Chili maturity SNP (Single Nucleotide Polymorphism) molecular marker and application thereof

Abstract

The invention discloses a chili maturity SNP (Single Nucleotide Polymorphism) molecular marker and the application thereof. The chili maturity SNP molecular marker comprises a sequence of SEQ ID NO.1as shown in the specification. By using a BSR-Seq (Bulked Segregant RNA-Seq) technique, a chromosome site controlling first flower nodes can be rapidly identified, and the chili maturity SNP molecularmarker is specifically developed for the site. By adopting the molecular marker and an amplification primer thereof, and with specific restriction endonuclease treatment, restriction endonuclease chilies can be rapidly and accurately screened at a cotyledon stage, molecular marker auxiliary selection upon chili maturity breeding can be achieved, the breeding selection efficiency can be improved,and the molecular marker has significant application values in chili maturity breeding.

Description

technical field

[0001] The invention relates to the field of molecular biology and plant molecular breeding, in particular to the pepper maturity SNP molecular marker and its application. Background technique

[0002] Pepper (Capsicum spp.) is an important economic crop of Solanaceae, which is widely planted all over the world. Pepper is one of the main vegetable crops in my country. The pepper plant is a symaxial branch structure, the main stem grows to a certain point and stops growing, and its top meristem is transformed into an inflorescence meristem. The lower part develops as a solitary flower, and the flowering marks the termination of the growth of the main stem. The lateral meristem grows and develops into a new branch, and soon the new branch stops growing, and the lateral meristem at the top of the new branch continues to replace the apical meristem. Repeat to form symaxial branches.

[0003] The insertion node of the first flower of pepper is called the initial...

Examples

Embodiment 1

[0044] The development of embodiment 1 capsicum ripeness SNP molecular marker

[0045] 1. Population material construction and genetic analysis

[0046] In the previous research process, the inventor found an early-maturing natural mutant strain in a population with normal maturity of pepper, and bred an early-maturing inbred line B with stable traits through multiple generations of selfing and directional selection. 9431 , depending on the planting environment, the first flowering node is 1-4 nodes. Take Early Pepper B 9431 As the female parent, late-maturing pepper (depending on the planting environment, the first flowering node is 13-18 nodes) A 145 F 1 Generation, F 1 Plants selfed to get F 2 group. Plant this F in spring 2017 2 There are 297 plants in the population, and the node position phenotype of flower initiation is measured by the number of true leaves (node ​​positions) between the cotyledon and the first flower on the main stem. The statistical results sho...

Embodiment 2

[0051] Example 2 Capsicum ripeness SNP molecular marker CSF2 in F 2 Application in the detection of colony plants

[0052] Extract parent B by CTAB method 9431 , parent A 145 , F 1 Plant (B 9431 ×A 145 ) and F 2 For plant DNA, the DNA concentration was detected with a Nanodrop spectrophotometer, and ddH was added 2 O Adjust all DNA concentrations to 100 ng / μL. Perform PCR according to the following conditions: Kangwei Century Biotechnology Co., Ltd. 2×Es Taq MasterMix (Dye) (CW0690) 5 μL, forward primer FFN1-5UF4 (10 μM) and reverse primer FFN1-e1R2 (10 μM) each 0.2 μL, 100ng 0.6 μL of DNA template per μL, add ddH 2 O to make up to 10 μL. PCR reaction program: pre-denaturation at 95°C for 2min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 40s, 38 cycles; extension at 72°C for 10min. The PCR reaction product was digested with restriction endonuclease TaiI: 0.65 μL of 10×Buffer R, 3.5 μL of PCR product, 0.1 μL of TaiI restriction endon...

Embodiment 3

[0053] Example 3 Application of Pepper Ripeness SNP Molecular Marker CSF2 in Identifying Pepper Ripeness Traits

[0054] 92 pepper (Capsicum annuum) germplasm materials (Table 1) with a variation range of 5-23 nodes of the first flowering node were selected, and their DNA was extracted by CTAB method, and the extra-early-maturing pepper B 9431 , Late Pepper A 145 and its F 1 Plant (B 9431 ×A 145 ) DNA was used as a control, and PCR was carried out according to the following conditions: the total reaction volume was 10 μL, including 5 μL of Kangwei Century Biotechnology Co., Ltd. 2×Es TaqMasterMix (Dye) (CW0690), forward primer FFN1-5UF4 (10 μM) and reverse primer 0.2 μL each of FFN1-e1R2 (10 μM), 0.6 μL of 30-100 ng / μL DNA template, add ddH 2 O to make up to 10 μL. PCR reaction program: pre-denaturation at 95°C for 2min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 40s, 38 cycles; extension at 72°C for 10min. The PCR reaction product was...

