Improving rice blast resistance and fragrance of rice maintainer through marker-assisted selection
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摘要:目的
优质抗病水稻保持系的获得是改良不育系品质与抗性的前提，利用分子标记辅助选择技术进行定向改良，可以加速优异水稻保持系的改良进程。
方法以‘恒丰B’、‘广8B’为轮回亲本，利用携带香味基因Badh2和抗稻瘟病基因Pita的‘B39’作为供体亲本，利用分子标记开展连续回交育种，定向改良‘恒丰B’、‘广8B’的品质和抗性性状。
结果通过多代回交选育，获得了14份BC3F2改良材料。利用改良单株系与对应的不育系测交，测保对应的小区不育等级为全不育和高不育；通过品质和抗性鉴定，改良后的目标株系香味评级为香味较浓；稻瘟病抗性均有进一步提高，经病圃鉴定表现为抗或高抗稻瘟病；稻米品质表现为米粒细长、低直链淀粉、低垩白；其他农艺经济性状与轮回亲本相似；改良后的株系遗传背景回复率在80%~90%之间，达到预期回复效果。
结论结合分子标记辅助选择的定向改良缩短了优质抗性保持系改良的育种进程，在较短时间内获得了在稻米品质、抗病性、香味性状上具有改良的新保持系，实现了‘恒丰B’、‘广8B’在香味性状和稻瘟病抗性方面的定向改良，为进一步协同改良三系杂交水稻的稻米品质和抗性提供了新的材料基础。
Abstract:ObjectiveThe acquisition of high quality disease-resistant rice maintainers is the premise of improving the quality and resistance of sterile lines. The molecular marker-assisted selection (MAS) can be used for oriented breeding and accelarating the breeding process of elite rice maintainers.
Method‘Hengfeng B’ and ‘Guang 8B’ were used as recurrent parents, and ‘B39’ carrying fragrance gene Badh2and rice blast resistance gene Pita was used as donor parent. The successive backcross breeding and MAS technology was combined to improve the quality and resistance traits of these two maintainer lines.
ResultThrough multi-generation backcross breeding, 14 samples of BC3F2 improved materials were obtained. The improved single-plant line was tested with the corresponding sterile line, and the sterility grades of the corresponding plot were fully sterile and high sterile. Through quality and resistance identification, most of improved target strains were rated as strong aroma. The resistance to rice blast significantly improved and the disease nursery identification showed that they were resistant or highly resistant to rice blast. Rice quality was characterized by slender rice grains with low amylose content and low chalkiness. Other agronomic and economic characteristics were similar to the recurrent parent. The genetic background recovery rates of most strains were 80% to 90% which met the estimated recovery effect.
ConclusionMAS shortens the process of trait improving for maintainer lines, helps acquiring improved maintainers with better quality, stronger blast resistance and fragrance, and realizes the oriented genetic improvement of ‘Hengfeng B’ and ‘Guang 8B’, which lays a foundation for further integrated improvement in rice quality and resistance of three-line hybrid rice.
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图 1 分子标记辅助选择(MAS)改良水稻保持系香味和稻瘟病抗性技术流程图
Figure 1. Process scheme of improving rice fragrance and blast resistance of rice maintainer through molecular marker-assisted selection(MAS)
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图 2 亲本和改良后代BC3F2株系Badh2基因分型毛细管电泳结果
M：DNA 标记；1：‘B39’；2：‘恒丰B’；3：‘广8B’；4~17：‘恒丰B’改良BC3F2株系；18~32：‘广8B’改良BC3F2株系
Figure 2. Genotyping of Badh2 of parents and the genetically-improved BC3F2 lines detected by capillary electrophoresis
M: DNA marker; 1: ‘B39’; 2: ‘Hengfeng B’; 3:‘Guang 8B’; 4−17: Improved BC3F2 lines from ‘Hengfeng B’; 18−32: Improved BC3F2 lines from ‘Guang 8B’
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图 3 亲本和改良后代BC3F2株系Pita基因分型HRM分析结果
灰色曲线表示位点含纯合有利等位基因(‘B39’为此基因型)；红色曲线表示位点不含抗性有利等位基因；蓝色曲线表示位点杂合
Figure 3. HRM genotyping of Pita of parents and the genetically-improved BC3F2 lines
