WO2018232563A1 - 小麦-长穗偃麦草抗赤霉病易位系的选育方法及分子标记 - Google Patents

小麦-长穗偃麦草抗赤霉病易位系的选育方法及分子标记 Download PDF

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WO2018232563A1
WO2018232563A1 PCT/CN2017/088985 CN2017088985W WO2018232563A1 WO 2018232563 A1 WO2018232563 A1 WO 2018232563A1 CN 2017088985 W CN2017088985 W CN 2017088985W WO 2018232563 A1 WO2018232563 A1 WO 2018232563A1
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seq
wheat
sequence
scab
molecular marker
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PCT/CN2017/088985
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French (fr)
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韩方普
石庆华
郭宪瑞
符书兰
王婧
郭翔
张晶
刘亚林
苏汉东
刘阳
冯超
袁静
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中国科学院遗传与发育生物学研究所
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  • the present disclosure relates to the field of molecular biology technology, and in particular to a method for breeding a wheat-long-buckthorn buckwheat-resistant scab line and a molecular marker thereof, and the present disclosure also relates to a primer for amplifying the molecular marker, The application of molecular markers in wheat genetic breeding.
  • Fusarium head blight (FHB) is a major disease that damages wheat production. It is a worldwide fungal disease caused by various Fusarium species. It mainly includes Fusarium graminearum Schw. In China, before the 1990s, wheat scab was mainly prevalent in the humid and rainy middle and lower reaches of the Yangtze River. With the changes of farming system, fertilizer and water conditions and climatic conditions, wheat scab showed warm and humid in China. The winter wheat area and the northeast spring wheat area in the middle and lower reaches of the Yangtze River gradually expanded toward the Huanghuai wheat area and the northern wheat area, and the area of the disease continued to expand.
  • FHB Fusarium head blight
  • the purpose of the present disclosure is to provide a method for breeding and molecular markers of a wheat-long-buckle buckwheat-resistant scab line, in order to solve at least some of the technical problems existing in the prior art mentioned above.
  • a method for breeding a wheat-long-buckthorn buckwheat-resistant scab line which comprises:
  • the earings of Elytrigia elongata were irradiated with 18Gy 60 Co- ⁇ , and the common wheat variety was pollinated in China, and the F1 generation seeds were harvested.
  • the long-eared buckwheat grass is the Israeli diploid long-eared buckwheat 7E ⁇ . End body attachment system;
  • the F1 generation seeds obtained by fluorescence in situ hybridization were used to identify seeds containing exogenous fragments of diploid E. longissima;
  • the disease-resistant strain was backcrossed with the common wheat variety Jimai 22 or Dwarf Kang58 for 3-4 generations, and then self-crossed to obtain a homozygous wheat-long-eared buckwheat-resistant scab resistance line.
  • the parental diploid E. longissima 7E ⁇ end-body addition line, the common wheat variety Chongchun, and the common wheat variety Jimai 22 or Dwarf anti-58 for backcrossing are all available to those skilled in the art through conventional means.
  • the obtained plant materials can be purchased from commercially available channels or imported from various breeding units or genebanks.
  • the operation of detecting the scab resistance of the F1 generation plant by the single flower instillation method is as follows: in the flowering period, the spikelets that are blooming near the middle of the ear are selected, and the Fusarium graminearum suspension is dropped into the place. The base of the spikelets is such that the plant receives infection from the bacterial suspension.
  • the concentration of the Fusarium graminearum suspension is 50,000 conidia/ml, and the instillation amount of the Fusarium graminearum suspension is 20 ⁇ l per instillation on each of the spikelets.
  • each of the spikelets infected with the bacterial suspension is continuously hydrated 3 days before the instillation.
  • the resistance of the plant to scab is detected after 21 days of instillation.
  • a molecular marker closely linked to a scab resistance gene in a wheat-long-buckle buckwheat resistance transgenic line which is one of the following molecules:
  • nucleic acid molecule having the sequence of SEQ ID NO. 1;
  • a primer pair for detecting the aforementioned molecular marker closely linked to a scab resistance gene which is one of the following primer pairs:
  • a primer pair consisting of a molecule having the sequence of SEQ ID NO. 4 and SEQ ID NO. 5, for detecting a nucleic acid molecule having the sequence of SEQ ID NO. 1;
  • a primer pair consisting of a molecule having the sequence of SEQ ID NO. 8 and SEQ ID NO. 9 for detecting a nucleic acid molecule having the sequence of SEQ ID NO.
  • kits for detecting the aforementioned molecular marker closely linked to a scab resistance gene which comprises the aforementioned primer pair.
