Codominance Functional Marker of Bacterial Blight Resistant Xa7 in Rice
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摘要:
目的 对水稻白叶枯病广谱抗性基因Xa7进行精准检测和世代跟踪，通过分子标记检测Xa7基因材料的纯合或杂合型。 方法 根据Xa7、xa7和无等位基因型序列的差异，通过Premier 5软件设计了含4条引物Xa7-F、Xa7-R、Xa7null-F、Xa7null-R的功能标记Xa7fun，且采用PCR方法分别对不同遗传资源材料进行分子标记特异性检测和验证，自然高温下于孕穗期对含Xa7基因的3份杂交改良系及亲本采用剪叶接种法接种7个白叶枯病菌菌株进行抗性鉴定，并于成熟期考察记录其农艺性状。 结果 分子标记特异性检测结果表明，Xa7基因纯合型材料R084可扩增出大小为91 bp的条带，无等位基因型材料Nip可扩增出大小为153 bp的条带，杂合体Nip/R084可扩增出大小为91 bp、153 bp的条带，共显性标记Xa7fun得到的电泳条带与引物设计时预测的目标片段完全吻合；18份不同类型的种质资源均未扩增出91 bp大小的功能条带，说明这些材料均不含Xa7基因；杂交改良系Ry-1、Ry-2、Ry-3均仅含有91 bp大小的功能条带，表明Xa7基因纯合；在高温下，华占对7个菌株表现为高感、中感或感病，R084除对菌株PX099感病外，对其余6菌株均为高抗、中抗或抗病，Ry-1对GDA2、HNA1-4、FuJ、GD1358、YN24等5个菌株为高抗或中抗，Ry-2对GDA2、GD1358、HNA1-4、PXO86、YN24等5个菌株为高抗或抗病，Ry-3对HNA1-4、FuJ、GDA2、GD1358、PXO86、YN24 等6个菌株均为高抗或中抗。因此，Xa7基因的渗入对华占改良系Ry-1、Ry-2、Ry-3的白叶枯病抗性有大幅度的提高；通过对3个改良系和亲本等农艺性状分析表明，3个改良系的生育期、株高、穗长、单株总粒数、结实率、千粒重介于R084和华占之间，Ry-1和Ry-3单株有效穗数显著高于华占和R084，Ry-2与华占、R084差异不显著，单株产量与华占或R084相比差异不显著。 结论 本研究开发的功能标记Xa7fun能够准确、高效地识别水稻Xa7基因纯合型、杂合型等，而且Xa7基因的渗入不会造成杂交改良系重要农艺性状变差，可在水稻白叶枯病抗性分子育种中推广应用。 Abstract:Objective A codominance functional marker of the broad-spectrum bacterial blight resistance gene,Xa7, of rice was identified for accurate detection, generation tracking, and differentiation between homozygous and hemizygous genotypes of the gene. Methods A potential functional marker containing 4 primers was designed using Premier 5 software and based on the differences on the sequences of Xa7, xa7, and allele-free genome.The molecular distinctness of the marker in different materials was verified by PCR.Three crossbreed lines of Xa7 and their parents were inoculated with 7 bacterial blight pathogens at booting stage to examine the affected agronomic traits at maturation. Results The homozygous R084 of Xa7 could be amplified into a 91 bp band and the Nip free of allele with a 153 bp band; while the heterozygote Nip/R084, 91 bp and 153 bp bands.The candidate codominance marker, Xa7fun, amplified fragments that matched the predicted target bands.No 91 bp fragment was amplified from 18 germplasms of varied types indicating a lack of Xa7 in them.Whereas Ry1, Ry2, and Ry3 had 91 bp band suggesting the inclusion of homozygous Xa7.Under an elevated temperature, Huazhan responded to the 7 bacterial blight pathogens as highly sensitive (HS), intermediate sensitive (MS), or sensitive (S); R084 to 6 of the 7 pathogens (HNA1-4, FuJ, GDA2, GD1358, PX086, and YN24) as highly resistant (HR), intermediate resistant (MR) or resistant (R); Ry-1 to 5 pathogens (GDA2, HNA1-4, FuJ, GD1358, and YN24) as HR or MR; Ry-2 to 5 pathogens (GDA2, GD1358, HNA1-4, PXO86, and YN24) as HR or R; and Ry-3 to 6 pathogens (HNA1-4, FuJ, GDA2, GD1358, PXO86, and YN24) as HR or MR.Therefore, the infiltration of Xa7 in the crossbred and improved lines RY-1, RY-2, and RY-3 significantly accentuated the blight resistance of Huazhan. Conclusion The homozygous or hemizygous Xa7 could be accurately differentiated by the currently identified codominance functional marker Xa7fun.The Xa7 introgression did not significantly alter the critical agronomic traits in the hybridization from generation to generation and could be safely applied in breeding bacterial leaf blight resistant rice varieties． -
Key words:
- Rice /
- bacterial blight /
- Xa7 gene /
- molecular maker /
- resistance
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图 2 梯度PCR检测Xa7fun分子标记
M为DNA marker(DL2000), 1～3分别为R084、 Nip、Nip/R084。
Figure 2. Detection of Xa7fun by gradient PCR
M: DNA marker (DL2000); 1-3: R084, Nip, and Nip/R084, respectively.