Grey lines represent the rice lines harboring elite homozygous allele(‘B39’has the genotype)，red lines represent the rice lines not carrying the elite allele, and blue lines represent the rice lines harboring heterozygous alleles
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图 4 ‘恒丰B’及其后代改良株系H2和H4穗型对比
Figure 4. Panicles of ‘Hengfeng B’ and its genetically improved line，H2 and H4
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图 5 ‘广8B’及其后代改良株系G4和G5穗型对比
Figure 5. Panicles of ‘Guang 8B’ and its genetically improved lines, G4 and G5
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图 6 ‘恒丰B’及其后代改良株系H2和H4株型对比
Figure 6. Plant types of ‘Hengfeng B’ and its genetically improved lines, H2 and H4
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图 7 ‘广8B’及其后代改良株系G4和G5株型对比
Figure 7. Plant types of ‘Guang 8B’ and its genetically improved lines，G4 and G5
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图 8 部分测保不育株花粉活力与株型调查结果
A：H2测保不育株花粉；B：G3测保不育株花粉；C：H2测保不育株株型；D：G3测保不育株株型
Figure 8. Pollen vigors and plant types of partial sterile lines
A: Pollen from sterile line maintained by H2; B: Pollen from sterile line maintained by G3; C: Plant type of sterile line maintained by H2; D: Plant type of sterile line maintained by G3
表 1 目标检测基因及引物序列
Table 1 Target genes and molecular marker sequences
分型方法
Genotyping method靶基因
Target
gene基因型
Allele表型 Phenotype 扩增产物/bp
Amplicon退火温度/℃
Annealing temperature正向/反向引物
Forward/reverse primer引物序列
Primer sequence毛细管电泳
Capillary electrophoresisBadh2 第7外显子
8 bp缺失
8 bp deletion
in the
7th exon香 Fragrant 360+215 57 正向 Forward GGGAGTTATGAA
ACTGGTAAAAAGA反向 Reverse AACCATAGGAG
CAGCTGAAATA无8 bp缺失
No 8 bp
deletion不香
Not fragrant368+188 57 正向 Forward CTTCCTTCAGG
TGTGCTAAACA反向 Reverse GAATGATGCTCA
AAGTGTCTTGA高分辨率溶
解曲线分析
HRMPita T 感 Sensitive 244+132 56 正向 Forward CCCAGGTTACAA
CTTACAAGGA反向 Reverse CTCTGAAGACG
TGAAGAGGATTG 抗 Resistant 244+160 56 正向 Forward CTTCTTTCTTT
CTCTGCCGTGG反向 Reverse TCATCAAGTCAG
GTTGAAGATGC
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表 2 ‘恒丰B’改良后代BC3F2稻瘟病抗性鉴定结果
Table 2 Rice blast resistance identification of the genetically-improved BC3F2lines from ‘Hengfeng B’
材料
Material来源
OriginPita基因分型
Genotyping of Pita阳江穗瘟感病级数
Disease grade of neck
blast in Yangjiang发病率/%
Incidence评价
EvaluationH1 BC3F2(恒丰B/B39) 纯合 Homozygous 3 10 抗 Resistant H2 BC3F2(恒丰B/B39) 纯合 Homozygous 3 5 抗 Resistant H3 BC3F2(恒丰B/B39) 纯合 Homozygous 1 5 高抗 Highly resistant H4 BC3F2(恒丰B/B39) 杂合 Heterozygous 3 20 抗 Resistant H5 BC3F2(恒丰B/B39) 纯合 Homozygous 3 10 抗 Resistant H6 BC3F2(恒丰B/B39) 纯合 Homozygous 3 20 抗 Resistant A1 恒丰B 无 None 5 20 中抗 Medium resistant A3 B39 纯合 Homozygous 1 1 高抗 Highly resistant
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表 3 ‘广8B’改良后代BC3F2稻瘟病抗性鉴定结果
Table 3 Rice blast resistance identification of the genetically-improved BC3F2 lines from ‘Guang 8B’
材料
Material来源
OriginPita基因分型
Genotyping of Pita阳江穗瘟病级
Disease grade of neck
blast in Yangjiang发病率/%
Incidence评价
EvaluationG1 BC3F2(广8B/B39) 纯合 Homozygous 3 5 抗 Resistant G2 BC3F2(广8B/B39) 纯合 Homozygous 1 5 高抗 Highly resistant G3 BC3F2(广8B/B39) 杂合 Heterozygous 3 20 抗 Resistant G4 BC3F2(广8B/B39) 纯合 Homozygous 3 20 抗 Resistant G5 BC3F2(广8B/B39) 纯合 Homozygous 3 10 抗 Resistant G6 BC3F2(广8B/B39) 纯合 Homozygous 3 10 抗 Resistant G7 BC3F2(广8B/B39) 杂合 Heterozygous 3 5 抗 Resistant G8 BC3F2(广8B/B39) 纯合 Homozygous 1 5 高抗 Highly resistant A2 广8B 无 None 3 20 抗 Resistant A3 B39 纯合 Homozygous 1 1 高抗 Highly resistant
