CN107119141B

CN107119141B - Breeding method and molecular marker of wheat-elytrigia elongata scab resistant translocation line

Info

Publication number: CN107119141B
Application number: CN201710462612.6A
Authority: CN
Inventors: 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
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2017-06-19
Filing date: 2017-06-19
Publication date: 2020-04-24
Anticipated expiration: 2037-06-19
Also published as: CN107119141A

Abstract

The invention discloses a breeding method and a molecular marker of a wheat-thinopyrum elongatum scab resistant translocation line. Meanwhile, the invention also provides a primer for amplifying the molecular marker and application of the molecular marker in wheat genetic breeding. The molecular marker is one of the following molecules: (1) nucleic acid molecule with sequence shown as SEQ ID NO. 1; or (2) a nucleic acid molecule with the sequence shown as SEQ ID NO. 2; or (3) a nucleic acid molecule with the sequence shown as SEQ ID NO. 3. The invention has important value for theoretical research and practical application of wheat genetic breeding.

Description

Breeding method and molecular marker of wheat-elytrigia elongata scab resistant translocation line

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a breeding method of a wheat-thinopyrum elongatum scab resistant translocation line and a molecular marker thereof, and also relates to a primer for amplifying the molecular marker and application of the molecular marker in wheat genetic breeding.

Background

Wheat (Triticum aestivum L.) is an important food crop in china, wheat scab (FHB) is a major disease that endangers wheat production, a worldwide fungal disease caused by many fusarium species, mainly including fusarium graminearum (fusarium graminearum L)m.graminearum Schw.). In China, before the 90 s of the 20 th century, wheat scab is popular mainly in the middle and lower reaches of the wet and rainy Yangtze river, and with the change of factors such as cultivation system, fertilizer and climate conditions, the wheat scab has a trend that the middle and lower reaches winter wheat areas of the warm and wet Yangtze river and the northeast spring wheat areas gradually expand to Huang-Hua wheat areas and northern wheat areas in China, and the disease occurrence area is continuously enlarged. The incidence rate of head of the. The incidence rate of the wheat head is 30-50% in the middle epidemic year, the yield is reduced by 5-15% (see the reference, Yaojinbao, Luweiloy, Chinese wheat scab resistant breeding research progress, Jiangsu agricultural science report, 2000, 16 (4): 242-248). In the prevalence of gibberellic disease in 2012, the main wheat producing areas such as Shandong, Henan, Anhui and Jiangsu are seriously attacked, and the yield loss of partial areas is as high as 301.5-1877.3kg/hm^-2(see references: Cheng shuang, Zhang Yong, Eridden, et al. harm to wheat scab and genetic improvement of resistance in China. Jiangsu agricultural science, 2012, 28 (5): 938-942). Wheat scab not only causes serious yield loss and quality influence on wheat production, but also produces mycotoxin mainly comprising Deoxynivalenol (DON), causes serious food safety problems and harms human and livestock health. Therefore, the breeding of new wheat scab resistant varieties and the excavation and utilization of new resistance resources are the key points of wheat breeding work.

Disclosure of Invention

In view of the above, the present invention provides a method for breeding a scab-resistant translocation line of Elytrigia elongata and a molecular marker thereof, so as to solve at least some of the technical problems in the prior art.

To achieve the above objects, according to one aspect of the present invention, there is provided a method for breeding a gibberellic disease resistant wheat-thinopyrum elongatum translocation line, comprising:

at 18Gy⁶⁰Co-gamma radiation of flowering ear of Elytrigia elongata (Elytrigia elongata) and pollination of common wheat variety in Chinese spring to harvest F1 generation seedsIs an annex line of an Israeli diploid elytrigia elongata 7E β end body;

identifying seeds containing the diploid thinopyrum elongatum exogenous fragment by detecting the obtained F1 generation seeds through fluorescence in situ hybridization;

planting identified seeds containing the diploid elytrigia elongata exogenous fragment, detecting the scab resistance of F1 generation plants by using a single-flower drip method in the flowering period, and selecting disease-resistant plants;

backcrossing the disease-resistant plant with common wheat variety Jimai 22 or Shibatang 58 for 3-4 generations, and then performing selfing to obtain the homozygous wheat-thinopyrum elongatum scab resistant translocation line.

