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作物学报 ›› 2023, Vol. 49 ›› Issue (3): 634-646.doi: 10.3724/SP.J.1006.2023.22005

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻半育突变体sfp10的生理特征分析及基因定位

李秋平1(), 张春龙1, 杨宏1, 王拓1, 李娟1, 金寿林1, 黄大军1, 李丹丹1,2,*(), 文建成1,*()   

  1. 1云南农业大学稻作研究所, 云南昆明 650201
    2云南省作物生产与智慧农业重点实验室, 云南昆明 650201
  • 收稿日期:2022-01-21 接受日期:2022-06-07 出版日期:2023-03-12 网络出版日期:2022-07-05
  • 通讯作者: 李丹丹,文建成
  • 作者简介:E-mail: qiupingyouxiang@163.com
  • 基金资助:
    云南省重大科技专项(202102AE090017);云南省基础研究计划面上项目(202201AT070263);云南省教育厅科学研究基金项目(2022J0280);云南省作物生产与智慧农业重点实验室开放基金课题(20210101)

Physiological characteristics analysis and gene mapping of a semi-sterility plant mutant sfp10 in rice (Oryza sativa L.)

LI Qiu-Ping1(), ZHANG Chun-Long1, YANG Hong1, WANG Tuo1, LI Juan1, JIN Shou-Lin1, HUANG Da-Jun1, LI Dan-Dan1,2,*(), WEN Jian-Cheng1,*()   

  1. 1Rice Research Institute of Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2Key Laboratory of Crop Production and Intelligent Agriculture of Yunnan Province, Kunming 650201, Yunnan, China
  • Received:2022-01-21 Accepted:2022-06-07 Published:2023-03-12 Published online:2022-07-05
  • Contact: LI Dan-Dan,WEN Jian-Cheng
  • Supported by:
    Major Science and Technology Project of Yunnan Province(202102AE090017);Basic Research General Program of Yunnan Province(202201AT070263);Science Research Fund Project of Yunnan Education Department(2022J0280);Open Fund Project of Key Laboratory of Crop Production and Intelligent Agriculture of Yunnan Province(20210101)

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摘要：

以水稻花粉半育性突变体lsm与籼稻93-11构建的高世代回交导入系semi-fertility plant 10 (sfp10)为研究对象, 与野生型93-11相比, 突变体在在株高、叶长、叶宽、分蘖数、花粉数量等农艺性状上均未发现显著差异, 但花粉育性却显著下降。花粉镜检及花粉发育后期的扫描电镜观察结果显示, sfp10突变体部分花粉在发育后期淀粉积累减少并最终败育。花粉发育相关生理指标检测结果表明, 突变体花药中脯氨酸和淀粉的含量显著下降; 蔗糖在突变体穗部上游组织(源叶、库叶、茎)中积累量显著增加, 但穗部含量却明显减少, 说明蔗糖到穗部的运输过程受到影响。遗传分析表明, sfp10突变体性状受1对隐性核基因控制, 基因初定位将突变位点定位于水稻10号染色体RM25389和RM25404之间的398 kb区间内, 该区间包含3个与蔗糖转运相关的基因和1个与淀粉合成相关的基因。本研究为进一步开展花粉半育性调控基因的精细定位、基因功能及调控机制的深入研究奠定基础。

关键词: 水稻, 半育性植株, sfp10, 基因定位, 淀粉积累

Abstract:

In this study, the advanced backcross population of semi-fertility plant 10 (sfp10) mutation constructed by rice pollen semi-fertile mutant lsm and indica rice cultivar 93-11 (wild-type, WT) was used as the research subjects. Compared to the WT, there were no significant differences in plant height, leaf length, leaf width, tiller number, pollen number and other agronomic traits, but pollen fertility reduced significantly. Pollen microscopy and scanning electron microscopy (SEM) at the late stage of pollen development showed that starch accumulation in some pollen of sfp10 decreased observably, eventually led to pollen abortion. Physiological indexes related pollen development revealed that the contents of proline and starch in pollen of the sfp10 mutant decreased significantly. Sucrose accumulation generally increased in the upstream tissues (source leaf, sink leaf, and stem) of panicle, but sucrose content decreased significantly in panicle, indicating that transport from sucrose to panicle was affected. Genetic analysis indicated that the sfp10 phenotype was controlled by a pair of recessive nuclear genes. Gene preliminary mapping located the mutant site into a 398 kb interval between RM25389 and RM25404 on rice chromosome 10, which contained 3 genes related to sucrose transport and 1 gene related to starch synthesis. This study laid a foundation for further research on fine mapping, gene function, and regulation mechanism of pollen semi-fertility regulation genes.

