Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (3): 533-547.doi: 10.16420/j.issn.0513-353x.2021-0100
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HUANG Jingjing1, LIU Wei2, LIU Yumei1, HAN Fengqing1, FANG Zhiyuan1, YANG Limei1, ZHUANG Mu1, ZHANG Yangyong1, LÜ Honghao1, WANG Yong1, JI Jialei1, LI Zhansheng1,*()
Received:
2021-03-08
Revised:
2021-11-08
Online:
2022-03-25
Published:
2022-03-25
Contact:
LI Zhansheng
E-mail:lizhansheng@caas.cn
CLC Number:
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.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2021-0100
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 | ||
BoRfo-6F | AGAGTTGCGAAGCAGGTT | 300 | 55 |
BoRfo-6R | CCATAAGTAATCTGGGTAGGCT | ||
BnRfo-AS2F | CATGCTTCGATCTCGTCCTTTA | 400 | 57 |
BnRfo-AS2R | TACGACATTGGGCCTACATGTC | ||
Bo138F | CCATGGACAATAATCTTAGT | 250 | 55 |
Bo138R | TAATGTGCATGAAGTTGAAG |
Table 1 Ogura CMS broccoli positive strain identification markers and its amplification information
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 | ||
BoRfo-6F | AGAGTTGCGAAGCAGGTT | 300 | 55 |
BoRfo-6R | CCATAAGTAATCTGGGTAGGCT | ||
BnRfo-AS2F | CATGCTTCGATCTCGTCCTTTA | 400 | 57 |
BnRfo-AS2R | TACGACATTGGGCCTACATGTC | ||
Bo138F | CCATGGACAATAATCTTAGT | 250 | 55 |
Bo138R | TAATGTGCATGAAGTTGAAG |
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 | |
LTSSR132-F | CCAGAAGGAACCAGCAAGAG | 250 ~ 750 |
LTSSR132-R | AAATGTCAATGCCACTGCAA | |
BoE134-F | CTCTTATTTCTTGTAGGGCTTTTA | 250 ~ 750 |
BoE134-R | CCGTTGGAGATGACTGACTG | |
LTSSR180-F | TGAATATGATAGCGGAGGGG | 100 ~ 500 |
LTSSR180-R | AACGGGGGAACGTTTAGATT | |
LTSSR188-F | AGACAGCGGAGACGGTAATG | 50 ~ 500 |
LTSSR188-F | CGATAGAGAGATGGGCAACG | |
LTSSR304-F | AAACGAACCACTTTGTTGCC | 250 ~ 500 |
LTSSR304-R | CTTCTGTTTCTTCTTCGCCG | |
LTSSR340-F | GCGACTTCCACTCCTCACTC | 100 ~ 500 |
LTSSR340-R | GAAGACAGCAAAGGACCAGC | |
LTSSR349-F | TTCAACGGCAAACATGTGAT | 250 ~ 750 |
LTSSR349-R | ACCCACCATTGAACAAGAGC | |
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 |
Table 2 Ogura CMS broccoli genetic diversity amplified markers
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 | |
LTSSR132-F | CCAGAAGGAACCAGCAAGAG | 250 ~ 750 |
LTSSR132-R | AAATGTCAATGCCACTGCAA | |
BoE134-F | CTCTTATTTCTTGTAGGGCTTTTA | 250 ~ 750 |
BoE134-R | CCGTTGGAGATGACTGACTG | |
LTSSR180-F | TGAATATGATAGCGGAGGGG | 100 ~ 500 |
LTSSR180-R | AACGGGGGAACGTTTAGATT | |
LTSSR188-F | AGACAGCGGAGACGGTAATG | 50 ~ 500 |
LTSSR188-F | CGATAGAGAGATGGGCAACG | |
LTSSR304-F | AAACGAACCACTTTGTTGCC | 250 ~ 500 |
LTSSR304-R | CTTCTGTTTCTTCTTCGCCG | |
LTSSR340-F | GCGACTTCCACTCCTCACTC | 100 ~ 500 |
LTSSR340-R | GAAGACAGCAAAGGACCAGC | |
LTSSR349-F | TTCAACGGCAAACATGTGAT | 250 ~ 750 |
LTSSR349-R | ACCCACCATTGAACAAGAGC | |
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 |
Table 3 Number of offspring(F1 generation)obtained by crossing Ogura CMS broccoli with fertility restoration line 18B592-1
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 |
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 | |||
---|---|---|---|---|---|---|---|---|
颜色 Color | 性状 Traits | 边缘 Margin | 叶缘波纹 Edge ripple | 叶面 Leaf foliage | ||||
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 |
Table 4 Agronomic traits of the Rfo transgenic offspring of Ogrua CMS broccoli
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 | |||
---|---|---|---|---|---|---|---|---|
颜色 Color | 性状 Traits | 边缘 Margin | 叶缘波纹 Edge ripple | 叶面 Leaf foliage | ||||
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 |
Fig. 1 Morphological characteristics of leaf(a),flower(b)and style(c)of the hybrid offspring of Ogura CMS broccoli(18-YX)and 18B592-1(F1 generation 19B832)
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 |
Table 5 The characteristics of Curd and floral organs in the Rfo transgenic offspring of Ogrua CMS broccoli
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 |
Fig. 2 Ogura CMS broccoli intermediate materials(a,c,d)and F1 generation(e)fertility performance during flowering(a)and pollen vigor identification(b、e)
Fig. 3 Observation of 19B833-1 chromosome behavior in hybrid offspring(F1 generation)of Ogrua CMS broccoli Ⅰ:Chromosome split equally;Ⅱ:Chromosome split unevenly;Ⅲ:Chromosome loss.
Fig. 5 Identification of relative DNA content of the Rfo transgenic offspring of Ogura CMS broccoli M1 represented the cell division cycle G0/G1 phase;M2 represented the cell division cycle G2/M phase;Fluorescence peak represents the number of nuclei.
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