Chromosome 6t-specific molecular marker of aegilops tauschii and application thereof

Abstract

The present application belongs to the field of crop molecular biology and genetic breeding, and particularly relates to a specific molecular marker of 6T chromosome of Aegilops sharonensis and application thereof. The molecular marker established by the present application can effectively detect whether the 6T chromosome of Aegilops sharonensis is contained in a wheat background. The specific molecular marker obtained by the present application can be used for screening and identifying the hybrid offspring of wheat-Aegilops sharonensis, and can also be applied to the assisted breeding of new wheat lines / varieties with resistance to powdery mildew, so as to improve the screening efficiency, shorten the breeding time and have great industrialization value.

Description

Technical Field

[0001] The invention belongs to the field of crop molecular biology and genetic breeding, specifically involving the 6T chromosome-specific molecular marker of Aegilops awnless and its application. Background Technology

[0002] The genus *Aegilops* is a wild relative of wheat, comprising 11 diploid, 10 tetraploid, and 2 hexaploid species (Gill & Friebe, 2002). *Aegilops* possesses an extremely diverse genomic resource, including D, S, U, C, N, and M (Kimber & Sears, 1987; Hammer, 1980; Kihara, 1954). Compared to *Rhizophora*, *Berberis*, and *Aegilops*, *Aegilops* is the most closely related genus to wheat (Schneider et al., 2008), making it possible to transfer agriculturally valuable genes to common wheat (Bowden, 1959). *Aegilops muticum* (2n=2x=14) is a unique species, not only due to its adolescent spikelet form (capable of floating on water) (Miranda et al., 2007), but also due to its unique T-genome and two types of cytoplasm (Ohta & Saruhashi, 1999), making it a valuable gene pool for wheat improvement. *Aegilops muticum* has been reported to contain a large number of genes controlling agronomic traits, such as disease resistance, pest resistance, and high protein content (Gill et al., 1983, Damalia & Pecetti, 1990, Dimov & Zaharieva, 1993), making it an excellent genetic resource for wheat improvement.

[0003] Aegilops species have been used to develop wheat-Aegilops hybrids, with the transfer and identification of powdery mildew resistance genes Pm12, Pm13, Pm32, Pm19, and Pm35 reported (Jia et al., 1996, Ceoloni et al., 1992, Lutz et al., 1995, Hsam et al., 2003, Miranda et al., 2007). However, distant hybridization between wheat and Aegilops awnless has been limited, with only partially diploid and chromosome-additional lines of wheat and Aegilops awnless exhibiting resistance to powdery mildew (Liuet et al., 2015). Nevertheless, no translocation lines of wheat and Aegilops awnless have been developed.

[0004] Research on chromosome molecular dynamics in *Aegilops awnless* is limited. Only Liu et al. (2015) used EST-STS and EST-SSR markers to study the 7T addition line of *Aegilops awnless*, identifying 13 pairs of specific molecular markers that can be used to track the 7T chromosome of *Aegilops awnless* against the background of wheat chromosomes. However, no studies have reported on the establishment of chromosome 6T-specific molecular markers for *Aegilops awnless*. Our previous research found that the Chinese Spring-*Aegilops awnless* double diploid TA8024 is immune to wheat powdery mildew, and the 6T addition line and 6TS.7AL translocation line in the BC1F3 generation of TA8024 and Mianyang 11 are immune to wheat powdery mildew. Other translocation and addition lines are highly susceptible to powdery mildew. This indicates that the 6T chromosome of *Aegilops awnless* contains a gene for resistance to powdery mildew. Therefore, developing chromosome 6T-specific molecular markers for *Aegilops awnless* is of great significance for screening and identifying materials from segregating hybrid populations and for breeding disease-resistant wheat lines / variety. Summary of the Invention

[0005] To address the lack of a specific PCR marker for the 6T chromosome of *Aegilops awnless* in existing technologies, this invention provides a rapid, effective, and convenient method for identifying whether a sample contains a specific molecular marker for the 6T chromosome of *Aegilops awnless*. Therefore, the objective of this invention is to establish a specific molecular marker for the 6T chromosome of *Aegilops awnless*, providing a new method for detecting *Aegilops awnless* 6T chromatin against a wheat background.

