Primer pair for detecting broccoli fruiting trait and its related application in breeding

Abstract

The application discloses a primer pair for detecting a broccoli fruiting trait and related application thereof in breeding, relates to the field of plant breeding, and comprises an upstream primer and a downstream primer with nucleotide sequences shown in SEQ ID NO:1 and 2 respectively. The primer pair can detect a molecular marker closely linked to the broccoli fruiting trait, and can be quickly and effectively applied to screening of a broccoli breeding material.

Description

Technical Field

[0001] This invention relates to the field of plant breeding, and more specifically, to primer pairs for detecting the fruit-setting trait of broccoli and their related applications in breeding. Background Technology

[0002] Broccoli (Brassica oleracea L. var. italica), also known as Chinese cabbage, belongs to the Brassica genus of the Brassicaceae family. Its nutritional value and health benefits are highly favored by consumers worldwide.

[0003] Currently, broccoli mainly uses male-sterile lines for seed production. During the seed production process, when the maternal parent has fertile cytoplasm, self-pollination or hybrid seed production is normal and the expected seed quantity can be obtained. However, when the maternal parent is converted to male-sterile cytoplasm, the original seed collection or hybrid seed production is abnormal, resulting in low seed yield. As a result, the yield of broccoli varieties cannot meet market demand, and the promotion and application of superior new varieties are limited.

[0004] There are few existing studies on the molecular regulatory mechanisms and gene mapping of agronomic traits in broccoli. Apart from self-incompatibility genes, research on genes related to broccoli seed setting is basically nonexistent.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide primer pairs for detecting the fruit-setting trait of broccoli and their related applications in breeding.

[0007] This invention is implemented as follows:

[0008] In a first aspect, embodiments of the present invention provide a primer pair comprising: an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer comprises the sequence shown in SEQ ID NO:1 or is at least 80% identical to the sequence shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer comprises the sequence shown in SEQ ID NO:2 or is at least 80% identical to the sequence shown in SEQ ID NO:2.

[0009] Secondly, embodiments of the present invention provide a kit comprising the primer pairs described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide a method for detecting the fruit-bearing trait of broccoli, which includes: performing PCR amplification on the DNA of the sample to be tested using the primers described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide the application of the primer pairs or kits described in the foregoing embodiments in the preparation of products for detecting the fruit set trait of broccoli.

[0012] Fifthly, embodiments of the present invention provide the application of primer pairs or kits as described in the foregoing embodiments in the screening of broccoli breeding materials or in the breeding of broccoli.

[0013] The present invention has the following beneficial effects:

[0014] The primer pairs described above can detect molecular markers closely linked to the fruit-setting trait of broccoli, and can be rapidly and effectively applied to the screening of broccoli breeding materials. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The hybridization results on the 7168 female parent (CMS) plant are shown; where A is a plant with fruit set, B is a plant without fruit set, and C-D are comparison images of seed pods with and without fruit set.

[0017] Figure 2 Breeding roadmap for maternal parent materials of broccoli hybrid 7168;

[0018] Figure 3 Polymorphic banding patterns of the N and Y pools of the 7168 parent (CMS) amplified using BoseedF / BoseedR primers;

[0019] Figure 4 Sequencing alignment of the N and Y pools of the 7168 parent (CMS) amplified using BoseedF / BoseedR primers;

[0020] Figure 5 Image showing gel amplification results of 130 individual plants of the 7168 parent (CMS) material using BoseedF / BoseedR primers;

[0021] Figure 6 Gel images of 152 individual plants (fertile cytoplasm) amplified from the original seed 7168 maternal parent (fertile cytoplasm) using BoseedF / BoseedR primers; where M: 100bp DNA Ladder;

[0022] Figure 7 Gel images of 310 individual plants of the original seed 7168 maternal parent (CMS) amplified using BoseedF / BoseedR primers; where M: 100bp DNA Ladder. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] On one hand, embodiments of the present invention provide a primer pair, comprising: an upstream primer and a downstream primer;

[0025] The nucleotide sequence of the upstream primer includes the sequence shown in SEQ ID NO:1 or has at least 80% identity with the sequence shown in SEQ ID NO:1;

[0026] The nucleotide sequence of the downstream primer includes the sequence shown in SEQ ID NO:2 or has at least 80% identity with the sequence shown in SEQ ID NO:2.

[0027] "Identity" means that two sequences are completely identical at the same site of nucleotide or amino acid residues. In some embodiments, the at least 80% may refer to any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%.

[0028] In some embodiments, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0029] On the other hand, embodiments of the present invention provide a kit comprising the primer pairs described in any of the foregoing embodiments.

[0030] In some embodiments, the kit further includes any one or more of the following: PCR detection reagents, DNA polymerase, and dNTPs.

[0031] On the other hand, embodiments of the present invention also provide a method for detecting the fruit-setting trait of broccoli, which includes: performing PCR amplification on the DNA of the sample to be tested using the primers described in any of the foregoing embodiments.