  • the kit further comprises one or more of a positive control, a negative control, a Taq enzyme, and Mg 2+ .
  • the present disclosure Compared with the prior art, the present disclosure achieves the following positive progress effects: the present disclosure newly discovers three molecular markers closely linked to the scab resistance gene in diploid E. longissima, which was not seen before the present disclosure.
  • the report is the first of its kind.
  • the newly discovered molecular markers can be used for wheat breeding, and the wheat-long-eared buckwheat resistance to scab resistance is successfully obtained, and the corresponding detection kit is developed.
  • the theoretical study and practical application of the present disclosure for wheat genetic breeding Both have important value.
  • Figure 1 shows the results of amplification of the molecular marker 7E ⁇ T-60 in Example 3 between each plant to be tested and the parent.
  • Fig. 2 shows the results of identification of scab resistance of each wheat-long-eared buckwheat translocation line to be tested in Example 3.
  • Fig. 3 shows the results of amplification of the molecular marker 7E ⁇ T-78 in each of the plants to be tested and the parent in Example 4.
  • Fig. 4 shows the results of amplification of the molecular marker 7E ⁇ T-107 in each of the plants to be tested and the parent in Example 5.
  • Elymus sibiricum has excellent traits lacking in common cultivated wheat and is an important wild germplasm resource for genetic improvement of common wheat.
  • the inventors of the present disclosure finally succeeded in obtaining a wheat-long-buckthorn buckwheat resistance transgenic line and its molecular markers, and developed primer pairs and reagents for detecting the molecular marker. box.
  • the common wheat variety Jimai 22 can be purchased commercially or imported from various breeding units or genebanks;
  • Common wheat variety Dwarf Kang 58 can be purchased commercially or imported from various breeding units or genebanks;
  • the DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;
  • the experimental methods used are all conventional methods unless otherwise specified, including genomic DNA extraction, fluorescence in situ hybridization, PCR amplification reactions, etc., refer to the prior art, such as "Molecular Cloning Experiment”. guide”.
  • the identified seeds containing the exogenous fragments of diploid E. longissima were obtained, and the wheat-long-eared buckwheat translocation plants with different lengths were obtained.
  • the flowering stage select each plant near the middle of the ear The flowering spikelets, the suspension of Fusarium graminearum is injected into the base of the spikelet, so that the plant receives the infection of the bacterial suspension, wherein the concentration of the Fusarium graminearum suspension is 50,000 conidia/ml, and the Fusarium graminearum suspension
  • the amount of liquid dripping was 20 ⁇ l per instillation on each spikelet.
  • three days before the instillation each spikelet infected with the bacterial suspension was continuously hydrated.
  • the disease-resistant strains high-resistant scab-fed wheat-long-eared buckwheat translocation plants
  • the common wheat variety Jimai 22 were backcrossed for 3 generations and then selfed to obtain homozygous wheat-long-eared buckwheat Scab line.
  • the backcross can also use the common wheat varieties such as the common wheat variety Dwarf Kang58, and can be returned back to 3 to 4 generations.
  • transcriptome sequence of the 7E ⁇ endpartite addition line of diploid E. longissima and the common wheat variety C. chinensis was sequenced, and 238 transcripts of 7E ⁇ specific expression of diploid E. longissima were screened, according to these specifically expressed transcripts. Further screening, 21 molecular markers of 7E ⁇ specific for diploid E. longissima were screened.
  • the 21 molecular markers were initially mapped using the wheat-long-eared buckwheat translocation lines of different lengths (obtained in the same manner as in Example 1), and the wheat-long-eared buckwheat translocation with high scab resistance was used.
  • the resistance and molecular markers of the wheat-long-spotted buckwheat translocation line of high-sensitivity scab were identified, and three molecular markers closely linked to scab resistance in the wheat-long-eared buckwheat translocation line were identified. 7E ⁇ T-60, 7E ⁇ T-78 and 7E ⁇ T-107, the sequences of which are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.
  • the primers for detecting the molecular marker 7E ⁇ T-60 were 7E ⁇ T-60 Primer F and 7E ⁇ T-60 Primer R, and their sequences are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
  • the process of detection is as follows:
  • the genomic DNA of the wheat-long-eared buckwheat translocation line was extracted, and the extracted genomic DNA was used as a template, and 7E ⁇ T-60 Primer F and 7E ⁇ T-60 Primer R were used as primer pairs.
  • PCR reaction after the reaction is completed, the PCR reaction product is detected by electrophoresis, and the corresponding 342 bp DNA band can be amplified to be a plant material containing the genetic resources of diploid Phyllostachys praecox.