The method is characterized in that a diploid elytrigia elongata 7E β end body additional line as a parent, a common wheat variety Chinese spring and a common wheat variety Jimai 22 or a dwarf antibody 58 for backcross belong to plant materials which can be obtained by the technicians in the field through conventional ways, such as being purchased from commercial sources and being introduced from various breeding units or germplasm libraries.

More preferably, the concentration of the fusarium graminearum suspension is 50,000conidia/ml, and the drip amount of the fusarium graminearum suspension is 20 μ l per ear drip.

More preferably, each of the spikelets infected with bacterial suspension is kept moist by water spray 3 days before instillation.

More preferably, the plants are tested for resistance to gibberellic disease 21 days after instillation.

As another aspect of the invention, a molecular marker closely linked with a scab resistance gene in a wheat-thinopyrum elongatum scab resistant translocation line is provided, wherein the molecular marker is one of the following molecules:

(1) nucleic acid molecule with sequence shown as SEQ ID NO. 1; or

(2) Nucleic acid molecule with sequence shown as SEQ ID NO. 2; or

(3) A nucleic acid molecule with a sequence shown as SEQ ID NO. 3.

As a further aspect of the present invention, there is provided a primer set for detecting the aforementioned molecular marker closely linked to a gibberellic disease resistance gene, which is one of the following primer sets:

(1) a primer pair consisting of molecules with sequences shown as SEQ ID NO.4 and SEQ ID NO.5 and used for detecting the nucleic acid molecule with the sequence shown as SEQ ID NO. 1; or

(2) A primer pair consisting of molecules with sequences shown as SEQ ID NO.6 and SEQ ID NO.7 and used for detecting the nucleic acid molecule with the sequence shown as SEQ ID NO. 2; or

(3) A primer pair consisting of molecules with sequences shown as SEQ ID NO.8 and SEQ ID NO.9, which is used for detecting the nucleic acid molecule with the sequence shown as SEQ ID NO. 3.

As a further aspect of the present invention, there is provided a kit for detecting the aforementioned molecular marker closely linked to a gibberellic disease resistance gene, which comprises the aforementioned primer pair.

Preferably, the kit further comprises a positive control, a negative control, Taq enzyme and Mg²⁺And the like.

As still another aspect of the present invention, there is provided a use of the aforementioned molecular marker closely linked to a gibberellic disease resistance gene in genetic breeding of wheat.

Compared with the prior art, the invention has the following positive progress effects: the invention newly finds three molecular markers which are closely linked with a scab resistance gene in the diploid elytrigia elongata, which is not reported before the invention, and belongs to the initiative. The newly found molecular marker can be used for wheat breeding, a wheat-elytrigia elongata scab resistant translocation line is successfully obtained, and a corresponding detection kit is developed at the same time.

Drawings

FIG. 1 shows the amplification results of the molecular marker 7E β T-60 in example 3 among the plants and parents to be tested.

FIG. 2 shows the results of the identification of gibberellic disease resistance of each of the wheat-thinopyrum elongatum translocation lines tested in example 3.

FIG. 3 shows the results of the amplification of the molecular marker 7E β T-78 in example 4 between each plant to be tested and the parent.

FIG. 4 shows the results of the amplification of the molecular marker 7E β T-107 in example 5 between each plant to be tested and the parent.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The inventor of the invention finds that the genetic resources in wheat species are limited, and many beneficial genes such as gibberellic disease resistance are lacked in the process of research and development. The genetic distance between the wheat kindred species and the common wheat variety is long, and the wheat kindred species is suitable for being used as a gene resource for genetic improvement of the common wheat. Therefore, it is possible to try to improve the current wheat variety by introducing a useful gene of a wild closely related species of wheat into a common cultivated wheat. Elytrigia elongata (Elytrigia elongata) is an important wild relative species of wheat, and is classified into three types, namely diploid (2 n-2 x-14, EE), tetraploid and decaploid. The elytrigia elongata has excellent properties which are lacked by common cultivated wheat and is an important wild germplasm resource for genetic improvement of common wheat. Through a series of research processes and application practices, the inventor of the invention successfully obtains a wheat-elytrigia elongata scab resistant translocation line and a molecular marker thereof, and develops a primer pair and a kit for detecting the molecular marker at the same time, thereby completing the invention.

