小麦抗条锈病基因YrC591分子标记的建立及应用
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摘要
我国条锈病危害普遍而严重，由于条锈病菌的高度变异性，小麦条锈病的
持续、有效控制问题一直没有解决。科学合理的应用抗锈基因选育抗病品种是
条锈病持续控制关键。
C591是源于印度的一个小麦品种，对当前流行校种抗性良好，又有很好的
农艺性状，以此为材料导入其它抗条锈基因，育成多基因抗性品种对小麦生产
意义重大。本研究的目的是用DNA分子标记技术，建立与小麦C591所含抗条锈
基因紧密连锁的分子标记，明确其在生产上的应用价值，辅助选育出含多个抗病
基因的小麦品种。
通过预备试验，设计出了适于C591的RAPD分析程序（94℃ 5分钟；94℃ 30
秒；36℃ 1分钟；0.3℃/秒至56℃；72℃延伸2分钟；36循环；72
℃ 7分钟）；PCR优化条件（25μ1反应体系：模板15ng；dNTPs150μM，Mg~(2+)2mM；
Taq聚合酶1U，引物0.4μM）。采用该程序供检测544个10碱基随机引物，
对C591×铭贤169 F2代抗感基因池（BSA）进行了RAPD分析，结果发现了与
YrC591紧密连锁的标记OPAN-19_(616)。经克隆、测序和引物设计转化成了SCAR
标记。
SC-OPAN19_(526)a 5’-GACCTTGATGAGTAAGTGTTATAC-3′
SC-OPAN19_(526)b 5’-CTTCTTCCAAGCATTGATAGGAGG-3′
经F2代分离单株的检测证实了SCAR标记的可靠性。
本研究还用已经获得的Yr8、Yr10抗条锈基因的SCAR标记对抗病育种材
料进行了辅助选择，筛选出具有以上两对抗条锈基因的材料。并建立了一套快
速、简易、实用的抗锈基因鉴定方法。
Stripe rust (yellow rust), caused by Puccinia striformis f. sp. tritici
West., is one of the most important diseases on wheat in all the wheat-
growing areas in the world, especially in China. Utilization of resistant
cultivars is the most economic, effective and environment-sound way to
control the disease. However, rapid reakdown of disease resistance
resulted from the use of one or very few simple genes in a large area and
then the disease prevailing is the problem puzzled breeders and
phytopathologist for many years. Molecular markers linked to resistance
gene can be very helpful to solve the problem.
The wheat cultivar C591, introduced from India, is resistant to
almost all the races of stripe rust in China at present. In this article the
bulked segregation analysis (BSA) was used to develop the molecular
makers linked to the resistance gene YrC59] in the cultivar.
At first, the reaction mix components and RAPD program were
optimized. The PCR mix in 25 ii 1 included: template DNA 1 5ng, dNTPs
150 i-i M, MgC12 2mM; Taq DNA Polymerase 1U, random primer 0.4
i-i M. The temperature profile was: 94C5min; then 940C 30secs, 360C
1mm, 0.30C/sec to 560C, 720C 2mm, for 36cycles; at last 720C 7mm.
Resistant DNA pool and susceptible DNA pooi were made up
respectively from 88 resistant F2 plants and 79 susceptible F2 plants
derived from a cross between the susceptible cultivar ingxian 169
and the cultivar 59 1 A total of 544 10-mer random primers had been
used to identify the RAPD markers linked to YrC59J gene. One out of
three polymorphic DNA fragments, i. e. OPAN-19616 amplified by the
primer OPAN- 19, was found closely linked to the gene YrC591.
According to the sequences of the DNA fragment OPAN- 1 6I6, a pair of
specific primers was designed. By using the specific primers the YrC591
gene can be detected effectively in wheat breeding materials.
Development of the SCAR marker for YrC591 gene will promote the use
of the resistance gene in wheat breeding for resistance to stripe rust.
Combined artificial inoculation and using the SCAR markers linked
to Yr8 and YrlO respectively, some plants with Yr8 and YrlO genes or
Yr8 and YrC59I genes were gained from the F2 and F3 segregating
population of CP93-6-19 X Compair, CP93-17-30 X Compair and CP93-
17-31 X Compair. The results showed that the markers and the technique
were usable and reliable.
