InDel molecular marker related to green leaf pigment content of non-heading Chinese cabbage and primer and application thereof

Abstract

The application provides an InDel molecular marker related to the green leaf pigment content of Brassica rapa, a primer of the InDel molecular marker and application, and the primer sequence of the InDel molecular marker is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The InDel molecular marker is designed and obtained according to the InDel mutation of an exon located in a BraC09g052220 gene. The primer pair of the InDel molecular marker provided by the application is significantly related to the green leaf color and the chlorophyll and carotenoid content of Brassica rapa, and can be used for molecular assisted breeding of the Brassica rapa and identification of high-quality germplasm resources, so that the creation of high-quality materials of the Brassica rapa and the new variety breeding process are accelerated.

Description

Technical Field

[0001] This invention belongs to the field of molecular-assisted breeding technology for non-heading Chinese cabbage, specifically involving an InDel molecular marker for the BraC09g052220 gene, its primers, and its application for assisting in the selection of green leaf color and its relationship with chlorophyll and carotenoid content in non-heading Chinese cabbage. Background Technology

[0002] Non-heading Chinese cabbage, a cruciferous vegetable, is rich in nutrients and is one of the most common leafy vegetables in my country. It boasts abundant leaf color resources, with green being the most common, which can be categorized into yellowish-green, light green, regular green, and dark green. The greenness of the leaves is mainly regulated by pigment content, such as chlorophyll and carotenoids. Meanwhile, chlorophyll and carotenoids are common nutrients that enhance human immunity, provide antioxidant effects, and protect the cardiovascular system. With the continuous improvement of people's living standards, the requirements for the color and nutritional value of vegetables are also increasing. Breeding vegetable varieties with high ornamental value and high nutritional quality is currently a market demand. In recent years, with the rapid development of molecular biology and genetic engineering technologies, more and more researchers are using molecular-assisted breeding to efficiently breed high-yield, high-quality, and highly resistant vegetable varieties. However, the available molecular markers for breeding non-heading Chinese cabbage varieties with green leaf traits are still lacking.

[0003] BraC09g052220, or BcSG1, is an important gene involved in chloroplast development. It regulates the development of the thylakoid membrane in chloroplasts and the expression of genes related to chloroplast development. Chloroplasts are the main site of chlorophyll and carotenoid biosynthesis, and their quantity and developmental state are closely related to pigment content. In the sg1 mutant of Arabidopsis thaliana, newly grown leaves are white, gradually changing from white to whitish-green as the plant grows, and finally turning green after three weeks. Therefore, the SG1 gene is correlated with green leaves in the early developmental stages.

[0004] To efficiently identify the green leaf color and chlorophyll and carotenoid content of non-heading Chinese cabbage, and to quickly breed green leaf varieties that meet market demands, it is urgent to develop a molecular marker closely related to green leaf color and chlorophyll and carotenoid content. Summary of the Invention

[0005] The purpose of this invention is to provide an InDel molecular marker for the BraC09g052220 gene in non-heading Chinese cabbage and its application. A recombinant inbred line population was constructed using *Wuta cabbage* (with dark green leaves) and *Erqing cabbage* (with yellow-green leaves) as parents. Resequencing was performed on the parents and the recombinant inbred line population to further identify mutation sites. Analysis revealed a mutation site in the exon region of BraC09g052220. An InDel molecular marker was developed for this mutation, and corresponding primers were designed. The marker was then used to identify the mutation in 36 non-heading Chinese cabbage varieties, with BraC09g052220 being the most prominent. The 9g052220 gene is the major gene for green leaves in non-heading Chinese cabbage. The non-heading Chinese cabbage varieties bred from these 24 core germplasm materials can be identified using this marker to determine the chlorophyll and carotenoid content. Therefore, the molecular marker obtained in this invention can be used as a marker-assisted breeding tool for these 24 core germplasm materials. It allows for rapid identification of leaf color and chlorophyll and carotenoid content in varieties bred based on these 24 core germplasm materials, reducing field screening work and having a very positive significance for efficient germplasm screening and breeding.

[0006] The technical solution of this invention is as follows:

[0007] The first objective of this invention is to provide the application of an InDel molecular marker closely linked to the pigment content trait of green leaves in non-heading Chinese cabbage in assisted breeding of this trait, wherein the nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4; the application is as follows:

[0008] (1) If the InDel molecular marker nucleotide sequence is the fragment shown in SEQ ID NO.3, the non-heading Chinese cabbage to be tested is determined to be a high pigment content variety;

[0009] (2) If the InDel molecular marker nucleotide sequence is the fragment shown in SEQ ID NO.4, the non-heading Chinese cabbage to be tested is determined to be a low pigment content variety.