In the following specific examples, the sources of experimental materials used are as follows:

an Israeli diploid elytrigia elongata 7E β end body addition line purchased from the American SmallGrains.

Common wheat varieties, namely Chinese spring, can be purchased in the market and also can be introduced from various breeding units or germplasm banks;

the common wheat variety Jimai 22 can be purchased in the market and also can be introduced from various breeding units or germplasm banks;

the common wheat variety dwarf 58 can be purchased in the market and can also be introduced from various breeding units or germplasm banks;

the DNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) Ltd;

the primer sequence was synthesized by seimer feishell science and technology (china) ltd;

sequencing was performed by Biotechnology Limited of Boxing Ke, Rui, Beijing;

other reagents for the test materials or instrumentation are shown as being routinely commercially available in the art unless otherwise specified.

In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the procedures including extraction of genomic DNA, fluorescence in situ hybridization, PCR amplification reaction, etc. can be referred to the prior art, such as "molecular cloning guidelines".

Example 1 obtaining of wheat-Elytrigia elongata scab resistant translocation line plant

At 18Gy⁶⁰Co-gamma radiation Israeli diploid elytrigia elongata 7E β end body attachment line flowering ear, and pollination is performed on Chinese spring wheat to obtain F1 generation seeds, and the obtained F1 generation seeds are detected through fluorescence in situ hybridization to identify the seeds containing the diploid elytrigia elongata exogenous segment.

Planting the identified seeds containing the diploid elytrigia elongata exogenous fragments to obtain the wheat-elytrigia elongata translocation line plants with different fragment lengths. In the flowering period, selecting small ears of each plant which are flowering close to the middle part of the ears, dripping fusarium graminearum suspension to the base parts of the small ears to enable the plants to be infected by the fusarium graminearum suspension, wherein the concentration of the fusarium graminearum suspension is 50,000conidia/ml, and the dripping amount of the fusarium graminearum suspension is 20 mu l per small ear. Each spikelet receiving bacterial suspension infection was kept moist by water spray 3 days before instillation. After 21 days of instillation, the plants were tested for resistance to gibberellic disease and disease-resistant plants were selected. In this example, 158 parts of a test plant sample of a wheat-elytrigia elongata translocation line is tested, and the results show that 21 parts of a wheat-elytrigia elongata translocation line with high scab resistance, 55 parts of a wheat-elytrigia elongata translocation line with medium scab resistance and 82 parts of a wheat-elytrigia elongata translocation line with high scab sensitivity are tested in 158 parts of the sample.

Backcrossing the disease-resistant plant (wheat with high resistance to scab-elytrigia elongata translocation line plant) with the common wheat variety Jimai 22 for 3 generations, and then carrying out selfing to obtain the homozygous wheat-elytrigia elongata scab translocation line.

Wherein, backcross can also use common wheat variety dwarf 58 and other wheat main cultivars, and can be performed for 3-4 generations.

Example 2 obtaining of molecular markers

And (3) carrying out transcriptome sequencing on the diploid elytrigia elongata 7E β end body addition line and common wheat variety Chinese spring, screening 238 transcripts specifically expressed by the diploid elytrigia elongata 7E β, and further screening 21 specific molecular markers of the diploid elytrigia elongata 7E β according to the transcripts specifically expressed.

The 21 molecular markers are preliminarily positioned by using wheat-elytrigia elongata translocation lines with different fragment lengths (obtained by the same method in example 1), and resistance molecular marker screening is carried out by using a wheat-elytrigia elongata translocation line with high scab resistance and a wheat-elytrigia elongata translocation line with high scab sensitivity, so as to identify and obtain three molecular markers 7E β T-60, 7E β T-78 and 7E β T-107 which are closely linked with scab resistance in the wheat-elytrigia elongata translocation line, and the sequences of the three molecular markers are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

Example 3 detection of molecular marker 7E β T-60 in plants to be tested

The primers for detecting the molecular marker 7E β T-60 are 7E β T-60 Primer F and 7E β T-60 Primer R, and the sequences of the primers are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.

