Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (5): 1351-1363.doi: 10.16420/j.issn.0513-353x.2025-0116
• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles
WANG Qingbiao1, WANG Yanping1, WU Xiangyu1, CHEN Ningjie1, CHI Ting1, HUA Dingzhu1, GE Ailing1, GUO Yu1, WEI Lei2,*(), ZHANG Li1,*(
)
Received:
2025-02-12
Revised:
2025-05-12
Online:
2025-05-25
Published:
2025-05-21
Contact:
WEI Lei, ZHANG Li
WANG Qingbiao, WANG Yanping, WU Xiangyu, CHEN Ningjie, CHI Ting, HUA Dingzhu, GE Ailing, GUO Yu, WEI Lei, ZHANG Li. Genetic Analysis and Preliminary Mapping of the Fertility Restoration Gene for NWB-CMS in Radish(Raphanus sativus)[J]. Acta Horticulturae Sinica, 2025, 52(5): 1351-1363.
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URL: https://www.ahs.ac.cn/EN/10.16420/j.issn.0513-353x.2025-0116
标记 Marker | 位置 Position | 正向引物(5′-3′) Forward primer | 反向引物(5′-3′) Reverse primer |
---|---|---|---|
Rf-117 | 16109162 | TCTCGCGGATGTAGTAAGCT | TTACGTGCCAATTATGTAGGAGAA |
Rf-6 | 17270936 | GCCACTATCACAATACTAGCATGA | ACTTGATTCTTGCAGAGTGATTGA |
Rf-8 | 18574289 | ACGCATTTTCGATGTGTGTACA | TGAGGGATATGATCTGCATTGTCA |
Rf-113 | 19086196 | TCTGGTCCAAGTCAGGAGGA | GGGGAGTGACAAGAACCTGA |
Rf-1 | 31297167 | CCACCTGTCTCGTTGAAGGA | TCTCAAGTCTGATTCCCCGC |
Rf-120 | 33851811 | CCACATACAGATTAACAGTGCCC | AGATTTCACTTTTGGGTTTTGGTTT |
Rf-119 | 34029651 | CCCGAGAAAGCCTAACTGCT | AAGGAACAAAGGCCAAGGGT |
Rf-122 | 37683242 | GGCATAGGTAGCAGGAGGC | TCCGTAAAACCCTAAACCGCA |
Rf-118 | 38202091 | AGTGACGTGGCACTCTCAAA | TTTATCGTCGGCAAAATTTGATGA |
Rf-10 | 38864737 | CTTTGCGCGGGTCTTTCAAA | ACACCTTGTGACTGAAGAGGA |
Rf-11 | 39147363 | TCCAATATATAATGGCTTTTGTGCAAA | TGAGATCGGCGGAAGACTTG |
Rf-14 | 40214027 | AAACTTACTTGTGTCGGAGATCC | CCTTCTAGAACATTTACTCTCCAGGT |
Rf-16 | 42080242 | GCCAAGACACAGTACGGTCA | CGAGTCAGGAAGCTGGTCAT |
Rf-128 | 42368813 | ACATCTCTGGGGAAAAAGCTTT | GCTATAGTCCCCGCATCAAGT |
Rf-131 | 40586659 | TGTTTTACCGTTACATTCAGTTTCAGT | CCAAAAGCCTATTCAACACTCGT |
Table 1 The polymorphic InDel primers used in this study
标记 Marker | 位置 Position | 正向引物(5′-3′) Forward primer | 反向引物(5′-3′) Reverse primer |
---|---|---|---|
Rf-117 | 16109162 | TCTCGCGGATGTAGTAAGCT | TTACGTGCCAATTATGTAGGAGAA |
Rf-6 | 17270936 | GCCACTATCACAATACTAGCATGA | ACTTGATTCTTGCAGAGTGATTGA |
Rf-8 | 18574289 | ACGCATTTTCGATGTGTGTACA | TGAGGGATATGATCTGCATTGTCA |
Rf-113 | 19086196 | TCTGGTCCAAGTCAGGAGGA | GGGGAGTGACAAGAACCTGA |
Rf-1 | 31297167 | CCACCTGTCTCGTTGAAGGA | TCTCAAGTCTGATTCCCCGC |
Rf-120 | 33851811 | CCACATACAGATTAACAGTGCCC | AGATTTCACTTTTGGGTTTTGGTTT |
Rf-119 | 34029651 | CCCGAGAAAGCCTAACTGCT | AAGGAACAAAGGCCAAGGGT |
Rf-122 | 37683242 | GGCATAGGTAGCAGGAGGC | TCCGTAAAACCCTAAACCGCA |
Rf-118 | 38202091 | AGTGACGTGGCACTCTCAAA | TTTATCGTCGGCAAAATTTGATGA |
Rf-10 | 38864737 | CTTTGCGCGGGTCTTTCAAA | ACACCTTGTGACTGAAGAGGA |
Rf-11 | 39147363 | TCCAATATATAATGGCTTTTGTGCAAA | TGAGATCGGCGGAAGACTTG |
Rf-14 | 40214027 | AAACTTACTTGTGTCGGAGATCC | CCTTCTAGAACATTTACTCTCCAGGT |
Rf-16 | 42080242 | GCCAAGACACAGTACGGTCA | CGAGTCAGGAAGCTGGTCAT |
Rf-128 | 42368813 | ACATCTCTGGGGAAAAAGCTTT | GCTATAGTCCCCGCATCAAGT |
Rf-131 | 40586659 | TGTTTTACCGTTACATTCAGTTTCAGT | CCAAAAGCCTATTCAACACTCGT |
父本种类 Male subspecies | 亲本来源 Parental origin | 世代 Generation | F:MF:S | χ2 | P | ||
---|---|---|---|---|---|---|---|
观测值Observed | 期望值Expected | ||||||
东亚大长萝卜 | YB-A × 短叶13 Duanye 13 | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
East Asia big long | YB-A × 象牙白 Xiangyabai | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