  • the PCR reaction procedure was as follows: predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute, running for 35 cycles, and finally extending at 72 ° C for 10 minutes.
  • the PCR amplification product can be stored at 4 °C.
  • the electrophoresis detection conditions of the PCR product were as follows: 8% polyacrylamide gel electrophoresis, voltage 200V, electrophoresis time was 1 hour, and buffer was 1X TBE. The results of electrophoresis detection are shown in Fig. 1. In Fig.
  • the lanes from left to right are Marker, Israel diploid long-eared buckwheat 7E ⁇ end-body addition line, common wheat variety Chinese spring, wheat-long-eared buckwheat grass
  • the translocation line is S166, S562, G415, G761, G514, S441, G408, S757, G1038, W219, G767, S1240, water, and the arrow indicates the amplified band of the molecular marker 7E ⁇ T-60.
  • Fig. 2 The results of the identification of scab resistance of each of the above lines are shown in Fig. 2.
  • Fig. 2 from left to right, the order of the 7E ⁇ endpartite addition line of the diploid E. longissima, the common wheat variety, Chinese spring, S166, S562, G415, G761, G514, S441, G408, S757, G1038, W219, G767, S1240.
  • S166, S562, G415, G761, G514 and S441 are high-resistance wheat-long-eared buckwheat translocation lines
  • G408, S757, G1038, W219, G767 and S1240 are high-sensitivity The wheat-long-eared buckwheat translocation line of scab, which is consistent with the test results of Figure 1.
  • the primers for detecting the molecular marker 7E ⁇ T-78 were 7E ⁇ T-78 Primer F and 7E ⁇ T-78 Primer R, and their sequences are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively.
  • the process of detection is as follows:
  • the genomic DNA of the wheat-long-eared buckwheat translocation line was extracted, and the extracted genomic DNA was used as a template to carry out PCR reaction with 7E ⁇ T-78 Primer F and 7E ⁇ T-78 Primer R as primer pairs. After the reaction, electrophoresis was carried out. The PCR reaction product is detected, and the corresponding 205 bp DNA band is a plant material containing the genetic resources of diploid Phyllostachys pubescens resistance.
  • the PCR reaction procedure was as follows: predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute, running for 35 cycles, and finally extending at 72 ° C for 10 minutes.
  • the PCR amplification product can be stored at 4 °C.
  • the electrophoresis detection conditions of the PCR product were as follows: 8% polyacrylamide gel electrophoresis, voltage 200V, electrophoresis time was 1 hour, and buffer was 1X TBE.
  • Fig. 3 The results of electrophoresis detection are shown in Fig. 3.
  • the lanes from left to right are Marker, Israel diploid long-eared buckwheat 7E ⁇ end-body addition line, common wheat variety Chinese spring, high resistance to scab Wheat-Elongated Bromegrass translocation lines (S166, S562, G415, G761, G514, S441) and wheat-long-eared buckwheat translocation lines (G408, S757, G1038, W219, G767, S1240) ), water, arrow indicates the amplified band of the molecular marker 7E ⁇ T-78.
  • the primers for detecting the molecular marker 7E ⁇ T-107 were 7E ⁇ T-107 Primer F and 77E ⁇ T-107 Primer R, and their sequences are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively.
  • the process of detection is as follows:
  • the genomic DNA of the wheat-long-eared buckwheat translocation line was extracted, and the extracted genomic DNA was used as a template to carry out PCR reaction with 7E ⁇ T-107 Primer F and 7E ⁇ T-107 Primer R as primer pairs. After the reaction, electrophoresis was carried out. The PCR reaction product is detected, and the corresponding 805 bp DNA band is a plant material containing the genetic resources of the diploid Phyllostachys pubescens resistance.
  • the PCR reaction procedure was as follows: predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 1 minute, running for 35 cycles, and finally extending at 72 ° C for 10 minutes.
  • the PCR amplification product can be stored at 4 °C.
  • the electrophoresis detection conditions of the PCR product were as follows: 1.5% agarose gel electrophoresis, voltage was 160 V, time was 40 minutes, and buffer was 1X TAE.
  • the results of electrophoresis detection are shown in Fig. 4. In Fig.
  • the lanes from left to right are Marker, Israel diploid long-eared buckwheat 7E ⁇ end-body addition line, common wheat variety Chinese spring, high resistance to scab Wheat-Elongated Bromegrass translocation lines (S166, S562, G415, G761, G514, S441) and wheat-long-eared buckwheat translocation lines (G408, S757, G1038, W219, G767, S1240) ), water, arrow indicates the amplified band of the molecular marker 7E ⁇ T-107.