technical field

[0001] The invention relates to the field of molecular biology and plant molecular breeding, in particular to the pepper maturity SNP molecular marker and its application. Background technique

[0002] Pepper (Capsicum spp.) is an important economic crop of Solanaceae, which is widely planted all over the world. Pepper is one of the main vegetable crops in my country. The pepper plant is a symaxial branch structure, the main stem grows to a certain point and stops growing, and its top meristem is transformed into an inflorescence meristem. The lower part develops as a solitary flower, and the flowering marks the termination of the growth of the main stem. The lateral meristem grows and develops into a new branch, and soon the new branch stops growing, and the lateral meristem at the top of the new branch continues to replace the apical meristem. Repeat to form symaxial branches.

[0003] The insertion node of the first flower of pepper is called the initial...

Examples

Embodiment 1

[0044] The development of embodiment 1 capsicum ripeness SNP molecular marker

[0045] 1. Population material construction and genetic analysis

[0046] In the previous research process, the inventor found an early-maturing natural mutant strain in a population with normal maturity of pepper, and bred an early-maturing inbred line B with stable traits through multiple generations of selfing and directional selection. 9431 , depending on the planting environment, the first flowering node is 1-4 nodes. Take Early Pepper B 9431 As the female parent, late-maturing pepper (depending on the planting environment, the first flowering node is 13-18 nodes) A 145 F 1 Generation, F 1 Plants selfed to get F 2 group. Plant this F in spring 2017 2 There are 297 plants in the population, and the node position phenotype of flower initiation is measured by the number of true leaves (node ​​positions) between the cotyledon and the first flower on the main stem. The statistical results sho...

Embodiment 2

[0051] Example 2 Capsicum ripeness SNP molecular marker CSF2 in F 2 Application in the detection of colony plants

[0052] Extract parent B by CTAB method 9431 , parent A 145 , F 1 Plant (B 9431 ×A 145 ) and F 2 For plant DNA, the DNA concentration was detected with a Nanodrop spectrophotometer, and ddH was added 2 O Adjust all DNA concentrations to 100 ng / μL. Perform PCR according to the following conditions: Kangwei Century Biotechnology Co., Ltd. 2×Es Taq MasterMix (Dye) (CW0690) 5 μL, forward primer FFN1-5UF4 (10 μM) and reverse primer FFN1-e1R2 (10 μM) each 0.2 μL, 100ng 0.6 μL of DNA template per μL, add ddH 2 O to make up to 10 μL. PCR reaction program: pre-denaturation at 95°C for 2min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 40s, 38 cycles; extension at 72°C for 10min. The PCR reaction product was digested with restriction endonuclease TaiI: 0.65 μL of 10×Buffer R, 3.5 μL of PCR product, 0.1 μL of TaiI restriction endon...

Embodiment 3

[0053] Example 3 Application of Pepper Ripeness SNP Molecular Marker CSF2 in Identifying Pepper Ripeness Traits

[0054] 92 pepper (Capsicum annuum) germplasm materials (Table 1) with a variation range of 5-23 nodes of the first flowering node were selected, and their DNA was extracted by CTAB method, and the extra-early-maturing pepper B 9431 , Late Pepper A 145 and its F 1 Plant (B 9431 ×A 145 ) DNA was used as a control, and PCR was carried out according to the following conditions: the total reaction volume was 10 μL, including 5 μL of Kangwei Century Biotechnology Co., Ltd. 2×Es TaqMasterMix (Dye) (CW0690), forward primer FFN1-5UF4 (10 μM) and reverse primer 0.2 μL each of FFN1-e1R2 (10 μM), 0.6 μL of 30-100 ng / μL DNA template, add ddH 2 O to make up to 10 μL. PCR reaction program: pre-denaturation at 95°C for 2min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 40s, 38 cycles; extension at 72°C for 10min. The PCR reaction product was...

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