InDel markers of new genes brhnt3.1 and brhnt3.2 for regulating leaf size development in brassica rapa and application thereof

Abstract

This invention belongs to the field of molecular genetics and breeding, and relates to a novel gene regulating the size of Chinese cabbage leaves. BrHINT3.1 and BrHINT3.2 The detection marker and its application in breeding. By combining BSA-seq (Bulked segregant analysis sequencing) technology with InDel markers, this invention successfully located a new gene related to the size of Chinese cabbage leaves. BrHINT3.1 and BrHINT3.2 This invention also designed a gene related to the size of Chinese cabbage leaves. BrHINT3.1 and BrHINT3.2 The relevant InDel markers, InDel-A06 and InDel-A09, were identified. PCR amplification of DNA from a Chinese cabbage genetic population was performed, followed by detection using agarose gel electrophoresis. The results showed that InDel-A06 and InDel-A09 could clearly identify plants with varying leaf sizes, and can be used for the breeding of high-yielding new varieties of Chinese cabbage.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and breeding, specifically involving a novel gene regulating leaf size and development in Chinese cabbage. BrHINT3.1 and BrHINT3.2 The detection markers and their application in breeding. Background Technology

[0002] Chinese cabbage ( Brassica rapa L. ssp. pekinensis Chinese cabbage (Chinese cabbage) is an important leafy vegetable native to my country, accounting for approximately 15% of the country's total vegetable production and consumption. It boasts diverse ecological types, a long supply period, rich nutrition, and high yield, playing a vital role in the "vegetable basket" project. In the past decade, my country's annual planting area for Chinese cabbage has reached 26-28 million mu (approximately 1.6-1.6 million hectares), ranking first in the world in both area and yield. As a leafy vegetable where the leaves themselves are an economic asset, the size and weight of the Chinese cabbage leaves are particularly important.

[0003] Leaves are the primary photosynthetic organs, directly influencing crop yield. Understanding the regulatory mechanisms of leaf size and development is crucial for food security and ecosystem turnover. Previous studies have shown a significant positive correlation between leaf size and Chinese cabbage yield, making it a key factor in yield traits and a core element of Chinese cabbage breeding objectives.

[0004] Leaf size and final morphology are complex traits controlled by multiple genes, and their regulation involves a complex gene network with multiple components and pathways, resulting from a high degree of integration between intrinsic genetic programs and external environmental signals. From a cellular level, plant leaf growth can be divided into two coordinated processes: cell proliferation and cell expansion. The regulation of cell proliferation consists of two very important regulatory modules: the ANT (Antigenesis) pathway and the TCP-GRF (Growth-Regulating Factor) pathway. Cell expansion is the dominant process in the later stages of leaf growth. ARL (ARGOS-LIKE) Genes are involved in organ growth dependent on cell expansion, changes caused by alterations in cell size rather than cell number; the TOR (Target of Rapamycin) signaling pathway is a global regulator that positively regulates cell expansion to control cell growth. Research on the leaf size and development of Chinese cabbage has made some progress, with overexpression of Chinese cabbage in Arabidopsis thaliana... BrARGOS , BrANT-1 , BrGRF5 Genes that can enhance cell proliferation and increase leaf size can be overexpressed in Chinese cabbage. BraAMMECR1 Genes can promote leaf cell proliferation; overexpression BrERF4 , BrPHYB , BrARP1 and BrDRM1Genes that inhibit cell expansion, resulting in smaller leaves; furthermore, overexpression of these genes in Chinese cabbage... BrHCYSTM1 The gene can increase leaf area, and heterologous expression of this gene in Arabidopsis can also increase leaf size. AtHCYSTM1 Overexpression of the gene in a deletion mutant can completely compensate for the mutant's defective phenotype.

[0005] Molecular markers are specific segments of DNA that reflect differences at the individual level among organisms. These differences are usually closely linked to or directly correspond to specific traits of crops (such as disease resistance, high yield, and quality). Molecular marker-assisted selection is one of the core technologies of modern biological breeding. By utilizing molecular breeding technology, breeding efficiency can be improved, the breeding cycle can be shortened, precision selection can be achieved, and traditional breeding problems can be overcome. Summary of the Invention

[0006] This invention identifies two novel genes in Chinese cabbage that are related to the regulation of leaf size development, and develops InDel markers InDel-A06 and InDel-A09 that are closely linked to these two genes, providing a basis for molecular-assisted breeding of Chinese cabbage varieties that regulate leaf size development using these markers, as well as for research on breeding Chinese cabbage varieties that regulate leaf size development.