  • the wheat-long-eared buckwheat translocation plant to be tested is detected by the primer pair of the present disclosure, and the trait of the plant capable of amplifying the molecular marker of the present disclosure is also resistant.
  • the traits of the plants that are unable to amplify the molecular markers of the present disclosure are also susceptible to susceptibility.
  • 342 bp, 205 bp or 805 bp can be amplified in the wheat-long-eared buckwheat-resistant scab resistance line corresponding to the molecular markers 7E ⁇ T-60, 7E ⁇ T-78 and The DNA band of 7E ⁇ T-107, the susceptible wheat-long-eared buckwheat translocation plant could not amplify the corresponding bands, which could be used to assist wheat breeding.
  • primer pairs used to amplify each molecular marker in the above embodiments can also be combined with other conventional detection reagents or materials such as a positive control, a negative control, a Taq enzyme, and a Mg 2+ .
  • a positive control a negative control
  • a Taq enzyme a tetrachloride
  • Mg 2+ a Mg 2+

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Abstract

提供了一种小麦－长穗偃麦草抗赤霉病易位系的选育方法及分子标记。还提供扩增该分子标记的引物、该分子标记在小麦遗传育种中的应用。所述分子标记是如下的分子之一：（1）序列如SEQ ID NO：1所示的核酸分子；或（2）序列如SEQ ID NO：2所示的核酸分子；或（3）序列如SEQ ID NO：3所示的核酸分子。

Description

小麦-长穗偃麦草抗赤霉病易位系的选育方法及分子标记 技术领域
本公开涉及分子生物学技术领域，具体涉及一种小麦-长穗偃麦草抗赤霉病易位系的选育方法及其分子标记，同时，本公开还涉及扩增该分子标记的引物、该分子标记在小麦遗传育种中的应用。
背景技术
小麦(Triticum aestivum L.)是中国重要的粮食作物，小麦赤霉病(fusarium head blight,FHB)是危害小麦生产的主要病害，其是由多种镰刀菌引起的一种世界性真菌病害，其中主要包括禾谷镰刀菌(Fusarium graminearum Schw.)。在我国，20世纪90年代以前，小麦赤霉病主要在湿润多雨的长江中下游地区流行，随着耕作制度、肥水条件和气候条件等因素的改变，小麦赤霉病在我国呈现出由温暖湿润的长江中下游冬麦区和东北春麦区逐渐向黄淮麦区和北方麦区扩展的趋势，该病害发生面积不断扩大。赤霉病大流行年份穗部发病率可达50％-100％，造成产量下降10％-40％，并且籽粒中积累大量真菌毒素，严重降低籽粒的品质。在中度流行年份小麦穗部发病率为30％-50％，产量下降5％-15％(见参考文献：姚金保，陆维忠.中国小麦抗赤霉病育种研究进展.江苏农业学报，2000，16(4)：242-248)。2012年赤霉病大流行中，山东、河南、安徽和江苏等小麦主产区发病严重，部分地区产量损失高达301.5-1877.3kg/hm-2(见参考文献：程顺和，张勇，别同德，等.中国小麦赤霉病的危害及抗性遗传改良.江苏农业学报.2012，28(5)：938-942)。小麦赤霉病不仅给小麦生产造成严重产量损失和品质影响，还产生以脱氧雪腐镰刀菌烯醇(DON)为主的真菌毒素，造成严重的食品安全问题，危害人畜健康。因此，小麦抗赤霉病新品种的选育和新抗性资源的挖掘与利用是小麦育种工作的重点。