The detection process is as follows:

extracting genome DNA of a plant to be detected of the wheat-elytrigia elongata translocation line, carrying out PCR reaction by taking the extracted genome DNA as a template and 7E β T-60 Primer F and 7E β T-60 Primer R as a Primer pair, and after the reaction is finished, detecting a PCR reaction product by electrophoresis, wherein a corresponding 342bp DNA strip can be amplified to obtain the plant material containing the diploid elytrigia elongata scab resistance genetic resource.

The PCR reaction system used is shown in the following Table 1:

TABLE 1 PCR reaction System of example 3

The reaction sequence for PCR was as follows: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 1 min, and running for 35 cycles; final extension at 72 ℃ for 10 min. The PCR amplification product can be stored at 4 ℃.

The electrophoresis detection conditions of the PCR products are as follows, 8% polyacrylamide gel electrophoresis, voltage 200V, electrophoresis time 1 hour, buffer solution 1 XTBE, electrophoresis detection results are shown in figure 1, each lane is sequentially marked with Marker, an annex line of Israeli long spike Elytrigia repens 7E β end body, a common wheat variety Chinese spring, a wheat-long spike Elytrigia repens translocation line to-be-detected line S166, S562, G415, G761, G514, S441, G408, S757, G1038, W219, G767, S1240 and water from left to right, an arrow indicates an amplification strip of a molecular Marker 7E β T-60, and as can be seen from figure 1, DNA strips corresponding to 342bp positions are amplified by S166, S562, G415, G761, G514 and S441, and G408, S757, G1038, W219, G767 and S1240 have no corresponding strips.

The gibberellic disease resistance identification results of the above-mentioned lines are shown in FIG. 2, an Israeli diploid thinopyrum elongatum 7E β end body addition line, common wheat varieties Chinese spring, S166, S562, G415, G761, G514, S441, G408, S757, G1038, W219, G767 and S1240 are arranged from left to right, and as can be seen from FIG. 2, S166, S562, G415, G761, G514 and S441 are wheat-thinopyrum elongatum translocation lines with high gibberellic disease resistance, and G408, S757, G1038, W219, G767 and S1240 are wheat-thinopyrum elongatum translocation lines with high gibberellic disease sensitivity, which is consistent with the detection results of FIG. 1.

Example 4 detection of the molecular marker 7E β T-78 in the plants to be tested

The primers for detecting the molecular marker 7E β T-78 are 7E β T-78 Primer F and 7E β T-78 Primer R, and the sequences of the primers are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7.

The detection process is as follows:

extracting genome DNA of a plant to be detected of the wheat-elytrigia elongata translocation line, carrying out PCR reaction by taking the extracted genome DNA as a template and 7E β T-78 Primer F and 7E β T-78 Primer R as a Primer pair, and after the reaction is finished, detecting a PCR reaction product by electrophoresis, wherein a corresponding 205bp DNA strip can be amplified to obtain the plant material containing the diploid elytrigia elongata scab resistance genetic resource.

The PCR reaction system used is shown in Table 2 below:

TABLE 2 PCR reaction System of example 4

Medicine and food additive	Volume (μ l)	Specification of
			10X Taq Buffer	2	1.8ml
dNTP	1	1ml(2.5mM each)
			Taq DNA polymerase	0.5	500U(2.5U/μl)
7EβT-78 Primer F	1
			7EβT-78 Primer R	1
Genomic DNA	1	100ng/μl
			ddH₂O	13.5
Total of	20

The conditions for the electrophoretic detection of the PCR products were as follows: 8% polyacrylamide gel electrophoresis, voltage 200V, electrophoresis time 1 hours, buffer 1X TBE.

The results of the electrophoresis are shown in FIG. 3, the lanes are Marker, Israeli diploid elytrigia elongata 7E β end body addition line, common wheat variety Chinese spring, high scab resistant wheat-elytrigia elongata translocation line (S166, S562, G415, G761, G514, S441) and high scab sensitive wheat-elytrigia elongata translocation line (G408, S757, G1038, W219, G767, S1240) from left to right, and the arrows indicate the amplification band of the molecular Marker 7E β T-78.