[0007] To utilize the above findings and apply them to specific Chinese cabbage breeding, this invention provides the following technical solution:

[0008] The first aspect of this invention is to provide a set of genes that specifically amplify and regulate the size development of Chinese cabbage leaves. BrHINT3.1 and BrHINT3.2 The primer set is characterized in that the InDel primer set is used to clone genes related to the regulation of leaf size development in Chinese cabbage. BrHINT3.1 and BrHINT3.2 Two closely linked InDels, wherein the primer set includes InDel-A06-F, whose nucleotide sequence is shown in SEQ ID NO:1; InDel-A06-R, whose nucleotide sequence is shown in SEQ ID NO:2; InDel-A09-F, whose nucleotide sequence is shown in SEQ ID NO:3; and InDel-A09-R, whose nucleotide sequence is shown in SEQ ID NO:4.

[0009] Furthermore, the primer combinations were used to amplify novel genes regulating leaf size development in Chinese cabbage. BrHINT3.1 and BrHINT3.2 The InDel molecular markers InDel-A06 and InDel-A09.

[0010] Furthermore, InDel-A06-F is used for amplification and... BrHINT3.1 Linked InDel-specific upstream primers;

[0011] Furthermore, InDel-A06-R is used for amplification and... BrHINT3.1 Linked InDel-specific downstream primers.

[0012] Furthermore, InDel-A09-F is used for amplification and... BrHINT3.2 Linked InDel-specific upstream primers;

[0013] Furthermore, InDel-A09-R is used for amplification and... BrHINT3.2 Linked InDel-specific downstream primers.

[0014] Furthermore, three bands will appear for each primer pair. For genes... BrHINT3.1 Due to the homozygous latent material Y1150-75 in BrHINT3.1 There is a 203bp insertion at the DNA level, so the band amplified by the primer set InDel-A06-F / R will show three possibilities: a homozygous recessive band (699bp), a homozygous dominant band (513bp), and a heterozygous band (699bp + 513bp); for the gene BrHINT3.2 Due to the homozygous dominant material 21-R16-9 in BrHINT3.2 There is a 36bp deletion at the DNA level, so the band amplified by the primer set InDel-A09-F / R will have three possibilities: homozygous recessive band (169bp), homozygous dominant band (133bp), and heterozygous band (169bp+133bp).

[0015] A second aspect of the present invention is to provide a kit for identifying whether Chinese cabbage has the ability to regulate leaf size development, characterized in that the kit comprises the primer combination described in the first aspect.

[0016] Furthermore, the kit also includes reagents required for PCR, including PCR Master Mix.

[0017] Furthermore, the concentration of each primer in the primer combination contained in the kit is 5~100 μmol / L, preferably, the concentration of each primer in the primer combination is 10 μmol / L.

[0018] The third aspect of this invention is a method for identifying whether Chinese cabbage has the ability to regulate leaf size development, characterized in that the method comprises the following steps:

[0019] 1) Extract DNA,

[0020] 2) Perform PCR amplification on the DNA sample using the primer combination described in the first aspect or the kit described in the second aspect.

[0021] 3) Band identification and analysis were performed using agarose gel electrophoresis.

[0022] Furthermore, in step 2), the PCR system is 10-50 μL, the DNA sample is 100-500 ng, and the amount of primers InDel-A06-F, InDel-A06-R, InDel-A09-F, and InDel-A09-R is 3-10 pmol.

[0023] Further, the PCR reaction procedure is as follows: (1) pre-denaturation at 95℃ for 5 min; (2) denaturation at 95℃ for 30 s, annealing at 56℃ for 30 min, extension at 72℃ for 30 s, BrHINT3.1 for a total of 25 cycles, BrHINT3.2 for a total of 35 cycles; (3) annealing at 72℃ for 2 min; (4) storage at 4℃.

[0024] A fourth aspect of the present invention is to provide the application of the primer combination described in the first aspect and the kit described in the second aspect in identifying the regulation of leaf size development in Chinese cabbage.

[0025] Furthermore, the application involves using the method described in the third aspect to detect DNA samples from Chinese cabbage.

[0026] The fifth aspect of this invention is to provide a novel gene for regulating leaf size and development in Chinese cabbage. BrHINT3.1 and BrHINT3.2 The InDel molecular markers InDel-A06 and InDel-A09 are characterized in that InDel-A06 is obtained by amplification using the primer set InDel-A06-F / R described in the first aspect, wherein InDel-A06-F is the upstream primer and InDel-A06-R is the reverse primer; and InDel-A09 is obtained by amplification using the primer set InDel-A09-F / R described in the first aspect, wherein InDel-A09-F is the upstream primer and InDel-A09-R is the reverse primer.