公开内容
(一)要解决的技术问题
本公开的目的是提供一种小麦-长穗偃麦草抗赤霉病易位系的选育方法及分子标记，以期解决上述现有技术中存在的至少部分技术问题。
(二)技术方案
为实现上述目的，作为本公开的一个方面，提供一种小麦-长穗偃麦草抗赤霉病易位系的选育方法，其包括：
用18Gy 60Co-γ辐射长穗偃麦草(Elytrigia elongata)正在开花的穗子，并给普通小麦品种中国春授粉，收获F1代种子，所述长穗偃麦草是以色列二倍体长穗偃麦草7Eβ端体附加系；
通过荧光原位杂交检测所得到的F1代种子，鉴定出含有二倍体长穗偃麦草外源片段的种子；
种植鉴定出的含有二倍体长穗偃麦草外源片段的种子，在扬花期用单花滴注法检测F1代植株的赤霉病抗性，选出抗病株；
将抗病株与普通小麦品种济麦22或矮抗58回交3-4代后再进行自交，得到纯合的小麦-长穗偃麦草抗赤霉病易位系。
其中，作为亲本的二倍体长穗偃麦草7Eβ端体附加系、普通小麦品种中国春以及用于回交的普通小麦品种济麦22或矮抗58都属于本领域技术人员通过常规途径即可获得的植物材料，如可以通过市售渠道购买得到，也可从各育种单位或种质库引进。优选地，所述用单花滴注法检测F1代植株的赤霉病抗性的操作如下：在扬花期，选取靠近穗中部正在开花的小穗，将禾谷镰刀菌悬液滴注到所述小穗的基部，使植株接受菌悬液的感染。
更优选地，所述禾谷镰刀菌悬液的浓度为50,000conidia/ml，所述禾谷镰刀菌悬液的滴注量为每个所述小穗上滴注20μl。
更优选地，在滴注前3天，接受菌悬液感染的每个所述小穗持续喷水保湿。
更优选地，在滴注21天后，检测植株对赤霉病的抗性。
作为本公开的另一个方面，提供一种小麦-长穗偃麦草抗赤霉病易位系中与赤霉病抗性基因紧密连锁的分子标记，其是如下的分子之一：
(1)序列如SEQ ID NO.1所示的核酸分子；或
(2)序列如SEQ ID NO.2所示的核酸分子；或
(3)序列如SEQ ID NO.3所示的核酸分子。
作为本公开的再一个方面，提供一种检测前述与赤霉病抗性基因紧密连锁的分子标记的引物对，其是如下的引物对之一：
(1)由序列如SEQ ID NO.4和SEQ ID NO.5所示分子组成的引物对，用于检测序列如SEQ ID NO.1所示的核酸分子；或
(2)由序列如SEQ ID NO.6和SEQ ID NO.7所示分子组成的引物对，用于检测序列如SEQ ID NO.2所示的核酸分子；或
(3)由序列如SEQ ID NO.8和SEQ ID NO.9所示分子组成的引物对，用于检测序列如SEQ ID NO.3所示的核酸分子。
作为本公开的又一个方面，提供一种检测前述与赤霉病抗性基因紧密连锁的分子标记的试剂盒，其包括前述的引物对。
优选地，所述试剂盒还包括阳性对照、阴性对照、Taq酶和Mg2+等中的一种或多种。
作为本公开的再又一个方面，提供一种前述与赤霉病抗性基因紧密连锁的分子标记在小麦遗传育种中的应用。
(三)有益效果
与现有技术相比，本公开取得了以下积极进步效果：本公开新发现二倍体长穗偃麦草中三个与赤霉病抗性基因紧密连锁的分子标记，这在本公开之前未见过报道，本公开属于首创。新发现的分子标记可以用于小麦的育种，并成功获得小麦-长穗偃麦草抗赤霉病易位系，同时开发出相应的检测试剂盒，本公开对于小麦遗传育种的理论研究和实际应用均有重要价值。
附图说明
图1显示实施例3中分子标记7EβT-60在各待测植株及亲本间的扩增结果。
图2显示实施例3中各待测小麦-长穗偃麦草易位系的赤霉病抗性鉴定结果。
图3显示实施例4中分子标记7EβT-78在各待测植株及亲本间的扩增结果。
图4显示实施例5中分子标记7EβT-107在各待测植株及亲本间的扩增结果。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开作进一步的详细说明。