Key words: rice, semi-fertility plant, sfp10, gene mapping, starch accumulation

附表1

用于sfp10基因定位的分子标记"

标记
Marker		 染色体
Chr.		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')		 扩增长度
Amplification length (bp)
RM24899 10 CACAAGTTCTGGGTCTCAGTGG AGAGGATCAACATCGCAATACC 280
RM24985 10 CACCTCCTCTCTCCTCTCTATCTTCC TATAAGGGCTTTCGGATGGATGG 262
RM25003 10 GATTGATCCGAGAGACAAATCC TCGATCAATAGTAGCAGCAGTAGG 116
RM25164 10 AAGAAGCGGACAGAAAGAAAGG AGAAGGAACGCACCCTAACG 368
RM25741 10 AAGGCCAGGCGGATTAGTGG AGAGGCTGGGTCCTTCCTCTCG 115
RM25810 10 GAAGGGCAGGAGGAGATAGTAGTAGG CGTCATTGTCGCCAGAAAGC 134
RM25273 10 ATCCAAGAACCATGTCGTTTCG TGCACAGTGGTAGCCAACTTATCC 345
RM5806 10 GAATGCTAATTGCGGTTGAAGC GGATCTTTCCTCCCAATCTTTGC 176
RM25425 10 CCAGCCCAAACAGCTCTTGC GGGCACTGTTTGTCTTTCTGTGC 155
mfp15 10 AGGGTAGAGTATGTCGGTGTTTCC CCGTGGCAGTAGCAGTAGGC 180
mfp41 10 GACACGGTTCTCCTTCCACTACC CGATGATAGCGTTAGCCATAACA 260
RM6142 10 CCTGCTTCTCCCTCCTGTACCC GCGAGCAAATACAGAGGCTACTACC 100
RM25348 10 CTCACGCCGATCAGAGCAAGC GCATCTCCATTCTACCACCCATTCG 283
RM25368 10 TATAGTTAAGGGAGCCACGCAAGC CCACCTCGTAAGAACATGGAGAACC 952
RM25379 10 CCCTTAGATTCCTGCAGCTTTCC AAGAGGGAATTGGAGGAATGAGC 216
RM25385 10 GCATCTCAAAGGAACTGACTGACC ATGAGATGCCTGCCATTGTACC 117
RM25389 10 GGAATGGGAGCTTCTGGCTAGG GGCGTGCACTGGCATAATTACC 471
RM25404 10 GCAACGGTTCTCCTTCCACTACC CCATGATAGCGTTAGCCATAAACG 265
RM5708 10 TGGTATCCATAACCTTGACAGC GTCACGTATCGAACAGTCTCC 249
RM24875 10 GATGAGCGAGGACAAGAATTAAGC GCTAGCTGATTCAGATCCCATCG 198
RM24956 10 TTCGTGTGCATTCCCACACATCC AAGGGTGAGGTGGCGGCTTAGG 298
RM25142 10 TGTGAGGAGAGCCATTTAGC GGGTTCCAGTCTTGAATTTAGC 206
RM25334 10 CCGTACACCGAGCTCATCACC CTTGGACAGCTTGAACGAGATCC 212
RM25359 10 CCTGCCGAAACCAACCTTATTCC GCGAAGGTAAAGCCCAATACTGTTCC 212
RM25362 10 CAGGAGCTCGCGGTTCATCG AGAGGAGGTTCTCCCTCGTGTGC 77
RM25365 10 TCTCGTCAACTTCGCTTGTATCG CGATTCGACTCTAAACAGTGATGC 155
RM25366 10 TCGGTCTCTGTGCCGTGATTAGG CACCAGCGCAGCAACTAACATCC 105
RM25375 10 TGTAGCTGCACATCTCCTTCAGC GCTCATCTCCAAGCTGCAGTCC 218
RM25376 10 GCGCTGACCTTCGGCACTTAGC CGTTCCGGATTTATCTGCTTGAGACC 447
RM25377 10 TTGGTTTCCTAGCTTGGCCTACG TAGAATGCGATGCGAGACAGTGG 160
RM25378 10 CCGTACGTTCAGGTATGTGTTTAAGG AGCAAACAGGCAGCATAAGAGG 400
RM25380 10 CGAGAACAGCTCCGAGATCAACC ATGGAGGCGTAGAACGGGATGC 111
RM8201 10 CCCACTATGCTGGTACACATCTTTCG CCTCTTCCGCTTCGCCTTCG 291
RM25386 10 GAGCACGAAAGCATGGGACAACC CGCAGAGCTCCAAGAAACACAGC 214
RM6868 10 GAGGTGAACATGCCGAGGAAGC GGCCGGAGTATATAGAACCCAAAGC 188
标记