[0027] The beneficial effects of this invention include:

[0028] 1) This invention successfully located two novel genes regulating leaf size development by employing BSA-seq (Bulked segregant analysis sequencing) technology. BrHINT3.1 and BrHINT3.2 .

[0029] 2) This invention is the first to disclose genes that regulate leaf size development. BrHINT3.1 and BrHINT3.2Related InDel molecular markers and their detection primers InDel-A06 and InDel-A09.

[0030] 3) This invention involves PCR amplification of DNA from Chinese cabbage and detection using agarose gel electrophoresis. The results show that InDel-A06 and InDel-A09 can clearly distinguish between homozygous and heterozygous materials, and can be used for the breeding of new varieties of Chinese cabbage that regulate leaf size development. Attached Figure Description

[0031] Figure 1 Phenotypic identification of parental materials 21-R16-9 and Y1150-75;

[0032] Figure 2 BSA-seq mapping results of leaf size-related genes;

[0033] Figure 3 candidate genes BrHINT3.1 and BrHINT3.2 Precise positioning;

[0034] Figure 4 BrHINT3.1 and BrHINT3.2 Cloning and sequence differences, with the primer sequences marked in purple boxes;

[0035] Figure 5 BraA09g004450.3.5C Cases where genotype and phenotype do not match;

[0036] Figure 6 The F2 population was validated using the markers InDel-A06 and InDel-A09;

[0037] Figure 7 The natural population was validated using the markers InDel-A06 and InDel-A09.

[0038] The biological material involved in this application, Chinese cabbage 21-R16-9, Y1150-75, is deposited at the Vegetable Research Institute of Henan Academy of Agricultural Sciences and Hebei Agricultural University. The applicant undertakes to provide the use of this biological material (issuance of samples) to any requester who meets the requirements of Article 28 of the Implementing Regulations of the Patent Law within 20 years from the date of this patent application. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0040] Example 1: Obtaining InDel

[0041] 1. Investigation of test materials and phenotypic traits

[0042] The parental materials Y1150-75 and 21-R16-9 are DH line materials, exhibiting highly significant size differences, and were used for constructing genetic populations. During the seedling stage, Y1150-75 showed obvious weaknesses in leaf size, plant height, and root length; during the rosette stage, Y1150-75 showed weak growth, with small and yellow leaves; and at harvest, Y1150-75 showed slow growth. Figure 1 ).

[0043] Inheritance patterns of leaf size development traits in Chinese cabbage: Phenotypic investigation of four generations (P1, P2, F1, and F2) revealed that the F2 population had 21,325 dominant plants and 1,415 weak plants, with a segregation ratio of 15:1 as determined by chi-square test. This indicates that leaf size is controlled by two dominant genes.

[0044] Table 1. Analysis of the genetic patterns of leaf size

[0045]

[0046] 2. Genomic DNA extraction

[0047] Genomic DNA was extracted from both parents and individual plants of the F1 and F2 generations using a modified CTAB method. The specific steps are as follows:

[0048] (1) Place 4 fresh leaves the size of a tube cap into a 2 mL centrifuge tube, and add a steel ball with a diameter of about 5 mm and 1000 μL of 2% CTAB buffer to each centrifuge tube;

[0049] (2) Vibrate the tissue at 30 r / s for 90 s on a tissue homogenizer to ensure that the sample is fully ground;

[0050] (3) Place the sample in a 65℃ drying oven for 1 hour, and invert it 2-3 times during the period to ensure that the sample is thoroughly mixed.

[0051] (4) Add 500 μL of isopropanol to each 1.5 mL centrifuge tube and pre-cool at -20℃ for 30-60 min;

[0052] (5) Cool the sample to room temperature, add 500 μL of chloroform (in a fume hood), mix by inversion and centrifuge at 12000 rpm for 10 min. Add 600 μL of supernatant to pre-cooled isopropanol, mix thoroughly and place in a -20℃ freezer for 30 min, then remove and centrifuge at 12000 rpm for 5 min;

[0053] (6) Discard the supernatant, add 500 μL of 75% ethanol to the centrifuge tube, and centrifuge at 12000 rpm for 2 min;

[0054] (7) Discard the supernatant, dry the DNA at room temperature, then add 100 μL of double-distilled water and vortex to completely dissolve the DNA. Determine the quality and concentration of the DNA using a Nano 300 micro spectrophotometer (Thermo Fisher Scientific, USA);