本公开的发明人在研发的过程中发现，小麦种内的遗传资源有限，缺少赤霉病抗性等很多有益基因。小麦近缘种属与普通小麦品种间遗传距离较远，适于作为普通小麦遗传改良的基因资源。因此，可以尝试将小麦野生近缘种属的有益基因导入普通栽培小麦以对目前的小麦品种进行改良。长穗偃麦草(Elytrigia elongata)是小麦的一个重要野生近缘种，有二倍体(2n＝2x＝14,EE)、四倍体和十倍体三种类型。长穗偃麦草具有普通栽培小麦所缺少的优良性状，是普通小麦遗传改良的重要野生种质资源。经过系列研究过程及应用实践，本公开的发明人终于成功得到了一种小麦-长穗偃麦草抗赤霉病易位系及其分子标记，同时发展出了检测该分子标记的引物对和试剂盒。
下述具体实施例中，所使用的实验材料的来源如下：
以色列二倍体长穗偃麦草7Eβ端体附加系购自美国SmallGrains.org资源库；
普通小麦品种中国春可市购，也可从各育种单位或种质库引进；
普通小麦品种济麦22可市购，也可从各育种单位或种质库引进；
普通小麦品种矮抗58可市购，也可从各育种单位或种质库引进；
DNA提取试剂盒购自天根生化科技(北京)有限公司；
引物序列由赛默飞世尔科技(中国)有限公司合成；
测序由北京睿博兴科生物技术有限公司完成；
其他的实验材料试剂或者仪器设备若未作特殊说明，均表示为本领域常规市售可得。
下述实施例中，所使用的实验方法若无特殊说明，表示均为常规方法，包括基因组DNA的提取、荧光原位杂交、PCR扩增反应等操作可参考现有技术，如《分子克隆实验指南》。
实施例1小麦-长穗偃麦草抗赤霉病易位系植株的获得
用18Gy 60Co-γ辐射以色列二倍体长穗偃麦草7Eβ端体附加系正在开花的穗子，并给中国春小麦授粉，得到F1代种子。通过荧光原位杂交检测所得到的F1代种子，鉴定出含有二倍体长穗偃麦草外源片段的种子。
种植鉴定出的含有二倍体长穗偃麦草外源片段的种子，即得到不同片段长度的小麦-长穗偃麦草易位系植株。在扬花期，选取各植株靠近穗中部 正在开花的小穗，将禾谷镰刀菌悬液滴注到小穗的基部，使植株接受菌悬液的感染，其中禾谷镰刀菌悬液的浓度为50,000conidia/ml，禾谷镰刀菌悬液的滴注量为每个小穗上滴注20μl。在滴注前3天，接受菌悬液感染的每个小穗持续喷水保湿。在滴注21天后，检测植株对赤霉病的抗性，选出抗病株。本实施例一共完成检测小麦-长穗偃麦草易位系待测植株样本158份，结果显示，在158份样本中，高抗赤霉病的小麦-长穗偃麦草易位系有21份，中抗赤霉病的小麦-长穗偃麦草易位系有55份，高感赤霉病的小麦-长穗偃麦草易位系有82份。
将抗病株(高抗赤霉病的小麦-长穗偃麦草易位系植株)与普通小麦品种济麦22回交3代后再进行自交，得到纯合的小麦-长穗偃麦草抗赤霉病易位系。
其中，回交也可以使用普通小麦品种矮抗58等小麦主栽品种，回交3～4代均可。
实施例2分子标记的获得
对二倍体长穗偃麦草7Eβ端体附加系和普通小麦品种中国春进行转录组测序，筛选出二倍体长穗偃麦草7Eβ特异表达的转录本238条，根据这些特异表达的转录本进行进一步筛选，筛选到二倍体长穗偃麦草7Eβ特异的分子标记21个。
利用不同片段长度的小麦-长穗偃麦草易位系(如实施例1中同样方法获得)对这21个分子标记进行初步定位，并且用高抗赤霉病的小麦-长穗偃麦草易位系和高感赤霉病的小麦-长穗偃麦草易位系进行抗性分子标记筛选，鉴定得到三个在小麦-长穗偃麦草易位系中与赤霉病抗性紧密连锁的分子标记7EβT-60，7EβT-78和7EβT-107，它们的序列分别如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示。
实施例3待测植株中分子标记7EβT-60的检测
检测分子标记7EβT-60的引物为7EβT-60 Primer F和7EβT-60 Primer R，它们的序列分别如SEQ ID NO.4和SEQ ID NO.5所示。
检测的过程如下：
提取小麦-长穗偃麦草易位系待测植株的基因组DNA，以所提取的基因组DNA为模板，以7EβT-60 Primer F和7EβT-60 Primer R为引物对进行 PCR反应，反应结束后，电泳检测PCR反应产物，能扩增出相应的342bpDNA条带的即为含有二倍体长穗偃麦草赤霉病抗性遗传资源的植物材料。
其中，所使用的PCR反应体系如下表1所示：
表1实施例3的PCR反应体系
药品 体积(μl) 规格
10X Taq Buffer 2 1.8ml
dNTP 1 1ml(2.5mM each)
Taq DNA聚合酶 0.5 500U(2.5U/μl)
7EβT-60 Primer F 1  
7EβT-60 Primer R 1  
基因组DNA 1 100ng/μl
ddH2O 13.5  
总计 20  
PCR的反应程序如下：94℃预变性5分钟、94℃变性30秒、60℃退火30秒、72℃延伸1分钟，运行35个循环；最后72℃延伸10分钟。PCR扩增产物可以在4℃保存。
