InDel marker for identifying green Chinese onion cytoplasm fertility based on chloroplast genome and application of InDel marker

Abstract

The invention belongs to the technical field of genetic breeding, and relates to an InDel marker for identifying green Chinese onion cytoplasm fertility based on a chloroplast genome and application of the InDel marker. The InDel marker is subjected to amplification detection by adopting a primer group, and the primer group comprises one or more of a first primer group, a second primer group, a third primer group and a fifth primer group; a pair of nucleotide sequences of the first primer group are as shown in SEQ ID NO.1-2; a pair of nucleotide sequences of the second primer group are as shown in SEQ ID NO.3-4; a pair of nucleotide sequences of the third primer group are as shown in SEQ ID NO.5-6; a pair of nucleotide sequences of the fourth primer group are as shown in SEQ ID NO.7-8; and a pair of nucleotide sequences of the fifth primer group are as shown in SEQ ID NO.9 to SEQ ID NO.10. The InDel marker provided by the invention can be used for identifying the cytoplasm fertility of the green Chinese onion, and the tedious procedure and process for extracting DNA can be omitted in the detection process, so that the detection process is simplified.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and relates to an InDel marker for identifying cytoplasmic fertility of green onion based on chloroplast genome and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Welsh onion (Allium fistulosum L.) is a vegetable crop. The flower organs of welsh onion are small, and artificial emasculation is not realistic. Using male sterile lines to breed and produce hybrids is the most effective technical measure to maintain variety characteristics, ensure seed quality, and reduce seed costs. Therefore, the research on male sterility of welsh onion is a very important hotspot both in applied basis and basic theoretical research.

[0004] Since green onions are biennial vegetables, the traditional method of breeding green onion male sterile lines and maintenance lines is time-consuming, labor-intensive and inefficient. The combination of conventional breeding and molecular marker-assisted breeding can effectively solve the key links of long breeding years and low selection efficiency.

[0005] There have been relevant reports on the research of molecular markers for cytoplasmic male sterility in onions. Gai Shupeng et al. used the sterile line and maintainer line of Zhangqiu onion as materials, performed RAPD marking on the cytoplasmic mitochondrial DNA, and obtained two RAPD markers that can identify the cytoplasmic types of some onion varieties (Gai Shupeng, Meng Xiangdong, Xu Lijuan. 2004. Research on molecular marker-assisted selection of male sterility in onion. Molecular Plant Breeding, 2: 223-228.; Gai Shupeng, Meng Xiangdong. 2004. Research on the conversion of RAPD markers of cytoplasmic male sterility sites in onion to SCAR markers. Journal of Laiyang Agricultural College, 21: 189-192; WANG, C., LI, HY, ZHANG, LY, PEI, YX and WANG, YQ 2013. Identification of an AFLP marker and conversion to a SCAR marker to identify cytoplasmic male-sterile or normal cytoplasmin Welsh onion (Allium fistulosum L.). Journal of Horticultural Science & Biotechnology, 88, 409-414.). However, these markers are all based on mitochondrial DNA, and since the extraction of mitochondrial DNA is relatively complicated, they are subject to certain limitations in practical application.

[0006] Chinese patent ZL201510296026.X developed the SCAR molecular marker with the total DNA of green onion as the template, Chinese patent ZL201510296026.X developed the dCAPS marker with the total DNA of green onion as the template, and Chinese patent ZL201710103250.1 developed the KASP marker with the total DNA of green onion as the template. The development and application of these markers effectively avoided the blindness of the screening of the maintenance line and improved the selection efficiency. However, the development of these markers is based on the difference of the mitochondrial genome of green onion. At the same time, the PCR reaction is carried out with the total DNA of green onion as the template, and then the cytoplasmic fertility is judged by electrophoresis. Although it is much faster than field identification, the extraction of total DNA of green onion is quite time-consuming and labor-intensive.

[0007] Cytoplasmic male sterility is common in the plant kingdom, and plant cytoplasmic male sterility is a typical cytoplasmic inheritance. Therefore, the research on plant cytoplasmic male sterility at the molecular level mainly focuses on the chloroplast genome and the mitochondrial genome. The research on cytoplasmic male sterility of green onions mainly focuses on mitochondria, while chloroplasts are plant-specific organelles. Like mitochondria, they are autonomous and are another type of genetic system outside the nucleus of higher plants. There are few studies on the relationship between green onion chloroplasts and cytoplasmic male sterility. Summary of the invention

[0008] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide an InDel marker for identifying the cytoplasmic fertility of green onion based on the chloroplast genome and its application. The InDel marker provided by the present invention can identify the cytoplasmic fertility of green onion, and can save the cumbersome procedures and processes of extracting DNA during the detection process, thereby simplifying the detection process.