radish | YB-A × 潍县青 Weixianqing | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
YB-A × YR Tengu | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × YR Kurama | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 西星5号 Xixing 5 | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 卫青萝卜 Weiqing | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 锦州五斤红 Jinzhou Wujinhong | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 北京花叶心里美 Beijing Huaye Xinlimei | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 板叶心里美 Banye Xinlimei | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
欧洲小萝卜 | YB-A × KP | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
European small | F2-1 | 114:24:10 | 12:3:1 | 0.65 | 0.72 | ||
radish | F2-2 | 238:68:29 | 12:3:1 | 4.23 | 0.12 | ||
BC1 | 136:74:84 | 2:1:1 | 2.33 | 0.31 | |||
YB-A × лΑТΑ | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 288:0:16 | 15:0:1 | 0.51 | 0.48 | |||
YB-A × NST601 | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 275:0:6 | 63:0:1 | 0.60 | 0.44 | |||
YB-A × DQG | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 282:0:1 | 63:1 | 2.69 | 0.10 | |||
YB-A × 红樱桃 Hongyingtao | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 266:0:4 | 63:0:1 | 0.01 | 0.91 | |||
YB-A × GiSi | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 287:0:19 | 15:0:1 | < 0.001 | 0.98 |
Table 2 Inheritance patterns of fertility restoration of NWB-CMS
父本种类 Male subspecies | 亲本来源 Parental origin | 世代 Generation | F:MF:S | χ2 | P | ||
---|---|---|---|---|---|---|---|
观测值Observed | 期望值Expected | ||||||
东亚大长萝卜 | YB-A × 短叶13 Duanye 13 | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
East Asia big long | YB-A × 象牙白 Xiangyabai | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
radish | YB-A × 潍县青 Weixianqing | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
YB-A × YR Tengu | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × YR Kurama | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 西星5号 Xixing 5 | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 卫青萝卜 Weiqing | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 锦州五斤红 Jinzhou Wujinhong | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 北京花叶心里美 Beijing Huaye Xinlimei | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
YB-A × 板叶心里美 Banye Xinlimei | F1 | 0:0:15 | 0:0:1 | 0 | 1 | ||
欧洲小萝卜 | YB-A × KP | F1 | 0:0:15 | 0:0:1 | 0 | 1 | |
European small | F2-1 | 114:24:10 | 12:3:1 | 0.65 | 0.72 | ||
radish | F2-2 | 238:68:29 | 12:3:1 | 4.23 | 0.12 | ||
BC1 | 136:74:84 | 2:1:1 | 2.33 | 0.31 | |||
YB-A × лΑТΑ | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 288:0:16 | 15:0:1 | 0.51 | 0.48 | |||
YB-A × NST601 | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 275:0:6 | 63:0:1 | 0.60 | 0.44 | |||
YB-A × DQG | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 282:0:1 | 63:1 | 2.69 | 0.10 | |||
YB-A × 红樱桃 Hongyingtao | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 266:0:4 | 63:0:1 | 0.01 | 0.91 | |||
YB-A × GiSi | F1 | 15:0:0 | 1:0:0 | 0 | 1 | ||
F2 | 287:0:19 | 15:0:1 | < 0.001 | 0.98 |
Fig. 1 Microscopical structure of anthers at mature pollen grain stages of F2 individuals(YB-A × KP) A:Fertile plant;B and C:Micro- fertile plant;D:Sterile plant. PS:Pollen sac;PG:Pollen grain. The red arrows indicate viable pollen grains stained darkly