PCR产物的电泳检测条件如下：8％聚丙烯酰胺凝胶电泳，电压200V，电泳时间为1小时，缓冲液为1X TBE。电泳检测的结果如图1所示，图1中，各泳道从左至右依次是Marker、以色列二倍体长穗偃麦草7Eβ端体附加系、普通小麦品种中国春、小麦-长穗偃麦草易位系待测株系S166、S562、G415、G761、G514、S441、G408、S757、G1038、W219、G767、S1240、水，箭头表示分子标记7EβT-60的扩增条带。从图1中可以看出，S166、S562、G415、G761、G514、S441扩增出对应于342bp位置的DNA条带，G408、S757、G1038、W219、G767、S1240无相应条带。
上述各株系的赤霉病抗性鉴定结果如图2所示，图2中，从左至右依次是以色列二倍体长穗偃麦草7Eβ端体附加系、普通小麦品种中国春、S166、S562、G415、G761、G514、S441、G408、S757、G1038、W219、G767、S1240。从图2中可以看出，S166、S562、G415、G761、G514和S441为高抗赤霉病的小麦-长穗偃麦草易位系，G408、S757、G1038、W219、G767和S1240为高感赤霉病的小麦-长穗偃麦草易位系，这与图1的检测结果一致。
实施例4待测植株中分子标记7EβT-78的检测
检测分子标记7EβT-78的引物为7EβT-78 Primer F和7EβT-78 Primer R，它们的序列分别如SEQ ID NO.6和SEQ ID NO.7所示。
检测的过程如下：
提取小麦-长穗偃麦草易位系待测植株的基因组DNA，以所提取的基因组DNA为模板，以7EβT-78 Primer F和7EβT-78 Primer R为引物对进行PCR反应，反应结束后，电泳检测PCR反应产物，能扩增出相应的205bpDNA条带的即为含有二倍体长穗偃麦草赤霉病抗性遗传资源的植物材料。
其中，所使用的PCR反应体系如下表2所示：
表2实施例4的PCR反应体系
药品 体积(μl) 规格
10X Taq Buffer 2 1.8ml
dNTP 1 1ml(2.5mM each)
Taq DNA聚合酶 0.5 500U(2.5U/μl)
7EβT-78 Primer F 1  
7EβT-78 Primer R 1  
基因组DNA 1 100ng/μl
ddH2O 13.5  
总计 20  
PCR的反应程序如下：94℃预变性5分钟、94℃变性30秒、60℃退火30秒、72℃延伸1分钟，运行35个循环；最后72℃延伸10分钟。PCR扩增产物可以在4℃保存。
PCR产物的电泳检测条件如下：8％聚丙烯酰胺凝胶电泳，电压200V，电泳时间为1小时，缓冲液为1X TBE。
电泳检测的结果如图3所示，图3中，各泳道从左至右依次是Marker、以色列二倍体长穗偃麦草7Eβ端体附加系、普通小麦品种中国春、高抗赤霉病的小麦-长穗偃麦草易位系(S166、S562、G415、G761、G514、S441)和高感赤霉病的小麦-长穗偃麦草易位系(G408、S757、G1038、W219、G767、S1240)、水，箭头表示分子标记7EβT-78的扩增条带。
实施例5待测植株中分子标记7EβT-107的检测
检测分子标记7EβT-107的引物为7EβT-107 Primer F和77EβT-107 Primer R，它们的序列分别如SEQ ID NO.8和SEQ ID NO.9所示。
检测的过程如下：
提取小麦-长穗偃麦草易位系待测植株的基因组DNA，以所提取的基因组DNA为模板，以7EβT-107 Primer F和7EβT-107 Primer R为引物对进行PCR反应，反应结束后，电泳检测PCR反应产物，能扩增出相应的805bp DNA条带的即为含有二倍体长穗偃麦草赤霉病抗性遗传资源的植物材料。
其中，所使用的PCR反应体系如下表3所示：
表3实施例5的PCR反应体系
药品 体积(μl) 规格
10X Taq Buffer 2 1.8ml
dNTP 1 1ml(2.5mM each)
Taq DNA聚合酶 0.5 500U(2.5U/μl)
7EβT-107 Primer F 1  
7EβT-107 Primer R 1  
基因组DNA 1 100ng/μl
ddH2O 13.5  
总计 20  
