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作物学报 ›› 2021, Vol. 47 ›› Issue (11): 2173-2183.doi: 10.3724/SP.J.1006.2021.02076

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻ptc1隐性核不育系的创制及其配合力分析

李京琳(), 李佳林, 李新鹏, 安保光, 曾翔, 吴永忠, 黄培劲, 龙湍*()   

  1. 海南波莲水稻基因科技有限公司, 海南海口 570203
  • 收稿日期:2020-11-15 接受日期:2021-03-19 出版日期:2021-11-12 网络出版日期:2021-04-06
  • 通讯作者: 龙湍
  • 作者简介:E-mail: 250864463@qq.com
  • 基金资助:
    中国水稻研究所水稻生物学国家重点实验室开放课题项目(20190203)

Creation and combining ability analysis of recessive genic sterile lines with a new ptc1 locus in rice

LI Jing-Lin(), LI Jia-Lin, LI Xin-Peng, AN Bao-Guang, ZENG Xiang, WU Yong-Zhong, HUANG Pei-Jing, LONG Tuan*()   

  1. Hainan Bolian Rice Gene Technology Co., Ltd., Haikou 570203, Hainan, China
  • Received:2020-11-15 Accepted:2021-03-19 Published:2021-11-12 Published online:2021-04-06
  • Contact: LONG Tuan
  • Supported by:
    Open Project of State Key Laboratory of Rice Biology Foundation of China Rice Research Institute(20190203)

摘要:

细胞质雄性不育系和光温敏雄性不育系是目前杂交水稻育种、制种中广泛利用的两种不育系, 然而这两种不育系分别具有组配不自由和育性不稳定的缺陷。隐性核雄性不育系可以克服上述缺陷, 创制、鉴定和利用细胞核雄性不育系将成为新一代杂交水稻技术的重要环节。本研究通过筛选9311的辐射诱变突变体库, 获得了一个无花粉型隐性核雄性不育突变体ptc1-2。图位克隆发现ptc1-2的突变位点位于9号染色体上, 是一段包含PTC1编码区在内259.37 kb的大片段缺失。共分离检测表明, 雄性不育表型是由ptc1-2突变位点造成的。通过分子标记辅助选择将ptc1-2突变位点回交转育至两系不育系C815S和三系保持系五丰B中, 在BC3F3世代分别获得与轮回亲本性状相似的隐性核不育系C815G和五丰G。配合力分析表明, C815G和五丰G分别具有与C815S和三系不育系五丰A基本相同的配合力水平。本研究为隐性核不育系的创制提供了新的基因和材料资源, 并证实了隐性核不育系代替现有三系和两系不育系的可行性。

关键词: 水稻, 雄性不育, ptc1, 图位克隆, 回交转育, 配合力分析

Abstract:

Cytoplasmic male sterile (CMS) lines and photoperiod/thermo-sensitive genic male sterile (PTGMS) lines are widely used as female parents in the commercial production of rice hybrid seeds. however, both systems have their intrinsic defects such as low usage of germplasm resources and unstable sterility. Recessive genic male sterile (GMS) lines, which overcome the problems of CMS and PTGMS lines, have played a key role in the development of next generation technologies for rice hybrid seed production. In this study, a GMS mutant ptc1-2 without pollen grains was identified from an irradiation-induced mutant library of 9311. Using a map-based cloning approach, a 257.37 kb deletion region was detected, which contained entire coding region of PTC1 on chromosome 9. PCR co-segregation analysis showed that the male sterility was completely associated with the ptc1-2 deletion region. The ptc1-2 deletion locus was then introgressed into a PTGMS line C815S and a CMS maintainer line Wufeng B by marker-assisted backcrossing. Corresponding GMS lines C815G and Wufeng G were obtained at BC3F3 generation, which showed the phenotypical similarity to the C815S and Wufeng B lines, respectively. Combining ability tests revealed that C815G and Wufeng G had the same combining ability as C815S and the CMS line Wufeng A in conventional field, respectively. These results indicated that ptc1-2 was a new allele of PTC1, which could be applied for breeding GMS lines and be the potential of GMS lines in rice hybrid seed production.

Key words: rice, male sterile, ptc1, map-based cloning, backcrossing, combining ability test

图1

ptc1-2突变体及野生型9311的表型 A: 灌浆期野生型9311和突变体ptc1-2的植株; B: 蜡熟期野生型9311和突变体ptc1-2的穗; C: 野生型9311和ptc1-2突变体的花器官; D: 野生型9311的花粉碘染; E: ptc1-2突变体植株的花粉碘染。标尺: 15 cm (A); 3 cm (B); 2 mm (C); 150 μm (D)。"

表1

本研究所用的部分引物"

