CN114921586B - Chili cytoplasmic male sterility restorer gene InDel mark, specific primer and application thereof - Google Patents

Chili cytoplasmic male sterility restorer gene InDel mark, specific primer and application thereof Download PDF

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CN114921586B
CN114921586B CN202210700780.5A CN202210700780A CN114921586B CN 114921586 B CN114921586 B CN 114921586B CN 202210700780 A CN202210700780 A CN 202210700780A CN 114921586 B CN114921586 B CN 114921586B
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p06gindel
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pepper
indel
restorer
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CN114921586A (en
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聂智星
朱为民
陈建瑛
宋云鹏
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Shanghai Academy of Agricultural Sciences
Hangzhou Institute of Agricultural Sciences
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Hangzhou Institute of Agricultural Sciences
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Abstract

The invention discloses an InDel marker of a hot pepper cytoplasmic male sterility fertility restorer gene, a specific primer and application thereof, wherein the InDel marker comprises P06gInDel-79 and P06gInDel-81, is positioned on a No. 6 chromosome of a hot pepper reference genome Zuna-1 V2.0, has corresponding physical positions of Chr06:215,419,754..215,420,003 and Chr06:215,498,219..215,498,426, and is used for detecting that the InDel marker is P06gInDel-79-F/P06gInDel-79-R and P06gInDel-81-F/P06gInDel-81-R respectively. By utilizing the InDel marking and screening method, a new pepper maintainer line and a new pepper restorer line can be rapidly screened from germplasm resources, so that the breeding process of pepper hybrid seeds is accelerated.