[0009] In order to achieve the above object, the technical solution of the present invention is:

[0010] In a first aspect, a primer set for amplifying an InDel marker comprises one or more of a first primer set, a second primer set, a third primer set, a fourth primer set, and a fifth primer set;

[0011] Wherein, a pair of nucleotide sequences of the first primer set are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively;

[0012] A pair of nucleotide sequences of the second primer set are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively;

[0013] A pair of nucleotide sequences of the third primer set are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0014] The nucleotide sequences of the fourth primer set are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively;

[0015] The pair of nucleotide sequences of the fifth primer set are shown as SEQ ID NO.9 and SEQ ID NO.10 respectively.

[0016] In a second aspect, an InDel marker for identifying the cytoplasmic fertility of green onions based on the chloroplast genome is provided. The InDel marker is amplified and detected using the primer set described in the first aspect of the present invention.

[0017] In a third aspect, a kit for detecting cytoplasmic fertility of green onions comprises the primer set described in the first aspect of the present invention.

[0018] In some embodiments, a premix for PCR reaction is also included. Specifically, the premix for PCR reaction is 2×PfuMasterMix; which includes Pfu DNA polymerase, dNTPs, MgCl 2 , reaction buffer, optimizer, stabilizer, etc.

[0019] In some embodiments, water is also included. The water used in the present invention is generally double distilled water (ddH 2 O).

[0020] In a fourth aspect, a use of the primer set described in the first aspect of the present invention, the InDel marker described in the second aspect of the present invention, or the detection kit described in the third aspect of the present invention in detecting the cytoplasmic fertility of green onion.

[0021] A fifth aspect is a method for detecting cytoplasmic fertility of green onion, comprising:

[0022] The genomic DNA of green onion leaves is used as a template and PCR amplification is performed using the primer set described in the first aspect of the present invention, and the cytoplasmic fertility of green onion is determined based on the amplified product.

[0023] In some embodiments, a DNA extraction-free PCR kit (PCR direct amplification kit) is used to collect samples to obtain a sample collection fluid, and PCR amplification is performed on the sample collection fluid.

[0024] In some embodiments, the reaction system during PCR amplification includes a sample collection solution, a premix for PCR reaction, and double distilled water.

[0025] In some embodiments, the amplification program for PCR amplification is:

[0026]

[0027] In a sixth aspect, a primer set according to the first aspect of the present invention, the InDel marker according to the second aspect of the present invention, or the detection kit according to the third aspect of the present invention is used in green onion breeding.

[0028] The beneficial effects of the present invention are:

[0029] The present invention successfully assembled the chloroplast genome of the male sterile line of Zhangqiu green onion and the corresponding maintenance line by Illumina HiSeq 4000 sequencing, developed an InDel marker for identifying cytoplasmic fertility based on sequence differences and verified it on multiple materials. The results show that the InDel molecular marker provided by the present invention can realize the identification of green onion cytoplasmic fertility. Since the InDel marker amplification product is short, the DNA quality requirements are low, and the sample quantity requirements are low, and highly degraded DNA can be amplified. Therefore, the present invention adopts a DNA extraction-free PCR kit (PCR direct amplification kit) for sample collection, which eliminates the cumbersome procedures and processes for extracting DNA and simplifies the detection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 This is a result diagram of the InDel marker development for identifying the fertility of the cytoplasm of green onion in the embodiment of the present invention; wherein M is a 50 bp DNA ladder, S is sterile cytoplasm, and N is fertile cytoplasm; S1 and N1 are marker 1, S2 and N2 are marker 2, S3 and N3 are marker 3, S4 and N4 are marker 4, and S5 and N5 are marker 5.

[0032] Figure 2 This is a diagram showing the results of the InDel marker verification for identifying the cytoplasmic fertility of Welsh onion in the embodiment of the present invention; wherein 1, 2, 3, 4, and 5 represent the five developed markers, and the lanes behind the markers are 980238A, 980238B, 980128A, 980128B, 200501A, 200501B, 201204A, 201204B, Lucongza No. 1 (F 1 )、Lucongza No. 5(F 1 )、Luchun Onion No. 1(F 1 )、Luqiucong No. 1(F 1 )、Luqiucong No. 2(F 1 ). DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] Example 1

[0035] Materials and Methods

[0036] The test materials used for marker development were the Zhangqiu green onion sterile line (ZQA) and the maintainer line (ZQB), and the materials used for marker verification were 4 pairs of sterile lines and maintainers (980238A / B, 980128A / B, 200501A / B, 201204A / B) and 5 hybrids (Lucongza 1, Lucongza 5, Luchuncong 1, Luqiucong 1, Luqiucong 2), all of which were bred by our research group. Among them, the sterile line is a stable sterile line bred by multi-generation backcrossing, which is highly consistent with its maintainer background.

[0037] The chloroplast genomes of Zhangqiu Welsh Onion ZQA and ZQB were sequenced using the Illumina Hiseq 4000 sequencing platform, and the sequences were assembled using bioinformatics analysis methods. The full length of the chloroplast genomes was 153109 bp (ZQA) and 153162 bp (ZQB), respectively. According to the insertion / deletion differential sites of the chloroplast genomes of the Zhangqiu Welsh Onion sterile line (ZQA) and the maintainer line (ZQB), InDel-labeled primers for identifying the cytoplasmic fertility of Welsh Onion were designed. There were 5 pairs of primers, and the nucleotide sequences were shown in Table 1. The total DNA of the sterile line (ZQA) and the maintainer line (ZQB) was amplified using these 5 pairs of primers. The specific steps are as follows:

[0038] (1) The total DNA extraction method of ZQA and ZQB materials was carried out using the rapid plant genome extraction kit produced by Beijing Tiangen Biochemical Technology Co., Ltd. The extraction method was referred to the instruction manual.