图 3 Xa7fun功能标记对部分水稻品种的分子检测
A中，M：DNA marker(DL2000)，+：R084，1～18对应水稻材料为长粒香、吉粳88、中花11、越光、绥粳4号、昆明小白谷、金23B、野香B、中浙2B、荃9311B、华占、农香39、五山丝苗、广恢390、R900、望恢006、黄华占、湘早籼45号。B中19～38为Y629的20个单株，C中39～58为Y192对应20个单株。
Figure 3. Molecular detection of Xa7fun in different rice varieties
In A, M: DNA marker(DL 2000), 1–18: Changlixiang, Jijing 88, Zhonghua 11, Yueguang, Suijing 4, Kunmingxiaobaigu, Jin 23B, Yexiang B, Zhongzhe 2B, Quan 9311B, Huazhan, Nongxiang 39, Wushansimiao, Guanghui 390, R900, Wanghui 006, Huanghuazhan, and Xiangzaoxian 45, respectively (A); 19-38: 20 individual plants of Y629 (B); 39-58: 20 individual plants of Y192 (C).
图 4 含Xa7基因高世代杂交后代的选育和分子检测
M：DNA marker(DL2000)，+：R084。A为Ry-1、Ry-2、Ry-3选育图，B为连锁标记M5检测结果，C为共显性功能标记Xa7fun检测结果。
Figure 4. Breeding of filial generations and molecule detection of Xa7
M：DNA marker(DL2000), +：R084. A: Breeding diagrams on Ry-1, Ry-2, and Ry-3; B: detection of linkage marker M5; C: detection of codominance functional marker Xa7fun.
图 5 白叶枯病菌株诱发期间的温度
Figure 5. Temperature at time of bacterial blight pathogen induction
表 1 Xa7fun标记引物序列
Table 1. Sequences of Xa7fun primers
引物名称
Primer name引物序列
Primer sequence(5′-3′)碱基数
Base number/bpXa7-R GCTCTTCAAGTGTGCGATGC 20 Xa7-F AGCCCTGACTGCTAAAACCA 20 Xa7null-R GTACATCATTGTCCCCACGG 20 Xa7null-F AACCGAGGGAGGTGATTTGC 20 
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表 2 含Xa7基因高世代杂交后代对7种白叶枯病菌株的抗性
Table 2. The filial generations of Xa7 gene to 7 bacterial leaf blight strains
品系 Lines FuJ GDA2 GD1358 HNA1-4 PXO86 PXO99 YN24 病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
Resistance病斑长度
Lesion
length/
cm抗性
ResistanceRy-1 3.7 MR 0.5 HR 5 MR 0.9 HR 10.2 MS 13.7 S 3.6 MR Ry-2 5.9 MS 0.4 HR 0.5 HR 0.5 HR 4.7 MR 16.3 S 4.1 MR Ry-3 3.5 MR 4.8 MR 4.5 MR 0.7 HR 4.9 MR 15.2 S 4.9 MR R084 2.1 R 0.2 HR 0.2 HR 0.3 HR 4.1 MR 15.3 S 1.7 R 华占
Huazhan24.6 HS 26.6 HS 12 MS 10.8 MS 8.9 MS 9.7 MS 19 S
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表 3 Xa7基因对高世代杂交后代农艺性状的影响
Table 3. Effects of Xa7 on agronomic traits of filial generation hybrids
品系
Lines播始历期
Days to flower/d株高
Plant height/cm单株有效穗数
Panicles per plant穗长
Panicle length/cm单株总粒数
Grains per plan结实率
Seed set/%千粒重
1000-grain weight/g单株产量
Yield per plant/gRy-1 73 117.3±2.08 Bc 15.5 ±1.00 Aa 22.2±0.05 BCb 1820.3±108.55 ABCbc 90.7±0.28 Aa 22.6±0.34 Bb 37.3±2.70 Aa Ry-2 73 119.3±2.52 ABbc 11.3 ±0.58 Bb 22.8±0.72 Bb 2336.0±321.38 Aa 89.1±0.79Aa 21.5±0.28 Bb 39.6±0.68 Aa Ry-3 77 123.0±2.65 Aab 15.3 ±0.50 Aa 23.0±0.15 Bb 1675.3±264.06 BCcd 90.2±0.40Aa 27.5±1.32 Aa 41.8±6.95 Aa R084 79 124.7±1.53 Aa 11.0 ±1.41 Bb 21.2±0.54 Cc 1312.3±128.39 Cd 90.0±1.63Aa 27.8±1.76 Aa 38.4±1.58 Aa 华占
Huazhan73 98.2±1.30 Cd 13.0 ±2.45 ABb 26.0±0.50 Aa 2208.0±146.08 ABab 84.2±1.05 Bb 20.1±0.28 Bb 39.7±1.37 Aa 不同大、小写字母分别表示同一品种在不同氮素水平下差异极显著（P＜0.01）或显著（P＜0.05）。
Data with different uppercase letters represent extremely significant differences (P＜0.01); those with different lowercase letters, significant differences (P＜0.05).
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