[0055] 3. New genes regulating leaf size and development in Chinese cabbage BrHINT3.1 and BrHINT3.2 Positioning

[0056] Ten parental lines 21-R16-9 and Y1150-75 were selected to construct two parental lines. From the F2 population, 30 highly dominant and 30 highly weak lines were selected to construct two mixed pools of dominant (bulk-robust) and weak (bulk-weak) progeny. After the sample DNA samples passed the quality test, BSA-seq was performed. After library construction, quality testing was performed. Once the quality met the requirements, paired-end 150 bp (PE150) sequencing was performed using the Illumina HiSeq platform. Using the Chinese cabbage reference genome (V3.5) as the reference sequence, the sequencing data was aligned with the reference genome using the MEM algorithm in BWA software, and reference data was extracted in SAM format. Then, the SAM files were converted to BAM format using samtools software, and the reads in the BAM files were sorted, and coverage and coverage depth were statistically analyzed. Based on the obtained BAM files, a gvcf file was first created for each sample using the HaplotypeCaller module in the GATK software package (v3.7). Then, the GenotypeGVCFs module was used to detect variations in all samples, including SNP and InDel variant sites. The Δ(SNP-index) value for each variant site was calculated using QTLseqr (R package), and its distribution across the genome was analyzed using a 2 Mb sliding window. Finally, the regions were located within the 23.05–25.71 Mb (2.66 Mb) and 1.37–3.73 Mb (2.36 Mb) regions on chromosome A06. Figure 2 Then, InDel and SNP markers were designed to further narrow down the regions, reducing them to 20.47 Kb (4 genes) and 22.43 Kb (6 genes), respectively. Figure 3 ).

[0057] 4. New genes regulating leaf size and development in Chinese cabbage BrHINT3.1 and BrHINT3.2InDel molecular markers InDel-A06 and InDel-A09

[0058] Cloning and sequencing of all genes within the two candidate regions revealed that only BrHINT3.1 , BrHINT3.2 and BraA09g004470.3.5C There is an insertion or deletion in the gene ( Figure 4 InDel markers were designed for insertions or deletions in three genes, and validated in a disadvantaged population (homozygous recessive). The results showed that... BrHINT3.1 , BrHINT3.2 The genotype and phenotype match 100%, and BraA09g004470.3.5C There were 3 weak plants whose genotypes did not match their phenotypes. Figure 5 Therefore, we speculate BrHINT3.1 , BrHINT3.2 This is a novel gene related to the regulation of leaf size development. The corresponding InDel gene is used as a reference for... BrHINT3.1 , BrHINT3.2 Tightly linked functional markers. Primer insertion:

[0059] InDel-A06-F:

[0060] 5'-GTCCGTGGCGAGTCTCCATGTCTC-3' (SEQ ID NO: 1);

[0061] InDel-A06-R:

[0062] 5'-CCCCAACAAAGCAGACATTTGCCCAC-3' (SEQ ID NO: 2);

[0063] InDel-A09-F:

[0064] 5'-CTCTCTTAATGTGCTGTCATTCTATGTAG-3' (SEQ ID NO: 3).

[0065] InDel-A09-R:

[0066] 5'-GCACTAAGTATTAAAGGGTTCAAGCATAG-3' (SEQ ID NO: 4).

[0067] Example 2: Validation using InDel primers in parental lines 21-R16-9, Y1150-75, their F2 populations, and natural populations.

[0068] 1. F2 group validation

[0069] 1) Add the components of the PCR reaction system to a 96-well plate. The reaction system is as follows:

[0070] 2 μL DNA (100 ng·μL) -1 ),

[0071] 5 μL PCR Master Mix

[0072] 0.5 μL primer F

[0073] 0.5 μL primer R

[0074] 2 μL ddH2O.

[0075] 2) The PCR reaction procedure is as follows:

[0076] (1) Pre-denaturation at 95℃ for 5 min;

[0077] (2) 95℃ denaturation for 30 s, 56℃ annealing for 30 min, 72℃ extension for 30 s, BrHINT3.1 for a total of 25 cycles, BrHINT3.2 for a total of 35 cycles;

[0078] (3) Refold at 72℃ for 2 min;

[0079] (4) Store at 4℃.