[0010] SEQ ID No. 3:

[0011] GCTATATTTTTTAAAGCCCACAAGTCTAAAATGAATCATTACAGACAGGATCCATGATCTTCATTCTCCTTCTTCCTCAGATCTTCTTACTGAACAAAGGGTTTGAAGGTTCATCACTCACCACAGTCATGTACTCTCTGGGAAGATTACAGCTTAATCACCTTCCCTTCACTCACACCTCTTCATCCTT

[0012] SEQ ID No. 4:

[0013] GCTATATTTTTTAAAGCCCACAAGTCTAAAATGAATCATTACAGACAGGATCTCTCTTTACTGAACAAAGGGTTTGAAGGTTCATCACTCACCACAATCACAATCATGTACTCCCTGGGAAGATTACAGCTTCATCACCAGCCAGCCCACCTTCCCTTCACTCACACCTCTTCATCCTT

[0014] Furthermore, the application also includes (3) if the InDel molecular markers shown in SEQ ID NO.3 and SEQ ID NO.4 are included at the same time, the non-heading Chinese cabbage to be tested is determined to be an intermediate pigment content variety.

[0015] A second objective of this invention is to provide the application of primer pairs for amplifying the aforementioned InDel molecular marker in assisted breeding of the trait of green leaf-related pigment content in non-heading Chinese cabbage.

[0016] Furthermore, the primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.2:

[0017] SEQ ID No.1:GCTATATTTTTTAAAGCCCAC;

[0018] SEQ ID No. 2: AAGGATGAAGAGGTGTGAGTG.

[0019] A third objective of this invention is to provide a kit for detecting the aforementioned InDel molecular markers for use in assisted breeding of the trait of green leaf-related pigment content in non-heading Chinese cabbage;

[0020] Preferably, the kit includes the primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2.

[0021] The fourth objective of this invention is to provide a method for assisted breeding of non-heading Chinese cabbage with green leaf pigment content traits, the method comprising the following steps:

[0022] (1) Extract total genomic DNA from the non-heading Chinese cabbage to be tested;

[0023] (2) The total genomic DNA of the non-heading Chinese cabbage to be tested was amplified by PCR using the InDel molecular marker primers shown in SEQ ID NO.1 and SEQ ID NO.2;

[0024] (3) Perform electrophoresis and / or sequencing on the PCR amplification products, and determine the content of green leaf-related pigments in non-heading Chinese cabbage based on the length differences of the amplified fragments and / or the sequencing results of the PCR products.

[0025] Furthermore, if the amplification product contains only a 192bp band and / or only the fragment with the sequence shown in SEQ ID NO.3, the non-heading Chinese cabbage to be tested is a high-pigment-content variety; if it contains only a 179bp band and / or only the fragment with the sequence shown in SEQ ID NO.4, the non-heading Chinese cabbage to be tested is a low-pigment-content variety; if it contains both 192bp and 179bp bands and / or simultaneously includes the InDel molecular marker shown in SEQ ID NO.3 and SEQ ID NO.3, the non-heading Chinese cabbage to be tested is a heterozygous individual and is an intermediate-pigment-content variety.

[0026] Further, the PCR amplification reaction system in step (2) is as follows: the total PCR amplification system is 10 μl, including 0.5 μl each of forward and reverse primers, 5 μl of Taq enzyme, 1 μl (50 ng) of DNA, and 3 μl of ddH2O; the PCR amplification reaction program in step (2) is: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 53℃ annealing for 15 s, 72℃ extension for 20 s, 30 cycles; 72℃ extension for 5 min, and storage at 10℃.

[0027] Further, the specific operation of electrophoresis in step (3) is as follows: load 5 μl onto a 2% agarose gel, electrophores at a constant voltage of 120V for 40 min, turn off the power, remove the gel, and perform UV color development.

[0028] Furthermore, the green leaf-related pigments described in this invention are total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids.