PCR的反应程序如下：94℃预变性5分钟、94℃变性30秒、60℃退火30秒、72℃延伸1分钟，运行35个循环；最后72℃延伸10分钟。PCR扩增产物可以在4℃保存。
PCR产物的电泳检测条件如下：1.5％琼脂糖凝胶电泳，电压为160V，时间为40分钟，缓冲液为1X TAE。电泳检测的结果如图4所示，图4中，各泳道从左至右依次是Marker、以色列二倍体长穗偃麦草7Eβ端体附加系、普通小麦品种中国春、高抗赤霉病的小麦-长穗偃麦草易位系(S166、S562、G415、G761、G514、S441)和高感赤霉病的小麦-长穗偃麦草易位系(G408、S757、G1038、W219、G767、S1240)、水，箭头表示分子标记7EβT-107的扩增条带。
在本公开其他的一些具体实施方式中，利用本公开的引物对检测待测小麦-长穗偃麦草易位系植株，对于能够扩增出本公开的分子标记的植株，其性状同样表现为抗赤霉病，对于不能扩增出本公开的分子标记的植株，其性状同样表现为感病。
由上述各实施例可以知晓，在小麦-长穗偃麦草抗赤霉病易位系中能够扩增出342bp、205bp或805bp对应于分子标记7EβT-60、7EβT-78和 7EβT-107的DNA条带，感病的小麦-长穗偃麦草易位系植株无法扩增出相应条带，从而可以辅助应用于小麦的育种。
另外，本领域技术人员应当理解的是，上述实施例中扩增各分子标记所使用的引物对，还可以和其他常规的检测试剂或材料，如阳性对照、阴性对照、Taq酶和Mg2+等中的一种或多种组合成检测试剂盒，应用于本公开中所述的与赤霉病抗性基因紧密连锁的分子标记的检测。
以上所述的具体实施例，对本公开的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (10)

  1. 一种小麦-长穗偃麦草抗赤霉病易位系的选育方法，其包括：
    用18Gy 60Co-γ辐射长穗偃麦草(Elytrigia elongata)正在开花的穗子，并给普通小麦品种中国春授粉，收获F1代种子，所述长穗偃麦草是以色列二倍体长穗偃麦草7Eβ端体附加系；
    通过荧光原位杂交检测所得到的F1代种子，鉴定出含有二倍体长穗偃麦草外源片段的种子；
    种植鉴定出的含有二倍体长穗偃麦草外源片段的种子，在扬花期用单花滴注法检测F1代植株的赤霉病抗性，选出抗病株；
    将抗病株与普通小麦品种济麦22或矮抗58回交3-4代后再进行自交，得到纯合的小麦-长穗偃麦草抗赤霉病易位系。
  2. 根据权利要求1所述的选育方法，其中，所述用单花滴注法检测F1代植株的赤霉病抗性的操作如下：在扬花期，选取靠近穗中部正在开花的小穗，将禾谷镰刀菌悬液滴注到所述小穗的基部，使植株接受菌悬液的感染。
  3. 根据权利要求2所述的选育方法，其中，所述禾谷镰刀菌悬液的浓度为50,000conidia/ml，所述禾谷镰刀菌悬液的滴注量为每个所述小穗上滴注20μl。
  4. 根据权利要求2所述的选育方法，其中，在滴注前3天，接受菌悬液感染的每个所述小穗持续喷水保湿。
  5. 根据权利要求2所述的选育方法，其中，在滴注21天后，检测植株对赤霉病的抗性。
  6. 一种小麦-长穗偃麦草抗赤霉病易位系中与赤霉病抗性基因紧密连锁的分子标记，其是如下的分子之一：
    (1)序列如SEQ ID NO.1所示的核酸分子；或
    (2)序列如SEQ ID NO.2所示的核酸分子；或
    (3)序列如SEQ ID NO.3所示的核酸分子。
  7. 一种检测权利要求6中所述的与赤霉病抗性基因紧密连锁的分子标记的引物对，其是如下的引物对之一：
    (1)由序列如SEQ ID NO.4和SEQ ID NO.5所示分子组成的引物对，用于检测序列如SEQ ID NO.1所示的核酸分子；或
    (2)由序列如SEQ ID NO.6和SEQ ID NO.7所示分子组成的引物对，用于检测序列如SEQ ID NO.2所示的核酸分子；或
    (3)由序列如SEQ ID NO.8和SEQ ID NO.9所示分子组成的引物对，用于检测序列如SEQ ID NO.3所示的核酸分子。
  8. 一种检测权利要求6中所述的与赤霉病抗性基因紧密连锁的分子标记的试剂盒，其包括权利要求7所述的引物对。
  9. 根据权利要求8所述的试剂盒，所述试剂盒还包括阳性对照、阴性对照、Taq酶和Mg2+中的一种或多种。
  10. 权利要求6中所述的与赤霉病抗性基因紧密连锁的分子标记在小麦遗传育种中的应用。
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