Example 5 detection of the molecular marker 7E β T-107 in plants to be tested

The primers for detecting the molecular marker 7E β T-107 are 7E β T-107 Primer F and 77E β T-107 Primer R, and the sequences of the primers are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.

The detection process is as follows:

extracting genome DNA of a plant to be detected of the wheat-elytrigia elongata translocation line, carrying out PCR reaction by taking the extracted genome DNA as a template and 7E β T-107 Primer F and 7E β T-107 Primer R as Primer pairs, and after the reaction is finished, detecting a PCR reaction product by electrophoresis, wherein a corresponding 805bp DNA band can be amplified to obtain the plant material containing the diploid elytrigia elongata scab resistance genetic resource.

The PCR reaction system used is shown in Table 3 below:

TABLE 3 PCR reaction System of example 5

Medicine and food additive	Volume (μ l)	Specification of
			10X Taq Buffer	2	1.8ml
dNTP	1	1ml(2.5mM each)
			Taq DNA polymerase	0.5	500U(2.5U/μl)
7EβT-107 Primer F	1
			7EβT-107 Primer R	1
Genomic DNA	1	100ng/μl
			ddH₂O	13.5
Total of	20

The electrophoresis detection conditions of the PCR products are that 1.5% agarose gel electrophoresis is carried out, the voltage is 160V, the time is 40 minutes, the buffer solution is 1 XTAE, the electrophoresis detection result is shown in figure 4, each lane comprises Marker, an annex line of the end body of Elytrigia elongata 7E β, an addition line of the Israeli diploid long spike, spring of common wheat variety, a wheat-Elytrigia elongata translocation line with high scab resistance (S166, S562, G415, G761, G514, S441) and a wheat-Elytrigia elongata translocation line with high scab sensitivity (G408, S757, G1038, W219, G767, S1240) from left to right, and an arrow indicates an amplification band of a molecular Marker 7E β T-107.

In other embodiments of the present invention, the primer pair of the present invention is used to detect a wheat-elytrigia elongata translocation line plant to be detected, wherein the trait of the plant capable of amplifying the molecular marker of the present invention is also manifested as scab resistance, and the trait of the plant incapable of amplifying the molecular marker of the present invention is also manifested as susceptibility to diseases.

The embodiments can know that 342bp, 205bp or 805bp DNA bands corresponding to molecular markers 7E β T-60, 7E β T-78 and 7E β T-107 can be amplified in the wheat-elytrigia elongata scab resistant translocation line, and the diseased wheat-elytrigia elongata translocation line plant cannot amplify corresponding bands, so that the method can be applied to wheat breeding in an auxiliary manner.

In addition, it will be understood by those skilled in the art that the primer pair used in the above examples to amplify each molecular marker can be combined with other conventional detection reagents or materials, such as positive control, negative control, Taq enzyme and Mg²⁺And the like, and is applied to the detection of the molecular marker which is closely linked with the gibberellic disease resistance gene in the invention.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

Breeding method and molecular marker of <120> wheat-elytrigia elongata scab resistant translocation line

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Claims

1. A molecular marker which is closely linked with a scab resistant gene in a wheat-thinopyrum elongatum scab resistant translocation line is one of the following molecules:

(1) nucleic acid molecule with sequence shown as SEQ ID NO. 1; or

(2) Nucleic acid molecule with sequence shown as SEQ ID NO. 2; or

(3) A nucleic acid molecule with a sequence shown as SEQ ID NO. 3.

2. A primer set for detecting the molecular marker closely linked to a gibberellic disease resistance gene as set forth in claim 1, which is one of the following primer sets:

3. A kit for detecting a molecular marker as set forth in claim 1 which is closely linked to a gibberellic disease resistance gene, comprising the primer pair set forth in claim 2.

4. The kit of claim 3, further comprising a positive control, a negative control, Taq enzyme and Mg²⁺One or more of (a).

5. Use of the primer pair of claim 2 in genetic breeding of wheat.