Marker		 染色体
Chr.		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')		 扩增长度
Amplification length (bp)
RM25407 10 TAGAAAGCCCACTTCCAACACG CCAGCTCGATACCCAAGTAAGTCC 400
RM14 1 CCGAGGAGAGGAGTTCGAC GTGCCAATTTCCTCGAAAAA 191
RM486 1 CCCCCCTCTCTCTCTCTCTC CCCCCCTCTCTCTCTCTCTC 104
RM315 1 GAGGTACTTCCTCCGTTTCAC AGTCAGCTCACTGTGCAGTG 133
RM579 1 TCCGAGTGGTTATGCAAATG AATTGTGTCCAATGGGCTGT 182
RM23 1 CATTGGAGTGGAGGCTGG GTCAGGCTTCTGCCATTCTC 145
RM211 2 CCGATCTCATCAACCAACTG CTTCACGAGGATCTCAAAGG 161
RM112 2 GGGAGGAGAGGCAAGCGGAGAG AGCCGGTGCAGTGGACGGTGAC 128
RM110 2 TCGAAGCCATCCACCAACGAAG TCCGTACGCCGACGAGGTCGAG 156
RM475 2 CCTCACGATTTTCCTCCAAC ACGGTGGGATTAGACTGTGC 235
RM6 3 GTCCCCTCCACCCAATTC TCGTCTACTGTTGGCTGCAC 163
RM156 3 GCCGCACCCTCACTCCCTCCTC TCTTGCCGGAGCGCTTGAGGTG 160
RM514 3 AGATTGATCTCCCATTCCCC CACGAGCATATTACTAGTGG 259
RM293 3 TCGTTGGGAGGTATGGTACC CTTTATCTGATCCTTGGGAAGG 207
RM143 3 GTCCCGAACCCTAGCCCGAGGG AGAGGCCCTCCACATGGCGACC 207
RM523 3 AAGGCATTGCAGCTAGAAGC GCACTTGGGAGGTTTGCTAG 148
RM503 3 CACCTTTCACACACACACAC GCCCCACTAACAAAACCAAG 268
RM401 4 TGGAACAGATAGGGTGTAAGGG CCGTTCACAACACTATACAAGC 283
RM471 4 ACGCACAAGCAGATGATGAG GGGAGAAGACGAATGTTTGC 106
RM273 4 GAAGCCGTCGTGAAGTTACC GTTTCCTACCTGATCGCGAC 207
RM334 5 GTTCAGTGTTCAGTGCCACC GACTTTGATCTTTGGTGGACG 182
RM163 5 ATCCATGTGCGCCTTTATGAGGA CGCTACCTCCTTCACTTACTAGT 124
RM440 5 CATGCAACAACGTCACCTTC ATGGTTGGTAGGCACCAAAG 169
RM136 6 GAGAGCTCAGCTGCTGCCTCTAGC GAGGAGCGCCACGGTGTACGCC 101
RM204 6 GTGACTGACTTGGTCATAGGG GCTAGCCATGCTCTCGTACC 169
RM589 6 ATCATGGTCGGTGGCTTAAC CAGGTTCCAACCAGACACTG 186
RM412 6 CACTTGAGAAAGTTAGTGCAGC CCCAAACACACCCAAATAC 198
RM527 6 GGCTCGATCTAGAAAATCCG TTGCACAGGTTGCGATAGAG 233
RM427 7 TCACTAGCTCTGCCCTGACC TGATGAGAGTTGGTTGCGAG 185
RM432 7 TTCTGTCTCACGCTGGATTG AGCTGCGTACGTGATGAATG 187
RM38 8 ACGAGCTCTCGATCAGCCTA TCGGTCTCCATGTCCCAC 250
RM296 9 CACATGGCACCAACCTCC GCCAAGTCATTCACTACTCTGG 123
RM206 11 CCCATGCGTTTAACTATTCT CGTTCCATCGATCCGTATGG 147
RM224 11 ATCGATCGATCTTCACGAGG TGCTATAAAAGGCATTCGGG 157
RM254 11 AGCCCCGAATAAATCCACCT CTGGAGGAGCATTTGGTAGC 165
RM473 11 TATCCTCGTCTCCATCGCTC AAGGATGTGGCGGTAGAATG 97
RM28778 12 CTTCATCACCGCCTCCGTTCC AGCTCTCTCCCGCTCTGGATGC 165
RM28802 12 GGAGGCTTAACTCAGCACTACTGG CATGCTCAGATGTGTTCACTTGG 179
RM28812 12 GGGAATTGAGAATCGACAGAAACC CCAGTACGTCAAACAGGGCTACG 195
RM28819 12 GAACGTCTCGTTCCCTATCACG TCCACTCACTCATCTCTCCTTGC 244
RM27545 12 GCAGGTTAGTTCACTCCATGTGC TTCGAAGGGTCTTGTGATGTATCC 375

