Primer pair for detecting seed setting character of broccoli and related application of primer pair in breeding

Abstract

The invention discloses a primer pair for detecting the seed setting character of broccoli and related application of the primer pair in breeding, and relates to the field of plant breeding, the primer pair comprises an upstream primer and a downstream primer of which the nucleotide sequences are sequentially shown as SEQ ID NO: 1-2, and the primer pair can be used for detecting molecular markers closely linked with the seed setting character of broccoli. The method can be quickly and effectively applied to screening of broccoli breeding materials.

Description

Technical Field

[0001] The present invention relates to the field of plant breeding, in particular to a primer pair for detecting the fruiting trait of broccoli and related applications thereof in breeding. Background Art

[0002] Broccoli (Brassica oleracea L.var.italica), also known as broccoli, belongs to the Brassica genus of the Cruciferae family. Its nutritional value and health benefits are favored by consumers all over the world.

[0003] At present, broccoli mainly uses male sterile lines for seed production. During the seed production process, it is common that when the female parent has fertile cytoplasm, self-pollination or hybrid seed production is normal and the expected seed quantity can be obtained. However, when the female parent is converted to 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 is difficult to meet market demand, and the promotion and application of excellent new varieties is limited.

[0004] There are few studies on the molecular regulation mechanism and gene location of broccoli agronomic traits. Except for the self-incompatibility gene, there is basically no research on the genes related to broccoli fruit set.

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

[0006] The purpose of the present invention is to provide a primer pair for detecting the fruiting trait of broccoli and its related application in breeding.

[0007] The present invention is achieved in that:

[0008] In the first aspect, an embodiment of the present invention provides 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] In a second aspect, an embodiment of the present invention provides a kit comprising the primer pair described in the preceding embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a method for detecting the fruit set trait of broccoli, comprising: using the primers described in the aforementioned embodiment to perform PCR amplification on the DNA of the sample to be treated.

[0011] In a fourth aspect, embodiments of the present invention provide use of the primer pair described in the preceding embodiments or the kit described in the preceding embodiments in the preparation of a product for detecting the fruit set trait of broccoli.

[0012] In a fifth aspect, embodiments of the present invention provide use of the primer pair described in the preceding embodiments or the kit described in the preceding 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 pair can detect molecular markers that are closely linked to the fruit set trait of broccoli and can be quickly and effectively applied to the screening of broccoli breeding materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The hybrid fruit set of 7168 female parent (CMS) plants; A is a picture of plant fruit set, B is a picture of plant non-fruit set, and C to D are comparison pictures of seed pod fruit set and non-fruit set;

[0017] Figure 2 It is the breeding roadmap for the female parent material of broccoli combination 7168;

[0018] Figure 3 The polymorphic banding pattern of the N pool and Y pool of the 7168 maternal (CMS) amplified by the BoseedF / BoseedR primers;

[0019] Figure 4 This is the sequencing alignment of the bands of the N pool and Y pool of the 7168 parent (CMS) amplified by the BoseedF / BoseedR primers;

[0020] Figure 5 This is the gel run result of 130 individual plants of 7168 maternal (CMS) material amplified by BoseedF / BoseedR primers;

[0021] Figure 6 This is the gel run of 152 individual plants of the original Peng 7168 mother plant (fertile cytoplasm) amplified by BoseedF / BoseedR primers; where M: 100bp DNA Ladder;

[0022] Figure 7 This is the gel run of 310 individual plants of the original seed Peng 7168 mother plant (CMS) amplified by BoseedF / BoseedR primers; where M: 100bp DNA Ladder. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0024] In one aspect, an embodiment of the present invention provides 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 is specifically at least 80% identical to 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 the nucleotides or amino acid residues in the same site of two sequences are exactly the same. In some embodiments, the at least 80% can refer to any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, or a range between any two of them.

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

[0029] On the other hand, an embodiment of the present invention provides a kit, which includes the primer pair described in any of the above embodiments.

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

[0031] On the other hand, an embodiment of the present invention further provides a method for detecting the fruit set trait of broccoli, comprising: using the primers described in any of the above embodiments to perform PCR amplification on the DNA of the sample to be treated.