[0006] This invention provides a chromosome-6T specific molecular marker for *Aegilops argyrophylla*, which is a combination of one or more of the following four primer pairs, with the following base sequences:

[0007] CINAU2570:

[0008] F: 5'-AGGTGGTGATTTCAGAGGCA-3', numbered SEQ ID NO.1;

[0009] R: 5'-CCGTGAAAAGCTAGACTCTGC-3', numbered SEQ ID NO.2;

[0010] CINAU2683:

[0011] F: 5'-AGATCTCTTTAGGGATGCGCT-3', numbered SEQ ID NO.3;

[0012] R: 5'-CCAACCTGTAAGCTGCGC-3', numbered SEQ ID NO.4;

[0013] CINAU1553:

[0014] F: 5'-CCTACCTCGTCCACCACAAG-3', numbered SEQ ID NO.5;

[0015] R: 5'-GCCGGATCTTCTTCATCACG-3', numbered SEQ ID NO.6;

[0016] TNAC1731:

[0017] F: 5'-TGTTGCTTTCAGAGCGAATTT-3', numbered SEQ ID NO.7;

[0018] R: 5'-TTGCTCCACCGAGATCACTAC-3', numbered SEQ ID NO.8;

[0019] The application of the Aegilops 6T chromosome-specific molecular marker described in this invention specifically involves using the Aegilops 6T chromosome-specific molecular marker to perform PCR amplification of the total genomic DNA of the test material, and to detect or assist in the detection of whether Aegilops 6T chromosome is present in the wheat background. This can be applied in the screening and identification of hybrid germplasm. After polyacrylamide electrophoresis, the PCR products were found to amplify a specific DNA band of 350 bp in length using primers CINAU2570, CINAU2683, and CINAU1553, which contains Aegilops 6T chromosome. After digestion of the PCR product amplified by primer TNAC1731 with restriction endonuclease TaqI and electrophoresis, a polymorphic band of approximately 750 bp in length was observed, which also contains Aegilops 6T chromosome.

[0020] Furthermore, the method for detecting or assisting in the detection of Aegilops 6T chromosome in wheat background includes the following steps:

[0021] (1) The total genomic DNA of the parents of wheat-Aegilops 6T addition line, 6TS.7AL translocation line and material without Aegilops 6T was used as a template. PCR amplification was performed on the wheat chromosome 6 using molecular markers that have been reported. The amplification products were detected by polyacrylamide gel electrophoresis.

[0022] (2) If specific DNA bands are found in the wheat-Aegilops 6T addition line and 6TS.7AL translocation line in the gel electrophoresis detection pattern of the template DNA to be tested, while the parental control does not have such bands, it indicates that the molecular marker can detect Aegilops 6T chromosome in the wheat genome background; if there is no difference between the wheat-Aegilops 6T addition line and 6TS.7AL translocation line and the parental control in terms of bands, it indicates that the molecular marker cannot detect Aegilops 6T chromosome in the wheat genome background.

[0023] The amplification characteristics of the four primer pairs are as follows: a 30 μL PCR reaction system consists of: 2.0 μL of 25 ng / μL template DNA, 0.3 μL of 5 U / μL Taq DNA polymerase, 200 μM of dNTPs, and Mg... 2+ Prepare 3.0 μL of 10×PCR buffer, 2 μL each of 10 μM upstream and downstream primers, and add sterile double-distilled water to bring the reaction volume to 30 μL.

[0024] The PCR amplification program is as follows: pre-denaturation at 94℃ for 3 min, followed by 35 cycles: denaturation at 94℃ for 45 s, annealing at 57℃ for 45 s, extension at 72℃ for 2 min, and final extension at 72℃ for 10 min, followed by storage at 4℃.