[0080] 3) Finally BrHINT3. The detection was performed using 1% agarose gel electrophoresis, and the results were obtained after 30 minutes. ​ Detection was performed using 2% agarose gel electrophoresis at 45 minutes. Three types of band results were observed: for genes... ​ Due to the homozygous latent material Y1150-75 in ​ There is a 203bp insertion at the DNA level, so the band amplified by the primer set InDel-A06-F / R will show three possibilities: a homozygous recessive band (699bp), a homozygous dominant band (513bp), and a heterozygous band (699bp + 513bp); for the gene ​ Due to the homozygous dominant material 21-R16-9 in ​ The DNA contains a 36bp deletion, so the band amplified by primer set InDel-A09-F / R will show three possibilities: homozygous recessive band (169bp), homozygous dominant band (133bp), and heterozygous band (619bp + 133bp). ​ ).

[0081] The results showed that the phenotype and genotype were consistent in the F2 population. The InDel-A06 and InDel-A09 markers could significantly distinguish between the two homozygous genotypes and also identify the heterozygous genotype. The genotype-phenotype concordance was 100%, indicating successful marker development. ​ ).

[0082] 2. Validation by natural populations

[0083] The same method was used to validate the results in 112 natural populations, and the results are as follows: ​ As shown, the genotypes and phenotypes of 112 populations were completely consistent, with a genotype-phenotype concordance rate of 100%. ​ ).

[0084] Table 2 Genotyping results of InDel-A06 and InDel-A09 markers in 112 natural populations

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A group of genes specifically amplified to regulate leaf size and development in Chinese cabbage. BrHINT3.1 and BrHINT3.2 The primer set for InDel is characterized by, The InDel primer set described above is used to clone genes that regulate the size development of Chinese cabbage leaves. BrHINT3.1 and BrHINT3.2 The InDel molecular markers InDel-A06 and InDel-A09, wherein the InDel primer set includes InDel-A06-F, whose nucleotide sequence is shown in SEQ ID NO:1; InDel-A06-R, whose nucleotide sequence is shown in SEQ ID NO:2; InDel-A09-F, whose nucleotide sequence is shown in SEQ ID NO:3; and InDel-A09-R, whose nucleotide sequence is shown in SEQ ID NO:

4.

2. The primer set according to claim 1, characterized in that, InDel-A06-F is a specific upstream primer for amplifying InDel linked to BrHINT3.1; InDel-A06-R is a downstream primer; InDel-A09-F is a specific upstream primer for amplifying InDel linked to BrHINT3.2; and InDel-A09-R is a downstream primer.

3. The primer set according to claim 1 or 2, characterized in that, For genes BrHINT3.1 Due to the homozygous latent material Y1150-75 in BrHINT3.1 There is a 203bp insertion at the DNA level, so the band amplified by the primer set InDel-A06-F / R will show three possibilities: a homozygous recessive band of 699bp, a homozygous dominant band of 513bp, and a heterozygous band of 699bp + 513bp; for the gene BrHINT3.2 Due to the homozygous dominant material 21-R16-9 in BrHINT3.2 There is a 36bp deletion at the DNA level, so the band amplified by the primer set InDel-A09-F / R will have three possibilities: a homozygous recessive band of 169bp, a homozygous dominant band of 133bp, and a heterozygous band of 169bp+133bp.

4. A kit for identifying whether Chinese cabbage has the ability to regulate leaf size development, characterized in that, The kit comprises the primer set as described in any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes reagents required for PCR, including PCR Master Mix.

6. A method for identifying whether Chinese cabbage has the ability to regulate leaf size development, characterized in that, The method includes the following steps: 1) Extract DNA, 2) Perform conventional PCR amplification on the DNA sample using the primer set described in any one of claims 1 to 3 or the kit described in claim 4. 3) Identify the PCR products using agarose gel electrophoresis.

7. The application of the primer set according to any one of claims 1 to 3 or the kit according to claim 4 in identifying the regulation of leaf size development in Chinese cabbage.

8. The application according to claim 7, characterized in that, The application described is to use the method described in claim 6 to detect DNA samples from Chinese cabbage.

9. A novel gene regulating the size and development of Chinese cabbage leaves BrHINT3.1 and BrHINT3.2 The InDel molecular markers InDel-A06 and InDel-A09 are characterized by, The InDel-A06 and InDel-A09 mentioned above are obtained by amplification using the primer set described in any one of claims 1 to 3, wherein InDel-A06-F is used for amplification and... BrHINT3.1 The specific upstream primer for linked InDel; wherein InDel-A06-R is the downstream primer; and InDel-A09-F is the primer for amplification and... BrHINT3.2 The specific upstream primer for InDel linkage; InDel-A09-R is the downstream primer.

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