[0032] In some embodiments, the detection method further includes: judging the detection results according to screening criteria; the screening criteria include: if the amplification product is a single band, the sample is judged to be of normal and firm type; if the amplification product is a double band, the sample is judged to be of non-firm type.

[0033] In some embodiments, the single strip is a 200bp single strip.

[0034] In some embodiments, the dual stripe is a dual stripe of 200bp and 214bp.

[0035] On the other hand, embodiments of the present invention provide the application of primer pairs as described in any of the foregoing embodiments or kits as described in any of the foregoing embodiments in the preparation of products for detecting the fruit set trait of broccoli.

[0036] In some embodiments, the product includes a reagent combination or a kit.

[0037] Furthermore, embodiments of the present invention also provide the application of primer pairs or kits as described in any of the foregoing embodiments in the screening of broccoli breeding materials or in the breeding of broccoli.

[0038] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0039] Example 1

[0040] 1. In 2021, a trial seed collection was conducted for the broccoli hybrid 7168, a cytoplasmic male sterile (CMS) variety. At the end of April, the hybridization and seed setting of the 7168 female parent (CMS) plants were investigated. A survey of three greenhouses revealed 248 plants with normal seed setting, while 137 plants exhibited abnormal seed setting, with no seeds contained in the seed pods. See the image showing normal seed setting. Figure 1 In section A, the plant failed to produce fruit (see image). Figure 1 See Figure B for a comparison of seed pods that are fertile and those that are not. Figure 1 C and D in the example.

[0041] Analysis suggests that the 7168 maternal parent (CMS) material may have undergone gene mutations or gene recombination related to fruit setting during the breeding process.

[0042] 2. The test material was the female parent of broccoli hybrid 7168 (CMS), which was obtained by backcrossing and breeding hybrid 7168 female parent (N) for 5 generations. The breeding roadmap is shown below. Figure 2 .

[0043] 3. Construct two mixed-pool screening primers for polymorphic markers tightly linked to the target cells.

[0044] Fifteen normally fruitful plants from 7168 parent (CMS) plants were randomly selected and their DNA was extracted and mixed into pool N. The DNA of 15 non-fruitful plants was extracted and mixed into pool Y.

[0045] The steps for extracting genomic DNA include: 1. Taking approximately 0.1g of leaf material into a 2mL centrifuge tube and adding 2 steel balls; 2. Adding 600-700μL of... 2. Grind the CTAB extraction buffer at 55-60 Hz for 100-120 seconds using a high-throughput grinder; incubate at 65℃ for 30-40 minutes, inverting and mixing 2-3 times during this period; remove the centrifuge tube and cool to room temperature; 3. Add an equal volume of chloroform:isoamyl alcohol (24:1) solution, cap the tube, gently invert it several times, and centrifuge at 12000 rpm for 10 minutes at room temperature; 4. Take the supernatant (about 500 μL), add 2 / 3V pre-chilled isopropanol, mix well, incubate at -20℃ for 30 minutes, centrifuge at 12000 rpm for 2 minutes at room temperature, and discard the supernatant; 5. Wash the precipitate 2-3 times with 70%-75% ethanol, and air dry upside down at room temperature; 6. Add an appropriate amount (about 100 μL) of ddH2O to dissolve the DNA, and store at -20℃ for later use.

[0046] Based on the whole genome of Braol_HDEM_V1.0 of cabbage, 300 pairs of SSR marker primers were designed using SSRhunter software, and polymorphic markers were screened (PCR amplification) in pools N and Y.

[0047] The PCR system is as follows: the total volume is 20 μL, including 10 μL of 2×Ftaq PCR Mix, 1.0 μL each of upstream and downstream primers (10 μmol / L), 1.0 μL of template DNA, and 7.0 μL of ddH2O.

[0048] The PCR reaction procedure is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; store at 12℃.

[0049] After amplification, the PCR amplification products were detected by electrophoresis using a 4% agarose gel at a constant voltage of 120V for 1.5 hours, and photographs were taken using a UV gel imaging system.

[0050] Among them, the BoseedF / BoseedR primer pair exhibited polymorphism in pool N and pool Y. Figure 3 The primer pair consists of two sequences: a single 200bp band (SEQ ID NO:3) for the viable band and a heterozygous band for the non-viable band, with 200bp (SEQ ID NO:3) and 214bp (SEQ ID NO:4) for the non-viable band. The primer pair sequence is as follows:

[0051] Upstream primer BoseedF: 5'-GCAACATCAAATCTGTAGTC-3' (SEQ ID NO:1);

[0052] Downstream primer BoseedR: 5'-GTAGCTATGTAAATGTACTG-3' (SEQ ID NO:2).