Description

Chili cytoplasmic male sterility restorer gene InDel mark, specific primer and application thereof
Technical Field
The invention belongs to the field of pepper breeding, and particularly relates to an InDel marker of a Cytoplasmic Male Sterility (CMS) fertility restorer gene of pepper, a specific primer and application thereof.
Background
China is the largest pepper producing country in the world, and more than 90% of pepper varieties are hybrid seeds. The three-line hybrid seed production system composed of CMS line, maintainer line and restorer line is one of the important methods for producing hybrid seed of hot pepper. The method can obviously reduce the production cost and improve the purity of the hybrid seeds. The fertility stability of the three-line hybrid is an important guarantee for large-area popularization, and the gene in the restorer plays a key role.
In the three-line cross breeding of hot pepper, the restoring line and the sterile line are the basis of the hybrid seed breeding. Selection of new restorer and sterile lines is usually performed by test crossing with an existing CMS line and observing the progeny F 1 The fertility of (c) is determined. This takes at least 2 growing seasons, and F 1 The fertility of the male parent may be influenced by environmental conditions, and the breeding efficiency is low and the breeding is accurateSex needs to be verified in different growth environments. In recent years, molecular marker-assisted selection has gradually been used effectively in breeding practice. On the basis of fine positioning of a restorer gene, a molecular marker coseparated with the fertility restorer gene is obtained, a new maintainer line and a restorer line can be screened from germplasm resources, and the newly screened maintainer line is used as a male parent to carry out continuous backcross on the original CMS line for more than 4 generations, so that a new sterile line can be obtained. The molecular marker assisted selection can save the breeding time and accelerate the seed selection process of pepper hybrids.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the problems, the invention aims to provide an InDel marker of a hot pepper cytoplasmic male sterility fertility restorer gene, a specific primer and application thereof.
The technical scheme is as follows: the purpose of the invention can be realized by the following technical scheme:
an InDel marker of a hot pepper cytoplasmic male sterility restorer gene comprises P06gInDel-79 and P06gInDel-81, is positioned on chromosome 6 of a hot pepper reference genome Zuna-1 V2.0, and has the corresponding physical positions of Chr06:215,419,754..215,420,003 and Chr06:215,498,219..215,498, 426.
The specific primer marked by the cytoplasmic male sterility restorer gene InDel of the capsicum is shown as follows:
InDel marker P06gInDel-79:
an upstream primer P06gInDel-79-F:5 'GAGGGTCTTCCTATGTTTGTCC-3', SEQ ID NO.1;
the downstream primer P06gInDel-79-R:5 'TGCACGAATGAACAGTAAACCA-3', SEQ ID NO.2;
InDel marker P06gInDel-81:
an upstream primer P06gInDel-81-F:5 'TCATTCCCTTTTTTCCAGCTCTG-3', SEQ ID NO.3;
the downstream primer P06gInDel-81-R:5 'ACAGGGTGTTATGGGTGATT-3' and SEQ ID NO.4.
A kit comprising the specific primer.
The application of the InDel marker, the specific primer or the kit in pepper molecular marker assisted breeding.
The application of the InDel marker, the specific primer or the kit in screening the pepper maintainer line and the pepper restorer line.
A method of screening pepper maintainer and restorer lines comprising the step of applying said InDel marker, said specific primers or said kit.
Further, the method for screening the pepper maintainer line and the pepper restorer line comprises the following steps of:
(1) Extracting genome DNA of a capsicum inbred line to be detected;
(2) Taking the genomic DNA extracted in the step (1) as a template, and carrying out PCR amplification by using the specific primer or the kit;
(3) Carrying out electrophoresis detection on the amplification product;
(4) If the banding patterns of the two InDel marked amplification products are the same as the banding pattern of the sterile line HZ1A at the same time, the selfing line of the pepper to be detected is a maintainer line; and if the band patterns of the two InDel marked amplification products are the same as the band pattern of the restoring line HZ1C at the same time, the pepper selfing line to be detected is the restoring line.
Specifically, in the step (4), 2 bands appear after the electrophoresis of the amplification product of P06gInDel-79 in the sterile line HZ1A, and 1 more band of about 200bp appears in the restorer line HZ1C; the P06gInDel-81 has only 1 band after electrophoresis in the sterile line HZ1A, and has 1 more band of about 120bp in the restorer line HZ 1C.
Has the advantages that: in traditional breeding, selection of new maintainer and restorer lines is typically carried out by test crossing with an existing sterile line and observing F 1 The fertility of the offspring is determined, but the time is longer, and the breeding efficiency of the method is lower. By utilizing the InDel marker and the screening method, a new maintainer line (backcrossing the existing sterile line for more than 4 generations continuously to obtain a new sterile line) and a restorer line can be rapidly screened in germplasm resources, so that the speed of the method is increasedAnd (5) a capsicum hybrid breeding process.
Drawings
FIG. 1 is an electrophoresis diagram of InDel markers P06gInDel-79 and P06gInDel-81 amplified in sterile line HZ1A, restorer line HZ1C and 13 inbred lines, wherein: a is the detection result of InDel mark P06gInDel-79 in sterile line HZ1A, restoring line HZ1C and 13 inbred lines; b is the detection result of an InDel marker P06gInDel-81 in the sterile line HZ1A, the restorer line HZ1C and 13 inbred lines; m:50bp Marker;1: HZ1A;2: HZ1C;3: p21204;4: p21238;5: p21239;6: p21241;7: p21243;8: p21244;9: p21246;10: p21270;11: p21273;12: p21215;13: p21240;14: p21247;15: p21274.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, which is to be given the full breadth of the claims appended hereto.
Example 1
Construction of (HZ 1A. Times. HZ 1C) F by using hot pepper cytoplasmic male sterile line HZ1A as female parent and restorer line HZ1C as male parent 2 And in the population, a fertility restorer gene of the sterile line HZ1A is positioned between InDel markers P06gInDel-66 and P06gInDel-89 on chromosome 6 by adopting a mixed grouping analysis method, the physical position is Chr06:215,097,259..215,631,069 (reference genome: zuna-1 V2.0), and the interval size is 533.81kb.
Within this localization interval, 2 newly developed InDel markers P06gInDel-79 and P06gInDel-81 cosegregated with the restorer gene. The 2 markers can be used as specific molecular markers for identifying the pepper restorer gene. P06gInDel-79 shows 2 bands after the electrophoresis of the amplification product in HZ1A, and 1 more band of about 200bp is shown in HZ 1C. P06gInDel-81 has only 1 band after the electrophoresis of the amplification product in HZ1A, and has about 1 more band of 120bp in HZ 1C.
Example 2
Screening of specific molecular marker for fertility restoring gene for new hot pepper maintainer line and restoring line
Biological material: 13 parts of pepper inbred line. 3: p21204;4: p21238;5: p21239;6: p21241;7: p21243;8: p21244;9: p21246;10: p21270;11: p21273;12: p21215;13: p21240;14: p21247;15: p21274.
Genomic DNA was extracted from young leaves using a DNA extraction kit (DP 321, tiangen Biochemical technology (Beijing) Ltd.).
PCR amplification was performed in 13 pepper inbred lines described above using InDel markers P06gInDel-79 and P06 gInDel-81. The specific primers used are as follows:
InDel marker P06gInDel-79:
an upstream primer P06gInDel-79-F:5 'GAGGGTCTTCCTATGTTTGTCC-3', SEQ ID NO.1;
the downstream primer P06gInDel-79-R:5 'TGCACGAATGAACAGTAAACCA-3', SEQ ID NO.2;
InDel marker P06gInDel-81:
an upstream primer P06gInDel-81-F:5 'TCATTCCCTTTTTTCCAGCTCTG-3', SEQ ID NO.3;
the downstream primer P06gInDel-81-R:5 'ACAGGGTGTTATGGGTGATT-3', SEQ ID NO.4.
The PCR reaction volumes were as follows: 5 mu L2 XHieff PCR Master Mix (10102 ES03, next saint Biotechnology (Shanghai) Pythiist Co., ltd.), 1 mu L10 mu M PCR upstream primer, 1 mu L10 mu M downstream primer and 20 ng template DNA, and finally adding ddH 2 O to 10. Mu.L.
The PCR reaction program is: 94. pre-denaturation at deg.C for 2 min; 94. 32 cycles of 30 s, 30 s of primer annealing temperature and 30 s of 72 ℃; final extension at 72 ℃ for 5 min; cooled to 12 ℃.
The amplification product was detected electrophoretically on 4% agarose gel at 120V for 50min, and photographed using an ultraviolet gel imager.
According to the PCR amplification results of InDel marks P06gInDel-79 and P06gInDel-81 in the 13 pepper inbred lines, the inbred line banding patterns of numbers 3-11 and 15 are the same as HZ1A, and the inbred line banding patterns are judged as a maintainer line of HZ1A (continuous backcross for multiple generations is a new sterile line), and the inbred line banding patterns of numbers 12-14 are the same as HZ1C, and the inbred line banding patterns are judged as a restorer line of HZ 1A.
The selfing line of pepper with the number of 3-15 is taken as male parent, HZ1A is taken as female parent to prepare hybrid combination, and F is observed 1 The fertility of (2) is judged, the inbred lines of 3-11 are maintainer lines of HZ1A, and the inbred lines of 12-15 are restorer lines of HZ 1A. The accuracy of identifying the pepper maintainer line and the pepper restorer line by 2 InDel markers P06gInDel-79 and P06gInDel-81 is 92.31 percent.
Sequence listing
<110> Shanghai city academy of agricultural sciences
Hangzhou Academy of Agricultural Sciences
<120> pepper cytoplasmic male sterility restorer gene InDel marker, specific primer and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagggtcttc ctatgtttgt cc 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgcacgaatg aacagtaaac ca 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcattccctt tttccagctc tg 22
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acaggggtgt tatgggtgat t 21