序号
No.
标记/基因名称
Marker/gene name
上游引物序列
Forward primer (5'-3')
下游引物序列
Reverse primer (5'-3')
扩增产物长度
Length of amplified products (bp)
用途
Purpose
1 RM7039 GCACATTTGCCATTCTACCG GCCTTCCAGTGAGGTGACTC 168 定位 Mapping
2 RM257 CAGTTCCGAGCAAGAGTACTC GGATCGGACGTGGCATATG 147 定位 Mapping
3 D9.168 CAATGTTAAAAGTGATGGGGTC GGGAGAGAGTGAGAGAATAGAG 171 定位 Mapping
4 D3102 ATCTCGCAGTTTACATGCAG ACGGAAATAAAATTCAGTTGGT 450 定位 Mapping
5 D3676 ATACTTATTGCGTTTCATGGTCA CTTGAATGGACTCGACGCTA 462 定位 Mapping
6 D5146 ATACACCAATGCCTAGCGGAA CTCTCCCCTCGGTGACCAGA 492 定位 Mapping
7 D461 CTATGTCATCAACTGCGTGCC ATTCAGTTCGTTGCCAACTCG 454 定位 Mapping
8 D27560 ACATCAGGTTCATCACGGAGC GTCCCCACCTTCGTCCACT 601 定位 Mapping
9 D27420 CATTCCAGAGCATATTGAAACGA ATTTACATCCATGACCCGTCT 527 定位 Mapping
10 D27580 TGGTTCATCGCTCTTCTTCGTT CATGCTGCCTCCATACGGGAA 606 定位 Mapping
序号
No.
标记/基因名称
Marker/gene name
上游引物序列
Forward primer (5'-3')
下游引物序列
Reverse primer (5'-3')
扩增产物长度
Length of amplified products (bp)
用途
Purpose
11 D27590 CTTTACCTATGTTCAAGCCTTCG CCTAACAAGGTCAGACGCATC 735 定位 Mapping
12 D27650 CTCTTGTTGTTCTTCATCGTCCT CATGCACCAGCAGCAACCA 451 定位 Mapping
13 D27820 ATGCGAGTCTATAAATTGCACCT GTCCATCAGCTCCGTCGAGA 468 定位 Mapping
14 1664SP1 GCACACTGCACGGCGACGTTTAGG 侧翼序列分离 Isolating flanking sequences
15 1664SP2 ACGATGGACTCCAGTCTGGCTGCCGTGGGAATTAGAGCAT 侧翼序列分离 Isolating flanking sequences
16 1664SP3 CCCTCCAGGAGATTGTCTAAAATTGACTTT 侧翼序列分离 Isolating flanking sequences
17 1664F1 CATCTCGCAGTTTACATGCAG ptc1-2标记 ptc1-2 markers
18 1664R1 AGTCTACTCGAGCTACTACCG ptc1-2标记 ptc1-2 markers
19 1664R2 CCATCTGAAACTAGTACTCCCA ptc1-2标记 ptc1-2 markers
20 Tms5 ATCCCACAAATCCTTTAGCA CCGTTATAGATAGACCCGAGA RsaI/329/414 tms5标记
tms5 markers

表2

ptc1-2突变体的遗传分析"

组合
Cross
F1结实率
Seed-setting rate of F1
(%)
F2 χ2(1:3) χ20.05
野生型植株数
No. of wild-type plants
突变体植株数
No. of mutant plants
ptc1-2/9311 89.2 312 92 0.953 3.841
ptc1-2/明恢63 ptc1-2/Minghui 63 88.1 351 107 0.570 3.841

图2

ptc1-2的图位克隆 A: ptc1-2位点粗定位在9号染色体长臂的RM7039和RM257两个标记间; B: ptc1-2基因精细定位至D3102和D3676之间, 物理距离为573 bp; C: ptc1-2位点包含从第15,324,556 bp到第15,583,926 bp共259,370 bp缺失, 该缺失片段包含整个PTC1基因。"

图3

ptc1-2位点侧翼序列的分离及共分离分析 A: ptc1-2位点两侧基因组序列分离第2轮TAIL-PCR扩增结果; B: ptc1-2突变体的共分离验证。雄性不育表型的F2植株扩增出的产物长546 bp, 而表型正常的F2植株扩增出的产物长811 bp或同时有546 bp和811 bp两条带。M为D2000 marker。"

表3

C815S、C815G、五丰A和五丰G的农艺性状分析"

性状
Trait
C815S C815G 五丰A
Wufeng A
五丰G
Wufeng G
抽穗期Heading date (d) 82.60±0.47 81.60±0.47 72.00±0.81 71.60±0.47
株高Plant height (cm) 67.50±1.75 70.60±3.13 68.60±1.02 72.30±2.59
总分蘖数Total tiller number 8.00±0.81 8.30±0.47 8.00±0.81 8.30±0.47
穗长Panicle length (cm) 23.43±0.16 22.96±0.09 23.26±0.12 23.83±0.20
穗粒数No.of spikelets per panicle 169.30±4.10 167.60±3.39 174.00±4.08 180.00±2.44
粒长Grain length (mm) 8.64±0.13 8.84±0.08 8.88±0.04 8.84±0.10
粒宽Grain width (mm) 2.76±0.10 2.60±0.06 2.78±0.10 2.82±0.07

