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作物学报 ›› 2019, Vol. 45 ›› Issue (2): 225-234.doi: 10.3724/SP.J.1006.2019.083033

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

两份玉米CMS-C恢复系的育性恢复力测定及恢复基因的分子标记定位

牟碧涛1,2,赵卓凡1,岳灵1,李川1,张钧3,李章波3,申汉3,曹墨菊1,*()   

  1. 1四川农业大学玉米研究所 / 农业部西南玉米生物学与遗传育种重点实验室, 四川成都 611130
    2宜宾市农业科学院食用菌蚕桑研究所, 四川宜宾 644000
    3内蒙古真金种业科技有限公司, 内蒙古鄂尔多斯 014300
  • 收稿日期:2018-04-16 接受日期:2018-10-08 出版日期:2019-02-12 网络出版日期:2018-11-05
  • 通讯作者: 曹墨菊
  • 基金资助:
    本研究由“十三五”国家重点研发计划项目资助(2016YFD0101206)

Identification of fertility restoration and molecular mapping of restorer genes in two maize restore lines of CMS-C

Bi-Tao MOU1,2,Zhuo-Fan ZHAO1,Ling YUE1,Chuan LI1,Jun ZHANG3,Zhang-Bo LI3,Han SHEN3,Mo-Ju CAO1,*()   

  1. 1 Maize Research Institute, Sichuan Agricultural University / Key Laboratory of Maize Biology and Genetics and Breeding of Southwest China, Ministry of Agriculture, Chengdu 611130, Sichuan, China
    2 Edible Fungus Sericulture Research Institute, Yibin Academy of Agricultural Sciences, Yibin 644000, Sichuan, China
    3 Inner Mongolia Zhenjin Seed S&T Co., Ltd, Ordos 014300, Inner Mongolia, China;
  • Received:2018-04-16 Accepted:2018-10-08 Published:2019-02-12 Published online:2018-11-05
  • Contact: Mo-Ju CAO
  • Supported by:
    This study was supported by the National Thirteenth Five-Year National Research and Development Program(2016YFD0101206)

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摘要：

为了发掘更多玉米C型不育胞质的强恢复系资源, 本研究对2份自交系Z16和7250-14-1进行了恢复能力的测定、恢复基因的遗传分析及恢复基因的分子标记定位。结果表明, Z16和7250-14-1对C黄早四、C478、C698-3和CMo17均表现为育性恢复, 而对C48-2则均表现为育性部分恢复。通过对玉米CMS-C不同亚组胞质测交鉴定发现, Z16对G48-2、EC48-2、ES48-2、RB48-2及类48-2均表现为不育性保持, 而7250-14-1对G48-2、EC48-2、ES48-2表现为育性部分恢复, 对RB48-2和类48-2则表现为不育性保持。Z16和7250-14-1对CMS-T不育系均表现为不育性保持, 而对CMS-S不育系则均表现为育性部分恢复。遗传分析显示, Z16对C478和C黄早四的育性恢复均受1对基因控制; 而7250-14-1对C黄早四及C478的育性恢复分别受1对基因及2对基因控制。利用(C黄早四×Z16)F2、(C黄早四×7250-14-1)F2群体分别对恢复基因进行分子标记定位, 其中Z16的恢复基因被定位于标记B-1至第8染色体短臂末端区域, 物理距离为494 kb; 7250-14-1的恢复基因被定位于第8染色体短臂的标记B-1和Chr8-86080之间, 物理距离为249 kb。该研究不仅为玉米CMS-C“三系”配套的生产利用提供了恢复基因资源, 也为玉米CMS-C恢复基因的克隆及恢复机制的研究奠定了一定基础。

关键词: 玉米, 细胞质雄性不育, 恢复基因, 分子标记定位

Abstract:

The objective of the present study was to identify novel and powerful restorer lines for CMS-C. So maize inbred lines Z16 and 7250-14-1 were crossed with both isonuclear alloplasmic and isoplasmic allonuclear CMS-C, CMS-T, and CMS-S male sterile lines. Self-cross and back-cross were conducted for some of the fertility restored F1 for genetic analysis and restorer gene mapping. Male fertility expression was investigated for all the F1, F2 and backcross populations, showing that Z16 and 7250-14-1 could restore the fertility for C Huangzaosi, C478, C698-3, and CMo17 completely, and partly restore the fertility for C48-2. Z16 could not restore the fertility for G48-2, EC48-2, ES48-2, RB48-2, and Lei48-2, while 7250-14-1 could partly restore the fertility for G48-2, EC48-2, and ES48-2, and maintain the sterility for RB48-2 and Lei48-2. Both Z16 and 7250-14-1 couldn’t restore the fertility of CMS-T, and partly restore the fertility for CMS-S. Genetic analysis showed that the fertility restoration was controlled by a pair of dominant genes for Z16 when crossed with C478 or C Huangzaosi. But for 7250-14-1, the fertility restoration was controlled by a pair of dominant genes for C Huangzaosi, and two pairs of complementary dominant genes for C478. Both of the restorer genes for Z16 and 7250-14-1 were mapped on the short arm of chromosome 8 by molecular markers. For Z16, it was mapped within a physical distance of 494 kb from the marker B-1 to the end of the chromosome, and for 7250-14-1, it was located between B-1 and Chr8-86080, with physical distance of 249 kb. This study not only provides some information for the practical application of Z16 and 7250-14-1, but also lays a foundation for the cloning and functional analysis of restorer genes.