[0032] In some embodiments, the detection method further comprises: judging the detection results according to screening criteria; the screening criteria comprise: if the amplified product is a single band, the sample is judged to be a normal fruiting type; if the amplified product is a double band, the sample is judged to be a non-fruiting type.

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

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

[0035] On the other hand, an embodiment of the present invention provides use of the primer pair described in any of the foregoing embodiments or the kit described in any of the foregoing embodiments in preparing a product for detecting the fruit set trait of broccoli.

[0036] In some embodiments, the product comprises a reagent set or kit.

[0037] In addition, the embodiments of the present invention also provide the use of the primer pair described in any of the above embodiments or the kit described in any of the above embodiments in the screening of broccoli breeding materials or the breeding of broccoli.

[0038] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0039] Example 1

[0040] 1. In 2021, we conducted a trial seed collection of broccoli combination 7168 cytoplasmic male sterility (CMS). At the end of April, we investigated the hybrid fruiting of 7168 female parent (CMS) plants. We found that 248 plants had normal fruiting in 3 sheds, and 137 plants had abnormal fruiting to the point that there was no seed in the seed pod. Figure 1 Middle A, the plant does not produce fruit. Figure 1 B in the figure shows the comparison of seed pods that are fruitful and those that are not fruitful. Figure 1 C and D in.

[0041] The analysis showed that the 7168 maternal (CMS) material may have undergone gene mutation or gene recombination related to fruiting during the breeding process.

[0042] 2. The test material is the broccoli combination 7168 female parent (CMS), which is the combination 7168 female parent (N) material as the recurrent parent backcross breeding 5 generations, breeding roadmap see Figure 2 .

[0043] 3. Construction of two mixed pools to screen polymorphic marker primers that are closely linked to fruiting

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

[0045] The steps of extracting genomic DNA include: 1. Take about 0.1g of leaves in a 2mL centrifuge tube and add 2 steel beads; 2. Add 600-700μL 2.0×CTAB extract, grind in a high-throughput grinder at 55-60Hz for 100-120s; keep warm at 65℃ for 30-40min, invert and mix 2-3 times during the process, take out the centrifuge tube and cool it to room temperature; 3. Add an equal volume of chloroform:isoamyl alcohol (24:1) solution, cover, gently invert the centrifuge tube several times, and centrifuge at 12000rpm for 10min at room temperature; 4. Aspirate the supernatant (about 500μL), add 2 / 3V pre-cooled isopropanol, mix, place at -20℃ for 30min, centrifuge at 12,000rpm for 2min at room temperature, and discard the supernatant; 5. Wash the precipitate 2-3 times with 70%-75% ethanol, invert and dry at room temperature; 6. Add an appropriate amount (about 100μl) of ddH2O to dissolve the DNA, and store at -20℃ for later use.

[0046] According to the Brassica oleracea genome Braol_HDEM_V1.0, 300 pairs of SSR marker primers were designed using SSRHunter software, and polymorphic markers were screened (PCR amplification) for N pool and Y pool.

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

[0048] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0049] After amplification, the PCR amplification products were detected by electrophoresis using 4% agarose gel at a constant voltage of 120 V for 1.5 h, and photographed using a UV gel imager.

[0050] Among them, the marker BoseedF / BoseedR primer pair showed polymorphism in the N pool and the Y pool ( Figure 3 ), the solid band type is a single band of 200 bp (SEQ ID NO: 3), and the non-solid band type is a hybrid band type, which are 200 bp (SEQ ID NO: 3) and 214 bp (SEQ ID NO: 4), respectively. The sequence of the primer pair is:

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

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

[0053] The fragments amplified from pools N and Y were recovered by gel excision and omega The purified DNA fragments were recovered by Gel Extraction Kit and cloned into pClone007 Simple Vector for single clone sequencing. The sequences obtained by sequencing were:

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

[0055] GCACATCAAATCTGTAGTCCATGTTACGATCATTGTAGACGCCAAAAGTTTCACGTTATCTGGAGTTGATATCGTAAAAGCCAATATGCTTAAATTAAATAACGCTAACAATCAAGAAACAGAAGCTGTAAGCTTCATCTCTCTCTCTCTCTCTATATATATATATATATATATATTTCTTCAGTACATTTACATAGCTAC.