Fig. 2 Distribution of ΔSNP/Indel index A:F_pool vs. S_pool;B:MF_pool vs. S_poll;C:F_pool vs. MF_pool. The scatter plot represents the raw values,and the black curve is the fitted value based on the sliding-window. A random permutation test was performed 1 000 times,with the red line indicating the 99% confidence level and the blue line indicating the 95% confidence level
极端池 Extreme pool | 位点 QTL | 起始 ~ 终止/Mb Start-End | 区间大小/Mb Region length | SNP和InDel数量 No. of SNPs and InDels | 基因数 No. of genes |
---|---|---|---|---|---|
F_pool vs. S_pool | Chr1:Rfn1.1 | 32.5 ~ 33.7 | 1.2 | 5 517 | 5 |
MF_pool vs. S_pool | Chr1:Rfn1.1 | 32.5 ~ 33.7 | 1.2 | 5 469 | 5 |
Chr9:Rfn9.1 | 12.2 ~ 13.3 | 1.1 | 34 167 | 128 | |
F_pool vs. MF_pool | Chr4:Rfn4.1 | 16.7 ~ 18.6 | 1.9 | 60 301 | 167 |
Chr4:Rfn4.2 | 31.9 ~ 33.1 | 1.2 | 28 059 | 55 | |
Chr4:Rfn4.3 | 34.3 ~ 37.9 | 3.6 | 54 362 | 106 |
Table 3 Preliminary mapping intervals for NWB-CMS fertility restoration genes and statistics within these intervals
极端池 Extreme pool | 位点 QTL | 起始 ~ 终止/Mb Start-End | 区间大小/Mb Region length | SNP和InDel数量 No. of SNPs and InDels | 基因数 No. of genes |
---|---|---|---|---|---|
F_pool vs. S_pool | Chr1:Rfn1.1 | 32.5 ~ 33.7 | 1.2 | 5 517 | 5 |
MF_pool vs. S_pool | Chr1:Rfn1.1 | 32.5 ~ 33.7 | 1.2 | 5 469 | 5 |
Chr9:Rfn9.1 | 12.2 ~ 13.3 | 1.1 | 34 167 | 128 | |
F_pool vs. MF_pool | Chr4:Rfn4.1 | 16.7 ~ 18.6 | 1.9 | 60 301 | 167 |
Chr4:Rfn4.2 | 31.9 ~ 33.1 | 1.2 | 28 059 | 55 | |
Chr4:Rfn4.3 | 34.3 ~ 37.9 | 3.6 | 54 362 | 106 |
Fig. 3 Genotypes of recombinant plants in the F2 population(YB-A×KP)using InDel markers within the Rfn4 localization interval The region between dashed lines indicates the refined interval for further gene mapping. F:Fertility;S:Sterility. Black boxes represent genotypes identical to the sterile line YB-A,white boxes correspond to the restorer line KP,and gray boxes indicate heterozygous genotypes
基因组 Genome | 位点/Mb Location | ||
---|---|---|---|
Rfn4.1 | Rfn4.2 | Rfn4.3 | |
ROD | 15.4 ~ 18.7 | 31.9 ~ 33.1 | 34.2 ~ 37.9 |
RS03 | 15.9 ~ 19.0 | 35.6 ~ 36.5 | 33.2 ~ 34.5 |
RS04 | 16.0 ~ 19.4 | 35.2 ~ 36.1 | 32.7 ~ 33.7 |
RS05 | 16.4 ~ 19.8 | 36.3 ~ 37.2 | 33.4 ~ 35.2 |
RS06 | 16.0 ~ 19.2 | 35.9 ~ 36.4 | 33.4 ~ 34.4 |
WK10039 | 18.7 ~ 23.7 | 29.0 ~ 29.2 | 19.1 ~ 23.7 |
XYB36-2 | 16.0 ~ 21.1 | 25.2 ~ 27.6 | 25.4 ~ 26.5 |
Table 4 Linear alignment of QTL regions for fertility restoration genes of NWB-CMS across genomes
基因组 Genome | 位点/Mb Location | ||
---|---|---|---|
Rfn4.1 | Rfn4.2 | Rfn4.3 | |
ROD | 15.4 ~ 18.7 | 31.9 ~ 33.1 | 34.2 ~ 37.9 |
RS03 | 15.9 ~ 19.0 | 35.6 ~ 36.5 | 33.2 ~ 34.5 |
RS04 | 16.0 ~ 19.4 | 35.2 ~ 36.1 | 32.7 ~ 33.7 |
RS05 | 16.4 ~ 19.8 | 36.3 ~ 37.2 | 33.4 ~ 35.2 |
RS06 | 16.0 ~ 19.2 | 35.9 ~ 36.4 | 33.4 ~ 34.4 |
WK10039 | 18.7 ~ 23.7 | 29.0 ~ 29.2 | 19.1 ~ 23.7 |
XYB36-2 | 16.0 ~ 21.1 | 25.2 ~ 27.6 | 25.4 ~ 26.5 |
Fig. 4 Collinearity and structural variation analysis within the Rfn4 mapped interval Yellow and blue lines represent inversions and duplications,respectively. The horizontal axis indicates the relative position of fragments in the Rfn4 mapped interval
Fig. 5 Genotyping of the Rf-118,Rf-119,Rf-120,Rf-122 marker within the Rfn4 fertility restoration locus of NWB-CMS in the F2-2 population The upper band represents the“aa”allele identical to YB-A,the lower band represents the“AA”allele identical to the restorer line KP,and the double band represents the heterozygous type“Aa”
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