[0025] The PCR amplification procedure also includes restriction enzyme digestion, specifically, primer TNAC1731 is digested with restriction enzyme TaqI.

[0026] In step (1), detection was performed using 10% non-denaturing polyacrylamide gel electrophoresis.

[0027] Polyacrylamide electrophoresis of the PCR products revealed that primers CINAU2570, CINAU2683, and CINAU1553 amplified specific DNA bands of 350 bp in the Chinese Spring-Aegilops duplex TA8024, the Chinese Spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F3 6T addition line, and the 6TS.7AL translocation line, respectively. These two specific bands could not be amplified in the parental controls (Chinese Spring, Mianyang 11, Mianyang 15) and negative controls (Jimai 22, Jimai 4075, and Jimai 6206) which do not contain Aegilops 6T chromosome. This suggests that these two specific bands may be specific fragments of the Aegilops 6T chromosome. Figure 1 ).

[0028] The PCR product amplified by primer TNAC1731 was digested with restriction endonuclease TaqI and then electrophoresed. The results showed that primer TNAC1731 / TaqI could obtain a polymorphic fragment of approximately 750 bp in the Chinese spring-Aegilops duploid TA8024, the Chinese spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F3 6T addition line, and the 6TS.7AL translocation line, but not in the parental controls Chinese spring, Mianyang 11, Mianyang 15, and the negative controls Jimai 22, Jimai 4075, and Jimai 6206. This indicates that these fragments are specific molecular markers of the Aegilops 6T chromosome. Figure 2 ).

[0029] The above four primer pairs can be used as specific molecular markers for the 6T chromosome of Aegilops awnless, and are designated as CINAU2570-IT, CINAU2683-IT, CINAU1553-IT and TNAC1731-PLUG, respectively.

[0030] Beneficial effects:

[0031] 1. This invention establishes a new marker for the 6T chromosome of *Aegilops awnless*, and provides a new method for detecting *Aegilops awnless* 6T chromosome in wheat background using this new marker. Because the 6T chromosome of *Aegilops awnless* contains a gene for resistance to wheat powdery mildew, the specific molecular marker obtained in this invention can be used for screening, identification, and assisted breeding of new wheat lines / variety resistant to powdery mildew in hybrid offspring.

[0032] 2. The selected specific molecular markers can be used to detect or assist in the detection of whether Aegilops awnless 6T chromosome is present in wheat background. They have high specificity and high detection accuracy, improve screening efficiency, shorten breeding time, and have positive industrialization value. Attached Figure Description

[0033] Figure 1 The electrophoresis results of the amplification products of primers CINAU2570(A), CINAU2683(B), and CINAU1553(C) in a 10% polyacrylamide gel are shown. The arrows indicate polymorphic bands. M = Marker (DL2000). Lanes 1-9 represent the following double diploid lines: Chinese Spring-Aegilops TA8024, Chinese Spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F3 6T addendum line, 6TS.7AL translocation line, Jimai 22, Jimai 4075, Jimai 6206, Mianyang 11, Mianyang 15, and Chinese Spring, respectively.

[0034] Figure 2 The image shows the electrophoresis results of the amplification product of primer TNAC1731 (digested with TaqI) on a 10% agarose gel. The arrows indicate polymorphic bands. M = Marker (DL2000). Lanes 1-9 represent the following double diploid lines: Chinese Spring-Aegilops TA8024, Chinese Spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F3 6T addition line, 6TS.7AL translocation line, Jimai 22, Jimai 4075, Jimai 6206, Mianyang 11, Mianyang 15, and Chinese Spring. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] Unless otherwise specified, the plant materials (Table 1) and reagents used in the following examples are commercially available.