[0053] Fragments amplified in the N-cell and Y-cell pools were gel-cleaved and recovered using omega-350. The purified DNA fragments were recovered using the Gel Extraction Kit. These fragments were cloned into a pClone007 Simple Vector and subjected to single-clone sequencing. The resulting sequences are as follows:

[0054] N-200bp (SEQ ID NO:3):

[0055] GCACATCAAATCTGTAGTCCATGTTACGATCATTGTAGACGCCAAAAGTTTCACGTTATCTGGAGTTGATATCGTAAAAGCCAATATGCTTAAATTAAATAACGCTAACAATCAAGAAACAGAAGCTGTAAGCTTCATCTCTCTCTCTCTCTCTATATATATATATATATATATATTTCTTCAGTACATTTACATAGCTAC.

[0056] Y-214bp (SEQ ID NO:4):

[0057] GCACATCAAATCTGTAGTCCATGTTACGATCATTGTAGACGCCAAAAGTTCACGTTCTCTGGAGTCGATATCGTAAAAGCCAATATGCTTAAATTAAATAACGCTAACAATCAAGAAACAGAAGCTGTAAGCTTCATCTCTCTCTCTCTCTCTCTCTATATATATATATATATATATATATATATATTTCTTCAGTACATTTACATAGCTAC.

[0058] See sequence alignment Figure 4 .

[0059] 4. Specificity verification of polymorphic marker primers BoseedF / BoseedR

[0060] A total of 130 leaf samples were collected from the 7168 female parent (CMS) plants in a single hybrid seed production greenhouse, and genomic DNA was extracted (DNA extraction method was the same as in step 3). The fruit set status of each individual plant was also investigated and recorded (see Table 1). The polymorphic marker primers BoseedF / BoseedR were used to detect the 130 samples (PCR system and PCR reaction procedure were the same as in step 3), and the amplification products were judged: if the amplification product was a single 200bp band, the sample was judged to be of normal fruit set; if the amplification product was a double band of 200bp and 214bp, the sample was judged to be of non-fruit set.

[0061] See results Figure 5 The molecular maps of 124 individual plants were verified to be consistent with the fruit set observation results, while 6 plants showed the opposite results, namely numbers 9, 51, 71, 92, 101 and 116. The matching degree was 95.4% (Table 1), indicating that the marker is closely linked to the fruit set-related gene in broccoli.

[0062] Table 1. Survey of hybridization fruit set of 130 individual plants from combination 7168 in 2021.

[0063]

[0064]

[0065]

[0066] Note: N indicates firm, Y indicates weak.

[0067] 5. Molecular-assisted screening and investigation of primary seed greenhouse materials using primers BoseedF / BoseedR.

[0068] The primary method for producing broccoli parent stock is artificial pollination by removing buds, therefore it is impossible to screen for non-fruiting plants of the 7168 female parent (CMS) through field surveys. Genomic DNA was extracted from plant leaves, and PCR amplification was performed on 311 individual plants of the 7168 female parent (CMS) and 152 individual plants of the 7168 female parent (fertile cytoplasm) using primers BoseedF / BoseedR. The PCR system and PCR reaction procedure were the same as in step 3.

[0069] The results showed that all 7168 maternal parents (fertile cytoplasm) from 152 individual plants were of the normal fruit-bearing type. Figure 6 Of the 310 individual plants and 7168 parent (CMS) materials, 189 were identified as having normal fruiting characteristics, and 121 were identified as having non-fruiting characteristics. Figure 7), and marked and harvested 191 plants with normal fruiting phenotype, which were planted in the fall of 2021 for hybrid seed production of combination 7168. In 2022, a survey was conducted on 1,435 individual plants in 3 greenhouses, of which 71 individual plants were non-fruiting phenotype (Table 2).

[0070] Table 2. Survey on Seed Setting Status of Hybridized Combination 7168 in 2022

[0071]

[0072] The molecular results showed a matching degree of 95.1%, consistent with the results of the initial marker validation, further indicating that BoseedF / BoseedR is a detection primer for molecular markers closely linked to genes related to broccoli fruit set.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer pair, characterized in that, It includes: Upstream and downstream primers; The nucleotide sequence of the upstream primer includes the sequence shown in SEQ ID NO:1; The nucleotide sequence of the downstream primer includes the sequence shown in SEQ ID NO:

2.

2. A reagent kit, characterized in that, It includes the primer pair as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes any one or more of the following: PCR detection reagents, DNA polymerase, and dNTPs.

4. A method for detecting the fruit-setting characteristics of broccoli, characterized in that, It includes: performing PCR amplification on the DNA of the sample to be tested using the primers described in claim 1; The detection method further includes: judging the detection results according to screening criteria; The screening criteria include: if the amplification product is a single band, the sample is judged to be of normal and firm type; if the amplification product is a double band, the sample is judged to be of non-firm type; the single band is a single band of 200bp; the double band is a double band of 200bp and 214bp.

5. The application of the primer pair as described in claim 1 in the preparation of products for detecting the fruit set trait of broccoli.

6. The application according to claim 5, characterized in that, The products include reagent combinations or kits.

7. The application of the primer pair as described in claim 1 or the kit as described in claim 2 or 3 in the screening of broccoli breeding materials or in the breeding of broccoli.

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