图1

野生型93-11和突变体sfp10的表型分析 A: 93-11和sfp10灌浆期的植株; B: 93-11和sfp10的花粉镜检形态; C: 93-11和sfp10的花粉数量、花粉育性比较, **表示在0.01水平差异显著; D: 93-11和sfp10成熟期的小穗和单穗籽粒(上: 实粒; 下: 空瘪粒)。"

表1

野生型93-11和突变体sfp10主要农艺性状的比较"

性状
Trait		 野生型93-11
Wild type 93-11		 突变体sfp10
sfp10 mutant		 相比93-11
Compared with 93-11 (%)
株高Plant height (cm) 109.10±4.75 111.10±4.23 1.83
分蘖数Tiller number 5.00±1.81 4.35±0.93 -13.00
剑叶长Swordleaf length (cm) 37.60±1.36 38.63±2.01 2.49
剑叶宽Swordleaf width (cm) 1.39±0.06 1.38±0.07 -0.72
生物产量Biological yield (g) 29.88±7.71 21.27±7.27 -28.81**
经济产量Economic yield (g) 12.37±3.26 7.98±2.74 -35.47**
结实率Seed setting rate (%) 92.84±2.65 59.34±8.79 -36.09**
千粒重1000-grain weight (g) 29.63±2.09 25.60±2.43 -13.58**
主穗粒数Grain number per panicle 131.15±20.08 60.40±11.86 -53.95**

图2

野生型93-11和突变体sfp10花粉发育后期的扫描电镜观察 A, B: 分别为93-11和sfp10花粉发育六期的花粉粒; C, D: 分别为93-11和sfp10花粉发育七期的花粉粒; E, F: 分别为93-11和sfp10花粉发育八期的花粉粒; G: 93-11和sfp10的花粉粒长; H: 93-11和sfp10的花粉粒宽。S6、S7、S8分别表示花粉发育六、七、八期, **表示在0.01水平差异显著。"

附表2

93-11与sfp10正常花粉粒形态分析"

时期
Stage		 花粉长度Pollen grain length (µm) 花粉宽度Pollen grain width (µm)
93-11 sfp10 93-11 sfp10
六期
Stage 6		 32.9 30.8 31.2 29.3
32.2 32.7 29.8 29.0
32.0 31.2 29.2 28.5
P值 P-value 0.28 0.15
七期
Stage 7		 39.9 40.7 38.8 37.4
40.4 39.9 39.1 38.9
40.2 37.7 39.8 38.7
P值 P-value 0.46 0.18
八期
Stage 8		 39.3 44.3 38.7 43.7
44.1 43.1 43.2 42.6
44.6 43.6 45.3 42.2
P值 P-value 0.59 0.84

图3

野生型93-11和突变体sfp10花粉发育相关生理指标 A: 93-11和sfp10花药中脯氨酸含量; B: 93-11和sfp10花药中淀粉含量; C~F: 93-11和sfp10源叶(C)、库叶(D)、茎部(E)、穗部(F)蔗糖含量。1: 孕穗期; 2: 抽穗期; 3: 开花期; 4: 灌浆期; **表示在0.01水平差异显著。"

表2

sfp10突变位点的遗传分析"

组合
Combination		 F2 χ²(3:1) χ²0.05
野生型株数 No. of wild type plants 突变型株数 No. of mutant type plants
Sfp10/93-11 297 210 72.03 3.84
93-11/Sfp10 439 47 60.09
合计Total 736 257 0.37

图4

sfp10突变表型的基因初定位 A: sfp10定位在10号染色体上11.91 Mb范围内; B: sfp10定位在1.79 Mb范围内; C: sfp10定位在398 kb范围内; D: 定位区间有31个预测基因。"

图5

sfp10定位区间基因的信息分析 A: sfp10定位区间基因的GO富集分析; B: sfp10定位区间基因的KEGG富集分析。"

表3

主要候选基因及功能注释"

参与过程
Participation process		 基因名称
Gene name		 基因注释
Gene annotation
氨基酸合成与代谢
Amino acid synthesis and metabolism		 Os10g0405100 Similar to serine/threonine-protein kinase NAK
Os10g0400100 Methionyl-tRNA synthetase, class Ia domain containing protein
Os10g0406300 Ascorbate oxidase promoter-binding protein
Os10g0400800 Similar to phenylalanyl-tRNA synthetase alpha chain
蔗糖转运
Sucrose transport		 Os10g0404500 Similar to sucrose transporter
Os10g0404533 Similar to sucrose transport protein SUT3
Os10g0404566 Similar to sucrose transporter
淀粉合成Starch synthesis Os10g0390500 Starch synthesis in developing seeds
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