微皱 Slightly wrinkling	5	有 Present
19B834-2	50	灰绿 Grayish green	卵圆 Ovate	波状 Undulant	大 Big	多皱 Much wrinkling	6	无 Absent
19B834-3	45	灰绿 Grayish green	倒卵圆 Obovate	波状 Undulant	中 Intermediate	皱 Wrinkling	5	无 Absent

样品名称 Sample name	花球大小/ cm Flower ball size	球色 Ball color	花蕾大小 Flower bud size	花冠开展度/ mm Corolla development	花瓣大小/ mm Petal size	花瓣颜色 Petal color	雌蕊长度/ mm Pistil length	蜜腺形态 Nectary morphology
18B592-1	18 × 19	绿 Green	大 Big	23 × 24	10 × 12	浅黄 Light yellow	13	饱满、粉多、黄色 Full,pink,yellow
19B711-1	20 × 20	绿 Green	大 Big	22 × 24	9 × 11	浅黄 Light yellow	10	饱满、粉多、黄色 Full,pink,yellow
19B711-2	15 × 15	深绿 Dark green	大 Big	25 × 27	12 × 11	黄 Yellow	8	饱满、粉多、黄色 Full,pink,yellow
19B809-6	12 × 12	深绿 Dark green	小 Small	16 × 16	9 × 7.5	浅黄 Light yellow	5	短丝状、粉少、淡黄色 Short filament,little powder,light yellow
19B831-1	14 × 14	深绿 Dark green	中 Intermediate	18 × 17	9 × 8	浅黄 Yellow	6	短丝状、粉少、淡黄色 Short filament,little powder,light yellow
19B833-1	15 × 15	深绿 Dark green	中 Intermediate	20 × 19	9 × 7	黄 Yellow	7	饱满、粉多、黄色 Full,pink,yellow
19B834-2	12 × 9	深绿 Dark green	大 Big	22 × 21	12 × 11	浅黄 Light yellow	12	饱满、粉多、黄色 Full,pink,yellow
19B834-3	12 × 10	深绿 Dark green	大 Big	19 × 18	8 × 7	浅黄 Light yellow	10	饱满、粉多、黄色 Full,pink,yellow

[1]	唐征, 徐谦, 张小玲, 刘庆, 朱世杨, 钟伟杰. 青花菜新品种‘瓯绿75’[J]. 园艺学报, 2021, 48(S2): 2835-2836.
[2]	任文静, 于海龙, 陈立, 张斌, 陈文迪, 方智远, 杨丽梅, 庄木, 吕红豪, 王勇, 季家磊, 张扬勇. 甘蓝Ogura CMS育性恢复基因Rfo的传递效率解析及育种应用[J]. 园艺学报, 2021, 48(11): 2197-2210.
[3]	张丽*，王庆彪，王艳萍. Ogura CMS育性恢复基因orf687在萝卜中的分布[J]. 园艺学报, 2020, 47(5): 864-874.
[4]	刘莉莉1，江汉民1，单晓政1，姚星伟1，文正华1，牛国保1，张小丽1，马云生2，孙德岭1,*. 青花菜新品种‘领秀3号’[J]. 园艺学报, 2018, 45(S2): 2747-2748.
[5]	王宇，蒋炜，闫凯，周雯文，王志敏，魏大勇，汤青林. 青花菜AGL18及HDA9与开花信号整合子互作研究[J]. 园艺学报, 2018, 45(12): 2383-2394.
[6]	李占省，张黎黎，张小丽，舒金帅，苏彦宾，孙继峰，刘玉梅*，方智远，杨丽梅，庄木，张扬勇，吕红豪. 转Cry1Ac青花菜回交后代鉴定方法研究[J]. 园艺学报, 2018, 45(1): 97-108.
[7]	王庆彪，张丽*，温常龙，杨静静. 萝卜Ogura-CMS 育性恢复基因Rfo 的高通量SNP 标记开发及其应用[J]. 园艺学报, 2017, 44(7): 1309-1318.
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Ogura CMS青花菜育性恢复材料创制及其遗传背景研究

Creation of Fertility Restorer Materials for Ogura CMS Broccoli and the Study of Its Genetic Background

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图/表 12

参考文献 36

相关文章 15

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