[0037] Unless otherwise specified, the PCR system and methods used in the following examples are the same as those described in the invention content section, and all primer synthesis was completed by Chengdu Ruixin Biotechnology Co., Ltd. Jimai 22, Jimai 4075, and Jimai 6206 were all independently bred by the applicant's research team. China Spring (CS), Mianyang 11, and Mianyang 15 were provided by the Crop Research Institute of Shandong Academy of Agricultural Sciences. The China Spring-Aegilops duploid, the China Spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F36T disomic addition line, and the China Spring-Aegilops / Mianyang 11 / / Mianyang 15BC1F36TS.7AL translocation line were progeny materials obtained from parental hybridization.

[0038] Table 1 Test Materials

[0039]

[0040] 1. Primer design and synthesis

[0041] The genes of wheat, rice, and distant wheat species, including *Aegilops awnlessii*, exhibit good collinearity, and sequence polymorphisms may exist among these three genes. Therefore, the design and amplification of intron targets and PCR-based landmark-specific gene primers are mainly based on the collinearity of species genes. This leads to the amplification of *Aegilops awnlessii* and wheat genomic DNA, and even the restriction enzyme digestion of the amplification products to establish *Aegilops awnlessii* chromosome-specific markers. The primers used are based on rice EST sequences.

[0042] 2. Primer screening and acquisition of chromosome 6T-specific DNA fragments from Aegilops spp.

[0043] To screen designed primers and preliminarily obtain chromosome 6T-specific DNA fragments from *Aegilops spp.*, *Chinese Spring*, *Mianyang 11*, and *Mianyang 15* were used as parental controls, and the *Chinese Spring*-*Aegilops spp.* double diploid TA8024 was used as a positive control. Intron-targeting and PCR-based landmark-specific gene primers were screened. The amplification characteristics of the PCR-based landmark-specific gene primers were as follows: a 30 μL PCR reaction system consisted of: 2.0 μL of 25 ng / μL template DNA, 0.3 μL of 5 U / μL Taq DNA polymerase, 200 μM dNTPs, and Mg2+. 2+ 3.0 μL of 10×PCR buffer, 2 μL each of 10 μM upstream and downstream primers, and sterile double-distilled water were added to bring the reaction volume to 30 μL. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, followed by 35 cycles: 94℃ denaturation for 45 s, 57℃ annealing for 45 s, 72℃ extension for 2 min, and a final extension at 72℃ for 10 min. The sample was then stored at 4℃.

[0044] The 30 μL PCR product was placed in a PCR tube. The PCR product amplified by TNAC1731 was digested with the restriction endonuclease TaqI (the digestion system contained 6.5 μL sterile double-distilled water, 3.0 μL 10×NEB buffer, 0.3 mL 10×BSA, and 0.2 μL restriction endonuclease; digestion temperature was 65℃; digestion time was 2 h). Both undigested and digested PCR products were electrophoresed on a 10% polyacrylamide gel using 10×Tris Gly electrophoresis buffer. 10 μL of the amplified product was electrophoresed at a constant voltage of 150 V for approximately 25 min, and then photographed.

[0045] Polyacrylamide electrophoresis of the PCR products revealed that primers CINAU2570, CINAU2683, and CINAU1553 amplified specific DNA bands of 350 bp in the *Aegilops chinensis*-*Aegilops lanceolata* double diploid, *Aegilops chinensis* / *Mianyang 11* / *Mianyang 15*BC1F3 6T addendum line, and 6TS.7AL translocation line, respectively. These two specific bands could not be amplified in the parental controls *Aegilops chinensis*, *Mianyang 11*, and *Mianyang 15*, or the negative controls *Jimai* 22, *Jimai* 4075, and *Jimai* 6206. Figure 1 (A, B, C) This indicates that these two specific bands may be specific segments of the 6T chromosome of *Aegilops argyrophylla*.

[0046] The PCR product amplified by primer TNAC1731 was digested with restriction endonuclease TaqI and then electrophoresed. The results showed that primer TNAC1731 / TaqI could obtain a polymorphic fragment of approximately 750 bp in the *Aegilops chinensis* double diploid, the *Aegilops chinensis* / Mianyang 11 / / Mianyang 15BC1F36T addition line, and the 6TS.7AL translocation line, but not in the parental controls *Aegilops chinensis*, *Mianyang 11*, *Mianyang 15*, and the negative controls Jimai 22, Jimai 4075, and Jimai 6206. This indicates that these fragments are specific molecular markers of the 6T chromosome of *Aegilops chinensis*. Figure 2 ).