持续、有效控制问题一直没有解决。科学合理的应用抗锈基因选育抗病品种是
条锈病持续控制关键。
C591是源于印度的一个小麦品种，对当前流行校种抗性良好，又有很好的
农艺性状，以此为材料导入其它抗条锈基因，育成多基因抗性品种对小麦生产
意义重大。本研究的目的是用DNA分子标记技术，建立与小麦C591所含抗条锈
基因紧密连锁的分子标记，明确其在生产上的应用价值，辅助选育出含多个抗病
基因的小麦品种。
通过预备试验，设计出了适于C591的RAPD分析程序（94℃ 5分钟；94℃ 30
秒；36℃ 1分钟；0.3℃/秒至56℃；72℃延伸2分钟；36循环；72
℃ 7分钟）；PCR优化条件（25μ1反应体系：模板15ng；dNTPs150μM，Mg~(2+)2mM；
Taq聚合酶1U，引物0.4μM）。采用该程序供检测544个10碱基随机引物，
对C591×铭贤169 F2代抗感基因池（BSA）进行了RAPD分析，结果发现了与
YrC591紧密连锁的标记OPAN-19_(616)。经克隆、测序和引物设计转化成了SCAR
标记。
SC-OPAN19_(526)a 5’-GACCTTGATGAGTAAGTGTTATAC-3′
SC-OPAN19_(526)b 5’-CTTCTTCCAAGCATTGATAGGAGG-3′
经F2代分离单株的检测证实了SCAR标记的可靠性。
本研究还用已经获得的Yr8、Yr10抗条锈基因的SCAR标记对抗病育种材
料进行了辅助选择，筛选出具有以上两对抗条锈基因的材料。并建立了一套快
速、简易、实用的抗锈基因鉴定方法。
Stripe rust (yellow rust), caused by Puccinia striformis f. sp. tritici
West., is one of the most important diseases on wheat in all the wheat-
growing areas in the world, especially in China. Utilization of resistant
cultivars is the most economic, effective and environment-sound way to
control the disease. However, rapid reakdown of disease resistance
resulted from the use of one or very few simple genes in a large area and
then the disease prevailing is the problem puzzled breeders and
phytopathologist for many years. Molecular markers linked to resistance
gene can be very helpful to solve the problem.
The wheat cultivar C591, introduced from India, is resistant to
almost all the races of stripe rust in China at present. In this article the
bulked segregation analysis (BSA) was used to develop the molecular
makers linked to the resistance gene YrC59] in the cultivar.
At first, the reaction mix components and RAPD program were
optimized. The PCR mix in 25 ii 1 included: template DNA 1 5ng, dNTPs
150 i-i M, MgC12 2mM; Taq DNA Polymerase 1U, random primer 0.4
i-i M. The temperature profile was: 94C5min; then 940C 30secs, 360C
1mm, 0.30C/sec to 560C, 720C 2mm, for 36cycles; at last 720C 7mm.
Resistant DNA pool and susceptible DNA pooi were made up
respectively from 88 resistant F2 plants and 79 susceptible F2 plants
derived from a cross between the susceptible cultivar ingxian 169
and the cultivar 59 1 A total of 544 10-mer random primers had been
used to identify the RAPD markers linked to YrC59J gene. One out of
three polymorphic DNA fragments, i. e. OPAN-19616 amplified by the
primer OPAN- 19, was found closely linked to the gene YrC591.
According to the sequences of the DNA fragment OPAN- 1 6I6, a pair of
specific primers was designed. By using the specific primers the YrC591
gene can be detected effectively in wheat breeding materials.
Development of the SCAR marker for YrC591 gene will promote the use
of the resistance gene in wheat breeding for resistance to stripe rust.
Combined artificial inoculation and using the SCAR markers linked
to Yr8 and YrlO respectively, some plants with Yr8 and YrlO genes or
Yr8 and YrC59I genes were gained from the F2 and F3 segregating
population of CP93-6-19 X Compair, CP93-17-30 X Compair and CP93-
17-31 X Compair. The results showed that the markers and the technique
were usable and reliable.
引文
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