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

[0057] GCACATCAAATCTGTAGTCCATGTTACGATCATTGTAGACGCCAAAAGTTCACGTTCTCTGGAGTCGATATCGTAAAAGCCAATATGCTTAAATTAAATAACGCTAACAATCAAGAAACAGAAGCTGTAAGCTTCATCTCTCTCTCTCTCTCTCTCTATATATATATATATATATATATATATATATTTCTTCAGTACATTTACATAGCTAC.

[0058] Sequence alignment see Figure 4 .

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

[0060] A total of 130 leaves of 7168 female parent (CMS) plants in a hybrid seed production shed were collected and genomic DNA was extracted (DNA extraction method is the same as step 3), and the fruiting situation of each plant was investigated and recorded (see Table 1). The polymorphic marker primers BoseedF / BoseedR were used for 130 material tests (PCR system and PCR reaction procedure are the same as step 3), and the amplification products were judged: if the amplification product is a single band of 200bp, the sample is judged to be a normal fruiting type; if the amplification product is a double band of 200bp and 214bp, the sample is judged to be a non-fruiting type.

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

[0062] Table 1 Survey on hybrid fruiting of 130 plants in combination 7168 in 2021

[0063]

[0064]

[0065]

[0066] Note: N for strong and Y for weak.

[0067] 5. Molecular assisted screening and investigation of original seed shed materials using primers BoseedF / BoseedR

[0068] Broccoli parent stock production is mainly artificial bud stripping pollination, so it is impossible to screen out the sterile plants of the 7168 mother parent (CMS) through field surveys. Collect plant leaves to extract genomic DNA, and use primers BoseedF / BoseedR to perform PCR amplification on 311 plants of the 7168 mother parent (CMS) and 152 plants of the 7168 mother parent (fertile cytoplasm). The PCR system and PCR reaction procedure are the same as step 3.

[0069] The results showed that all 152 individual plants of 7168 female parent (fertile cytoplasm) materials were of normal fruiting band type ( Figure 6 ), 7168 maternal (CMS) materials of 310 plants were screened out, 189 of which were normal fruit-bearing type and 121 were non-fruit-bearing type ( Figure 7), and 191 plants with normal fruiting phenotype were marked and harvested, and planted in the autumn of 2021 for combination 7168 hybrid seed production. In 2022, a total of 1435 plants in three greenhouses were surveyed, of which 71 plants showed a non-fruiting phenotype (Table 2).

[0070] Table 2. Survey on the fruiting status of hybrid seed production of combination 7168 in 2022

[0071]

[0072] The molecular result matched 95.1%, which was consistent with the result of the initial verification of the marker, further indicating that BoseedF / BoseedR were the detection primers for detecting molecular markers that were closely linked to genes related to fruiting in broccoli.

[0073] 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 pair, characterized in that: It includes: Upstream primer and downstream primer; The nucleotide sequence of the upstream primer includes the sequence shown in SEQ ID NO: 1 or is specifically at least 80% identical to the sequence shown in SEQ ID NO: 1; 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.

2. A kit, characterized in that: It comprises the primer pair according to claim 1.

3. The kit according to claim 2, characterized in that The kit further comprises: any one or more of PCR detection reagent, DNA polymerase and dNTP.

4. A method for detecting the fruiting trait of broccoli, characterized in that: It includes: The primers described in claim 1 are used to perform PCR amplification on the DNA of the sample to be treated.

5. The detection method according to claim 4, characterized in that: The detection method further comprises: judging the detection result according to the screening criteria; The screening criteria include: if the amplified product is a single band, the sample is judged to be a normal fruiting type; if the amplified product is a double band, the sample is judged to be a non-fruiting type.

6. The detection method according to claim 5, characterized in that: The single band is a single band of 200 bp.

7. The detection method according to claim 5, characterized in that: The double bands are double bands of 200 bp and 214 bp.

8. Use of the primer pair according to claim 1 or the kit according to claim 2 or 3 in preparing a product for detecting the fruit set trait of broccoli.

9. The use according to claim 8, characterized in that: The products include reagent sets or kits.

10. Use of the primer pair according to claim 1 or the kit according to claim 2 or 3 in screening broccoli breeding materials or breeding broccoli.

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