[0029] Furthermore, the non-heading Chinese cabbage is selected from at least one of the following 24 core germplasms of non-heading Chinese cabbage, or from at least one of the non-heading Chinese cabbage varieties bred using the 24 core germplasms of non-heading Chinese cabbage as parents:

[0030]

[0031]

[0032] Beneficial effects:

[0033] The molecular marker developed in this invention has the ability to detect the green leaf color and chlorophyll and carotenoid content of non-heading Chinese cabbage. Using this marker, we can accurately identify the chlorophyll and carotenoid content and reliably predict leaf color in a large number of non-heading Chinese cabbage varieties at the seed stage. Compared with traditional breeding methods, this technology significantly shortens the breeding time and improves the accuracy of identification. Attached Figure Description

[0034] Figure 1 The molecular marker developed for BraC09g052220 was identified as a band in *Solanum tuberosum* and *Echinopsis pilosula*. The W-type band is the molecular marker cloned in *Solanum tuberosum*, and the E-type band is the molecular marker cloned in *Echinopsis pilosula*.

[0035] Figure 2 Correlation analysis of molecular marker genotypes developed for BraC09g052220 and chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, carotene, and xanthophyll in 36 samples from 24 species. Detailed Implementation

[0036] The present invention will now be described in detail and comprehensively through specific embodiments. It should be understood that these embodiments are only a part of the many possible embodiments of the present invention, and not all of them. For those skilled in the art, all other embodiments that can be derived based on these embodiments without innovative work are included within the protection scope of the present invention.

[0037] Primers were developed by Beijing Qingke Biotechnology Co., Ltd., and the non-heading Chinese cabbage varieties used in the following examples were all from the Chinese cabbage systems biology laboratory of the College of Horticulture, Nanjing Agricultural University.

[0038] Example 1: Obtaining the BraC09g052220 gene, which is related to the green leaf color and chlorophyll and carotenoid content of non-heading Chinese cabbage.

[0039] (1) QTL positioning of green leaf color

[0040] In this embodiment, resequencing was performed on a recombinant inbred line population constructed using *Brassica rapa* and *Brassica juncea* as parents. High-density linkage maps were constructed, and QTL mapping was performed using leaf color (chlorophyll and carotenoid content) obtained from repeats at three locations. The QTL mapping results showed that leaf color is mainly controlled by two major loci, primarily located on chromosomes A07 and A09. Further fine mapping of these two major loci was performed using the F2 population constructed from *Brassica rapa* and *Brassica juncea*, ultimately narrowing down the QTL loci on chromosomes A07 and A09 to 170.25 kb and 191.41 kb, respectively. Annotation using a non-heading Chinese cabbage database revealed that the *BraC09g052220* gene exists within the 191.41 kb region of chromosome A09.

[0041] (2) Acquisition of the BcSG1 gene

[0042] The full-length sequence of the BraC09g052220 gene was downloaded from the non-heading Chinese cabbage database. Cloning primers for this gene were designed using SnapGeneViewer and sent to Qingke Biotechnology Co., Ltd. for primer synthesis.

[0043] PCR amplification was further performed using synthesized primers. The 20ul amplification system consisted of 10ul of high-fidelity enzyme, 1ul of forward primer, 1ul of reverse primer, 1ul of *Hydrocotyle sibthorpioides* / *Hydrocotyle sibthorpioides* genomic DNA, and 7ul of sterile water. The reaction program was 98℃ for 5min, 98℃ for 10s, 55℃ for 15s, 72℃ for 1min and 20s, and 72℃ for 5min, for 35 cycles.

[0044] The obtained PCR product was stained with 3.3 μL of 6x loading buffer (Takara) and separated by 1.2% agarose gel electrophoresis. A band was obtained at the 1000 bp position. The gene was recovered by gel cutting and agarose gel recovery kit (Sangon Biotech).

[0045] (3) The gene was constructed on a T vector and sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain the BraC09g052220 gene sequence in *Solanum tuberosum* and *Solanum lycopersicum*.

[0046] Example 2: Obtaining the molecular marker of the BraC09g052220 gene, which is related to the green leaf color and chlorophyll and carotenoid content of non-heading Chinese cabbage.

[0047] This embodiment mainly compares the BraC09g052220 gene sequences obtained from *Brassica napus* and *Brassica rapa* in Example 1. Three InDel variant sites were found in the exon region of the BcSG1 gene. Through population variation information analysis in Example 1, it was found that these three InDel sites are linked. Based on the position of these three InDel markers, a 200bp molecular marker was designed using the full-length BraC09g052220 gene downloaded from the non-heading Chinese cabbage database as a reference sequence. Primer sequences as shown in SEQ ID No. 1 and SEQ ID No. 2 were designed using SnapGene Viewer.