Key words: maize, cytoplasmic male sterility, restorer gene, molecular mapping

表1

玉米同核异质、同质异核不育系"

C型不育系
CMS-C		 T型不育系
CMS-T		 S型不育系
CMS-S		 C型亚组不育系
CMS-C subgroup
CMo17 TMo17 SMo17 G48-2
C698-3 T698-3 S698-3 EC48-2
C黄早四
C Huangzaosi		 ES48-2
C478 RB48-2
C48-2 类48-2 Lei 48-2

图1

部分测交组合的育性表现 I: 花药不外露; II: 花药外露比例为0~25%; III: 花药外露比例为25%~50%; IV: 花药外露比例为50%~75%; V: 花药外露比例为75%~100%。"

表2

CMS-C不育系与Z16测交后代的育性鉴定结果"

组合
Combination		 雄穗育性等级 Fertility grade of tassel 花粉可染率 Pollen staining rate
I II III IV V 可染
Fully staining (%)		 半染
Semi-staining (%)		 不染
Not staining (%)
CMo17×Z16 0 0 0 0 28 98.6 0 1.4
C698-3×Z16 0 0 0 0 25 96.7 0 3.3
C黄早四×Z16 C Huangzaosi×Z16 0 0 0 0 29 97.4 0 2.6
C478×Z16 0 0 0 0 28 97.8 0 2.2
C48-2×Z16 0 4 24 0 0 39.0 26.9 34.1
G48-2×Z16 28 0 0 0 0 0 0 100
EC48-2×Z16 30 0 0 0 0 0 0 100
ES48-2×Z16 29 0 0 0 0 - - -
RB48-2×Z16 28 0 0 0 0 - - -
类48-2×Z16 Lei48-2×Z16 28 0 0 0 0 - - -

表3

CMS-T、CMS-S不育系与Z16测交后代的育性鉴定结果"

组合
Combination		 雄穗育性等级Fertility grade of tassel 花粉可染率Pollen staining rate
I II III IV V 可染
Fully staining (%)		 半染
Semi-staining (%)		 不染
Not staining (%)
TMo17×Z16 28 0 0 0 0 — — —
T698-3×Z16 29 0 0 0 0 — — —
SMo17×Z16 0 5 24 0 0 22.0 12.1 65.9
S698-3×Z16 30 0 0 0 0 29.2 33.3 37.5

表4

CMS-C不育系与7250-14-1测交后代的育性鉴定结果"

组合
Combination		 雄穗育性等级Fertility grade of tassel 花粉可染率Pollen staining rate
I II III IV V 可染
Fully staining (%)		 半染
Semi-staining (%)		 不染
Not staining (%)
CMo17×7250-14-1 0 0 0 0 28 96.6 3.3 0
C698-3×7250-14-1 0 0 0 0 29 91.9 2.7 5.4
C黄早四×7250-14-1
C Huangzaosi×7250-14-1		 0
 0
 0
 0
 30
 98.8
 0
 1.2
C478×7250-14-1 0 0 0 0 29 97.8 0 2.2
C48-2×7250-14-1 0 0 29 0 0 83.3 11.1 5.6
G48-2×7250-14-1 0 0 28 0 0 94.1 0 5.9
EC48-2×7250-14-1 0 0 28 0 0 22.6 25.0 52.4
ES48-2×7250-14-1 0 2 26 0 0 96.6 0 3.4
RB48-2×7250-14-1 29 0 0 0 0 0 0 100
类48-2×7250-14-1
Lei48-2×7250-14-1		 28
 0
 0
 0
 0
 0
 0
 100

表5

CMS-T、CMS-S不育系与7250-14-1测交后代的育性鉴定结果"

组合
Combination		 雄穗育性等级Fertility grade of tassel 花粉可染率Pollen staining rate
I II III IV V 可染
Fully staining (%)		 半染
Semi-staining (%)		 不染
Not staining (%)
TMo17×7250-14-1 29 0 0 0 0 — — —
T698-3×7250-14-1 29 0 0 0 0 — — —
SMo17×7250-14-1 0 0 0 29 0 14.1 15.3 70.6
S698-3×7250-14-1 0 0 0 28 0 43.9 43.9 12.2