图4

C815G和五丰G的表型 A: 抽穗期C815S和C815G的植株; B: C815S和C815G的小穗; C: C815S和C815G花器官; D: C815S的花粉碘染; E: C815G的花粉碘染; F: 抽穗期五丰A和五丰G的植株; G: 五丰A和五丰G的小穗; H: 五丰A和五丰G花器官; I: 五丰A的花粉碘染; J: 五丰G的花粉碘染。标尺: 10 cm (A, F); 0.5 cm (B, C, G, H); 200 μm (D, E, I, J)。"

表4

C815G和五丰G配制的杂交组合的表型"

性状
Trait
C815S/+ C815G/+ 五丰A/+
Wufeng A/+
五丰G/+
Wufeng G/+
抽穗期Heading date (d) 84.60±4.84 84.30±4.98 81.45±5.84 82.13±5.78
株高Plant height (cm) 103.80±5.43 103.50±5.47 113.29±4.88 108.92±5.47*
总分蘖数Total tiller number 12.69±1.55 12.96±1.03 11.13±1.22 11.42±1.21
穗长Panicle length (cm) 23.15±0.62 23.23±0.89 23.18±1.12 23.29±0.97
穗粒数No.of spikelets per panicle 186.61±16.36 186.45±15.42 201.75±24.63 196.96±18.33
结实率Seed-setting rate (%) 83.90±4.70 83.92±3.72 87.83±5.21 88.38±4.79
粒长Grain length (mm) 8.94±0.36 8.94±0.40 8.88±0.25 8.90±0.27
粒宽Grain width (mm) 2.56±0.20 2.56±0.19 2.58±0.13 2.59±0.15
千粒重1000-grain weight (g) 24.43±1.91 24.35±1.99 24.92±2.04 24.97±2.00
单株产量Grain yield per plant (g) 52.44±9.03 53.93±9.27 54.05±9.24 51.75±5.04

表5

C815S和C815G的田间配合力方差分析"

性状
Trait
变因Variance
区组
Block
组合Combination 父本
Male parent
母本
Female parent
父本×母本
Male × female
误差
Error
自由度DF 3 29 14 1 14 87
抽穗期Heading date 16.03** 99.92** 126.39 2.7 80.4** 1.4
株高Plant height 128.76** 122.89** 208.11** 2.7** 46.25 26.8
总分蘖数Total tiller number 5.34 7.23** 10.2* 2.13 4.64* 2.47
穗长Panicle length 1.14 2.43** 2.58 0.18 2.43** 0.56
穗粒数No. of spikelets perpanicle 462.34* 1046.13** 1018.12 0.71 1148.8** 142.64
结实率Seed-setting rate 0.005* 0.004** 0.003 0.001 0.004** 0.001
粒长Grain length 0.03 0.59** 0.54 0.0007 0.69** 0.03
粒宽Grain width 0.02 0.16** 0.15 0.0001 0.18** 0.01
千粒重1000-grain weight 0.24 15.82** 21.19* 0.23 11.56** 0.3
单株产量Grain yield per plant 1279.36** 348.89** 486.62* 66.11 231.37** 37.54

表6

五丰A和五丰G的田间配合力方差分析"

性状
Trait
变因Variance
区组
Block
组合Combination 父本
Male parent
母本
Female parent
父本×母本
Male × female
误差
Error
自由度DF 3 29 14 1 14 87
抽穗期Heading date 39.56** 140.12** 170.42 14.01 118.83** 3.24
株高Plant height 395.38** 130.89** 178.40** 571.60** 51.89** 50.23
总分蘖数Total tiller number 9.95** 6.23** 6.85 2.64 5.86** 2.11
穗长Panicle length 5.07** 4.55** 4.83 0.37 4.57** 0.78
穗粒数No. of spikelets per panicle 702.16** 1973.93** 2169.24 686.89 1870.55** 136.50
结实率Seed-setting rate 0.003** 0.01** 0.01 0.001 0.01** 0.001
粒长Grain length 0.59** 0.29** 0.29 0.01 0.30** 0.07
粒宽Grain width 0.10** 0.08** 0.05 0.01 0.13 0.02
千粒重1000-grain weight 1.14 16.88** 21.29 0.08 13.68** 0.57
单株产量Grain yield per plant 500.55** 234.86** 256.85 158.86 218.30** 70.25

表7

田间一般配合力相对效应值"

性状
Trait
C815S C815G 五丰A
Wufeng A
五丰G
Wufeng G
抽穗期Heading date 0.1776 -0.1776 -0.4177 0.4177
株高Plant height 0.1447 -0.1447 1.9643 -1.9643
总分蘖数Total tiller number -1.0399 1.0399 -0.2967 0.2967
穗长Panicle length -0.1653 0.1653 -0.2403 0.2403
穗粒数No. of spikelets per panicle 0.0411 -0.0411 1.2001 -1.2001
结实率Seed-setting rate 0.0795 -0.0795 -0.3093 0.3093
粒长Grain length -0.0280 0.0280 -0.0984 0.0984
粒宽Grain width 0.0391 -0.0391 -0.2902 0.2902
千粒重1000-grain weight 0.1784 -0.1784 -0.1012 0.1012
单株产量Grain yield per plant -1.4929 1.4929 2.1750 -2.1750
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