Claims (3)

1. The application of a specific primer in screening a pepper maintainer line and a pepper restorer line is characterized in that the sequence of the specific primer is as follows:
InDel marker P06gInDel-79:
an upstream primer P06gInDel-79-F:5 'GAGGGTCTTCCTATGTTTGTCC-3', SEQ ID NO.1;
the downstream primer P06gInDel-79-R:5 'TGCACGAATGAACAGTAAACCA-3', SEQ ID NO.2;
InDel marker P06gInDel-81:
an upstream primer P06gInDel-81-F:5 'TCATTCCCTTTTTTCCAGCTCTG-3', SEQ ID NO.3;
the downstream primer P06gInDel-81-R:5 'ACAGGGTGTTATGGGTGATT-3' and SEQ ID NO.4.
2. Use of a kit for screening pepper maintainer and restorer lines, wherein the kit comprises the specific primers of claim 1.
3. A method for screening pepper maintainer and restorer lines, which comprises the step of using the specific primers of claim 1 or the kit of claim 2.
CN202210700780.5A 2022-06-20 2022-06-20 Chili cytoplasmic male sterility restorer gene InDel mark, specific primer and application thereof Active CN114921586B (en)

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CN101748195A (en) * 2008-12-10 2010-06-23 上海市农业科学院 Creation method of sweet pepper SSR finger-print and application thereof
CN104561297B (en) * 2014-12-29 2017-06-16 浙江省农业科学院 A kind of SSR molecular marker method and its kit for detecting hot pepper male sterile Restore gene
CN111518944B (en) * 2020-05-29 2022-05-31 中国农业科学院蔬菜花卉研究所 Chili cytoplasmic male sterility restoring gene related InDel marker D6-26, specific primer and application thereof

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