表6

不育系与自交系Z16杂交后代的育性调查结果"

组合
Combination		 可育株
No. of fertile plants		 不育株
No. of sterile plants		 总株数
Total No. of
plants		 理论比例
Theoretical
ratio		 χ2 年份和地点
Year and place
(C黄早四×Z16)F2
(C Huangzaosi×Z16) F2		 287 115 402 3:1 1.33 2016景洪 Jinghong
299 106 405 3:1 0.15 2017温江 Wenjiang
1820 616 2436 3:1 0.05 2017崇州 Chongzhou
(C黄早四×Z16)×黄早四
(C Huangzaosi×Z16)×Huangzaosi		 33 36 69 1:1 0.06 2016温江 Wenjiang
98 107 205 1:1 0.20 2017温江 Wenjiang
(C478×Z16)F2 251 107 358 3:1 2.15 2017温江 Wenjiang
(C478×Z16)×478 134 135 269 1:1 0 2017温江 Wenjiang

表7

不育系与自交系7250-14-1杂交后代的育性调查结果"

组合
Combination		 可育株
No. of fertile plants		 不育株
No. of sterile plants		 总株数
Total No. of
plants		 期望比例
Theoretical
ratio		 χ2 年份、地点
Year and place
(C黄早四×7250-14-1)F2
(C Huangzaosi×7250-14-1) F2		 271 85 356 3:1 0.12 2016景洪 Jinghong
319 104 423 3:1 0.02 2017温江 Wenjiang
1386 475 1861 3:1 0.14 2017崇州 Chongzhou
(C黄早四×7250-14-1)×黄早四
(C Huangzaosi×7250-14-1) ×Huangzaosi		 23 22 45 1:1 0 2016温江 Wenjiang
124 141 265 1:1 0.55 2017温江 Wenjiang
(C478×7250-14-1)F2 163 110 273 9:7 1.19 2016温江 Wenjiang
243 159 392 9:7 1.44 2017温江 Wenjiang
(C478×7250-14-1)×478 59 191 247 1:3 0.37 2017温江 Wenjiang

附表1

用于Z16恢复基因定位的多态性引物"

引物
Primer		 引物类型
Primer type		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
Chr8-86080 InDel CGTCGTTGAGGTGAGAAGAG CTCCGAACCTGATCCGAGTA
B-2 InDel ACGAATACGATACGTAGCCA GTGAATCTGCGGTGAACAAA
B-6-1 InDel GGATGGAATATATAAAGTTTGCT GGCTCATTACCTTGGTGTCA
B-1 InDel GATCGTTCCGGCCCAAGAAG TAGCCGTGGAGTTGGTAGCC
m-1 SSR CATTGACCGGGGTAGGAAGT CATTGACCGGGGTAGGAAGT
TIDP5557 InDel CATGAGATCAACGGGATGC AGTAGAGATCCGGGAGGTGG
Chr8-1330080 InDel CCAAGTTGGATACAACGACAGA AGAAGCAACGTCTGCAGGAT
IDP8573 InDel CGAGTCAGTTGCTTACGGG AATTGCCGAGTGGATACAGG

图2

自交系Z16所含Rf基因在第8染色体上的连锁图谱"

图3

自交系7250-14-1所含Rf基因在第8染色体上的连锁图谱"

附表2

用于7250-14-1恢复基因定位的多态性引物"

引物
Primer		 引物类型
Primer type		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
m-10 SSR AGCGCTCGATTCCTGTAGTG GGGTGTCGTTGGTTGGGATT
Chr8-86080 InDel CGTCGTTGAGGTGAGAAGAG CTCCGAACCTGATCCGAGTA
B-2 InDel ACGAATACGATACGTAGCCA GTGAATCTGCGGTGAACAAA
B-6-2 InDel CCAATGTTTTGATGGAAGTCCT AATTGCCATGTTCTTACCTGT
B-1 InDel GATCGTTCCGGCCCAAGAAG TAGCCGTGGAGTTGGTAGCC
Chr8-398180 InDel GCCAGTTCGGAGACAGGAT ACCGCCATCCAATTAACAAG
IDP8319 InDel TTGACCCTCCTGTTACGTGC GAGCATGGACCACATGACC
IDP500 InDel CACTGCCGTAGAGTAGTGCG GGCTTCAAGATCAGTCCG
IDP7866 InDel GGACGAAGCGATCGAGTACC AGATGAGGGAAGTGAGCAGC
Chr8-1330080 InDel CCAAGTTGGATACAACGACAGA AGAAGCAACGTCTGCAGGAT
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