[0039] (2) PCR reaction was carried out on a TC-XP-D gene amplification instrument produced by Bio-Rad. The target fragment was obtained by adding 10 μL of 2×PfuMasterMix and two primers (10 μmol·L -1 ) 1.0 μL each, sample DNA 1.0 μL, ddHO 2 O to 20 μL; the reaction program was: 94°C pre-denaturation for 2 min; 94°C denaturation for 30 s, 55°C annealing for 1 min, 72°C extension for 1 min, 35 cycles; 72°C extension for 5 min, and storage at 4°C.

[0040] (3) Electrophoresis and staining detection: The PCR amplification products were separated by 8% non-denaturing polyacrylamide gel electrophoresis for 1 hour. After the gel was taken out, it was stained and developed by silver staining. The results were as follows: Figure 1 shown.

[0041] (4) Sequencing: Sequencing was performed after gel recovery. The measured sequence sizes are shown in Table 1.

[0042] Table 1 Primer sequences and product sizes of marker development designs

[0043]

[0044]

[0045] Verification of the marker: To test the stability and reliability of the InDel marker, other materials selected and bred by our research group were used for testing and verification. The testing steps are as follows:

[0046] (1) Collect samples from fresh green onion leaves using a DNA extraction-free PCR kit (PCR direct amplification kit);

[0047] (2) PCR reactions were performed on a TC-XP-D gene amplification instrument produced by Bio-Rad. The target fragment was obtained by adding 10 μL of 2×PfuMasterMix and two primers (10 μmol L -1 ) 1.0 μL each, sample collection solution 1.0 μL, ddHO 2 O is added to 20 μL; the reaction procedure is:

[0048]

[0049] (3) Electrophoresis and staining detection: The PCR amplification products were separated by 8% non-denaturing polyacrylamide gel electrophoresis for 1 hour. After the gel was taken out, it was stained and developed by silver staining. The development results were as follows: Figure 2 As shown,

[0050] (4) Sequencing: Sequencing was performed after gel recovery. The fragment sizes of the sequencing results are shown in Table 2.

[0051] Table 2 Verification results of individual plants of cytoplasm types of different materials of green onion

[0052]

[0053] Figure 2 The results in Table 2 show that the sizes of the fragments amplified from all sterile materials and hybrid combinations are consistent with the ZQA results, and the fragments amplified from the maintainer materials are consistent with the ZQB results. This result shows that the five InDel markers of the present invention can all be used for cytoplasmic fertility screening of green onion.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer set for amplifying an InDel marker, characterized in that: including one or more of a first primer set, a second primer set, a third primer set, a fourth primer set, and a fifth primer set; Wherein, a pair of nucleotide sequences of the first primer set are shown as SEQ ID NO.1 and SEQ ID NO.2 respectively; A pair of nucleotide sequences of the second primer set are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively; A pair of nucleotide sequences of the third primer set are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively; The nucleotide sequences of the fourth primer set are shown as SEQ ID NO.7 and SEQ ID NO.8 respectively; The pair of nucleotide sequences of the fifth primer set are shown as SEQ ID NO.9 and SEQ ID NO.10 respectively.

2. An InDel marker for identifying cytoplasmic fertility of green onion based on chloroplast genome, characterized in that: The InDel marker is amplified and detected using the primer set described in claim 1.

3. A kit for detecting cytoplasmic fertility of green onion, characterized in that: Comprising the primer set of claim 1.

4. The detection kit according to claim 3, characterized in that: The method further comprises a premix for PCR reaction; preferably, the premix for PCR reaction is 2×PfuMasterMix; or, further comprises water.

5. Use of the primer set according to claim 1, the InDel marker according to claim 2, or the detection kit according to claim 3 or 4 in detecting the cytoplasmic fertility of green onion.

6. A method for detecting cytoplasmic fertility of green onion, characterized in that: include: The genomic DNA of green onion leaves is used as a template and PCR amplification is performed using the primer set described in the first aspect of the present invention, and the cytoplasmic fertility of green onion is determined based on the amplified product.

7. The detection method according to claim 6, characterized in that: A DNA extraction-free PCR kit was used to collect samples to obtain sample collection fluid, and PCR amplification was performed on the sample collection fluid.

8. The detection method according to claim 6, characterized in that: The reaction system during PCR amplification includes a sample collection solution, a premix for PCR reaction, and double distilled water.

9. The detection method according to claim 6, characterized in that: The amplification program for PCR amplification is:

10. Use of the primer set according to claim 1, the InDel marker according to claim 2, or the detection kit according to claim 3 or 4 in green onion breeding.

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