[0047] Based on the above method steps, a chromosome-6T specific molecular marker of Aegilops argyracea was obtained, and its base sequence is shown in Table 2 below.

[0048] Table 2. Chromosome-specific markers of Aegilops argyracea and related information.

[0049]

[0050] Note: "—" indicates that the PCR product can be polymorphic without enzyme digestion.

[0051] The above description is only a preferred embodiment of this patent. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of this patent, and these improvements and substitutions should also be considered within the scope of protection of this patent.

Claims

1. The application of a primer set specifically targeting the 6T chromosome of *Aegilops awnless* in detecting or assisting in the detection of *Aegilops awnless* 6T chromosome in wheat background, characterized in that... PCR amplification was performed using a primer set containing specific molecular markers for the Aegilops 6T chromosome. The PCR products were then subjected to polyacrylamide electrophoresis. If primers CINAU2570, CINAU2683, and CINAU1553 amplified specific DNA bands of 350 bp, then the wheat background contained the Aegilops 6T chromosome. The PCR products amplified by primer TNAC1731 were digested with the restriction endonuclease TaqI and then electrophoresed. If a specific DNA band of approximately 750 bp was observed, then the wheat background contained the Aegilops 6T chromosome. The primer combination is as follows: CINAU2570: F: 5'-AGGTGGTGATTTCAGAGGCA-3'; R: 5'-CCGTGAAAAGCTAGACTCTGC-3'; CINAU2683: F: 5'- AGATCTCTTTAGGGATGCGCT-3'; R: 5'-CCAACCTGTAAGCTGCGC-3'; CINAU1553: F: 5'- CCTACCTCGTCCACCACAAG-3'; R: 5'-GCCGGATCTCTTCATCACG-3'; TNAC1731: F: 5'-TGTTGCTTTCAGAGCGAATTT-3'; R: 5'-TTGCTCCACCGAGATCACTAC-3'.

2. The application according to claim 1, characterized in that, The detection or auxiliary detection steps are as follows: (1) Using the total genomic DNA of the wheat background line that may contain the 6T chromosome of Aegilops awnless as a template, PCR amplification was performed on it using the primer combination of the specific molecular marker of Aegilops awnless 6T chromosome as described in claim 1. The amplification products were detected by polyacrylamide electrophoresis. (2) If the gel electrophoresis pattern of the template DNA to be tested contains the same specific DNA band as the positive control but the negative control does not contain the same band, it indicates that the genome of the wheat background line to be tested contains the 6T chromosome of Aegilops awnless; if the gel electrophoresis pattern of the template DNA to be tested does not contain the same specific DNA band as the positive control, it indicates that the genome of the wheat background line to be tested does not contain the 6T chromosome of Aegilops awnless; the positive control is Chinese spring-Aegilops awnless double diploid; the negative control is Chinese spring wheat.

3. The application according to claim 2, characterized in that, The PCR amplification system consisted of: 2.0 μL template DNA (25 ng / μL), 0.3 μL Taq DNA polymerase (5 U / μL), 200 μM dNTPs, and Mg2+. 2+ 3.0 μL of 10×PCR buffer, 2 μL each of 10 μM upstream and downstream primers, and sterile double-distilled water were added to bring the reaction volume to 30 μL. The PCR amplification program was as follows: pre-denaturation at 94℃ for 3 min; followed by 35 cycles: denaturation at 94℃ for 45 s, annealing at 57℃ for 45 s, extension at 72℃ for 2 min; and final extension at 72℃ for 10 min, followed by storage at 4℃.

4. The application according to claim 2, characterized in that... In step (1), 10% polyacrylamide electrophoresis was used for detection.

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