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表 4 亲本和改良后代BC3F2株系的主要农艺性状
Table 4 Main agronomic characteristics of parents and the genetically-improved BC3F2 lines
材料
Material株高/cm
Plant
height剑叶
长/cm
Leaf length剑叶
宽/cm
Leaf width单株穗质量/g
Panicle
weight
per plant有效穗数
Effective panicle number穗长/cm
Panicle length每穗粒数
Grains per spike结实率/%
Seed setting rate长宽比
Grain length-to-width ratio千粒质量/g
1 000-seed weight恒丰B 108.6±0.07 37.3±2.78 2.3±0.06 27.2±1.42 9.0±0.00 22.4±0.33 180.4±8.80 80.3±0.67 4.5±0.02 19.7±0.23 H1 109.8 36.8 2.3 28.5 7 20.95 207.25 79.32 4.56 19.79 H2 108.7 38.5 2.3 29.1 7 24.12 199.42 81.15 4.33 19.56 H3 109.8 39.8 2.2 31.2 8 24.01 201.17 79.65 4.35 19.96 H4 111.3 35.8 2.4 27.6 8 20.62 197.25 81.02 4.34 19.71 H5 112.3 36.7 2.3 27.5 7 23.63 210.71 80.62 4.27 20.09 H6 106.5 40.6 2.3 29.8 7 21.71 198.60 80.31 4.32 20.11 广8B 109.9±0.75 37.3±0.34 1.9±0.03 30.8±2.52 7.6±0.33 22.2±0.43 206.5±3.40 73.0±1.15 4.1±0.12 15.8±0.03 G1 110.9 37.4 1.8 34.1 8 25.95 207.11 74.15 4.16 14.63 G2 111.3 38.5 1.9 28.8 8 23.36 196.75 75.03 4.14 14.93 G3 112.3 36.7 1.9 31.6 7 22.97 187.00 71.98 4.20 15.24 G4 111.2 35.7 2.0 32.8 9 22.33 195.08 71.65 4.06 15.61 G5 113.7 36.5 1.8 27.9 8 22.44 214.22 72.81 4.08 17.08 G6 112.8 38.5 1.8 28.5 8 21.83 202.88 76.01 4.12 16.08 G7 110.6 39.2 2.1 29.2 9 22.22 196.75 69.98 4.04 15.96 G8 112.4 37.5 1.9 28.9 8 23.14 189.64 71.65 4.16 16.15 B39 98.9±1.02 41.1±0.50 1.9±0.03 24.3±0.73 8.6±0.33 22.9±0.64 166.4±24.88 79.0±8.51 3.7±0.77 19.2±0.19
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表 5 亲本和改良后代BC3F2株系的稻米品质性状
Table 5 Rice quality traits of parents and the genetically-improved BC3F2 lines
材料
Material垩白粒率/%
Chalky grain percentage垩白度/‰
Chalk
degree糙米率/%
Brown rice rate精米率/%
Milled rice ratio整精米率/%
Head yieldw(直链淀
粉)/%
Amylose
content胶稠度/mm
Gel consistency碱消值
Alkali spreading value香味性状
Fragrance恒丰B 4.3±0.16 13.8±0.12 73.8±0.48 62.7±0.61 50.1±0.64 18.6±0.41 61.7±0.26 5.7±0.17 不香 H1 4 13.67 72.97 63.14 50.91 18.54 63.4 5.74 香 H2 3 12.57 74.55 63.25 49.67 17.56 62.9 5.68 香 H3 3 14.52 74.96 63.21 51.34 19.45 63.7 5.64 香 H4 4 11.54 73.53 64.50 50.29 18.21 61.7 5.24 香 H5 5 13.24 71.97 61.34 51.98 17.96 62.8 5.36 香 H6 5 12.47 72.58 62.25 50.47 18.47 63.7 5.28 香 广8B 3.9±0.64 19.7±0.25 71.6±0.67 61.8±0.94 51.5±0.47 17.9±0.28 63.8±0.54 5.6±0.23 不香 G1 4 18.45 71.45 61.47 51.74 17.58 64.5 5.91 香 G2 3 17.21 70.86 60.57 51.02 17.12 65.3 5.64 香 G3 4 16.84 70.67 60.97 50.86 18.56 64.1 5.17 香 G4 5 19.57 71.96 61.56 50.34 18.23 65.8 5.87 香 G5 3 16.58 71.54 61.32 51.21 17.56 64.7 5.63 香 G6 4 18.59 71.61 61.87 51.23 16.87 65.9 5.67 香 G7 4 15.34 70.34 60.25 50.67 17.54 66.7 5.85 香 G8 3 18.57 72.47 62.19 51.11 16.98 64.8 5.47 香 B39 3.1±0.25 12.9±0.64 75.4±0.23 64.9±0.47 53.8±0.96 16.4±0.84 65.9±0.78 5.4±0.36 香
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表 6 部分改良BC3F2株系遗传背景回复率
Table 6 Genetic background reversion rate of some genetically-improved BC3F2 lines
材料
Material遗传背景回复率/%
Genetic background
reversion rate杂合SNP位点
比例/%
Percentage of
heterozygous SNP lociH1 85.64 3.16 H2 83.71 5.19 H3 86.96 2.17 G1 89.14 3.67 G2 87.23 2.96 G3 89.16 1.39 G4 81.29 4.61 平均 Average 86.16 3.31
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