[0048] Using the BraC09g052220 gene sequence obtained from *Solanum tuberosum* and *Erqing* in Example 1, sequence information from primers SEQ ID No. 1 and SEQ ID No. 2 was extracted, and SEQ ID No. 3 (192 bp) and SEQ ID No. 4 (179 bp) sequences were obtained from *Solanum tuberosum*.

[0049] Example 3: Application of the molecular marker BraC09g052220 gene, which is related to the green leaf color and chlorophyll and carotenoid content of non-heading Chinese cabbage.

[0050] This embodiment mainly uses the InDel molecular marker primers designed in Example 2 to identify 36 non-heading Chinese cabbage core germplasm samples.

[0051] (1) Pigment content determination

[0052] The contents of chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, xanthophyll, and carotene were determined in 36 non-heading Chinese cabbage core germplasms. About 0.1g of mature leaves were taken, ground with liquid nitrogen, and 1ml of anhydrous ethanol and acetone (1:1) extraction solution was added. The mixture was incubated in the dark on a horizontal shaker for 24h. After centrifugation, the supernatant was diluted by half and the absorbance was measured at 470nm, 474nm, 485nm, 642nm, 649nm, and 665nm using an ELISA reader. The results were repeated three times.

[0053] (2) Genotyping

[0054] DNA of 36 core germplasms of non-heading Chinese cabbage (Table 1) was extracted by the CTAB method. The 36 core germplasm materials include the core germplasm materials for the breeding of most non-heading Chinese cabbage varieties. Among them, the most common germplasm materials in the middle and lower reaches of the Yangtze River are Suzhouqing, Yangzhouqing, Aijiaohuang, and Shanghaiqing. In order to better identify the chlorophyll and carotenoid contents between the varieties bred from the same germplasm material, common varieties of Suzhouqing, Yangzhouqing, Aijiaohuang, and Shanghaiqing collected from the market were included in this example to identify the chlorophyll and carotenoid contents between the varieties bred from the same germplasm material. PCR amplification was carried out according to the molecular marker primers developed in Example 2. The PCR amplification system was 5 μl Taq mix, 0.5 μl primer such as SEQ ID.No.1, 0.5 μl primer such as SEQ ID.No.2, 1 μl DNA, and 3 μl sterile water. The PCR amplification program was 95°C for 3 min, 95°C for 15 s, 53°C for 15 s, 72°C for 20 s, 72°C for 5 min, 30 cycles, extension at 72°C for 5 min, and preservation at 10°C.

[0055] In the table, the W type refers to the genotype detecting a 192 bp band, which is a variety with high pigment content; the E type refers to the genotype detecting a 179 bp band, which is a variety with low pigment content. The high-generation inbred lines used in the example are highly pure lines without heterozygous types, but they appear in the F1 after the hybridization of Wuta-cai and Erqing. The pigment content of its F1 shows an intermediate type.

[0056] Table 1

[0057]

[0058]

[0059] The 5 μl PCR amplification product was electrophoresed on a 2% agarose gel at a constant voltage of 120 V for 40 min, and the band genotype was read by ultraviolet visualization ( Figure 1 ).

[0060] (3) Result analysis

[0061] Among the identification results of 36 non-heading Chinese cabbage germplasm samples, it was found that the chlorophyll and carotenoid contents of 24 core germplasms could be identified by this molecular marker. The amplified band size was consistent with the amplification result of Wuta-cai, that is, 192 bp, which was a variety with high chlorophyll and carotenoid contents. The amplified band size was consistent with the amplification result of Erqing, that is, 179 bp, which was a variety with low pigment content. The correlation coefficient between the molecular marker and the pigment content was as high as 0.91 - 0.93. The molecular marker developed by BcSG1 was significantly correlated with the pigment content (P < 0.05).

[0062] Among them, the families containing 12 W genotypes had an average chlorophyll a content of 49.21±5.80 mg / 100g, an average chlorophyll b content of 22.48±8.48 mg / 100g, an average total chlorophyll content of 87.71±10.91 mg / 100g, an average xanthophyll content of 7.37±3.25 mg / 100g, an average carotene content of 10.52±5.06 mg / 100g, and an average carotenoid content of 18.23±2.70 mg / 100g.

[0063] The family containing 24 families with the same E genotype had an average chlorophyll a content of 24.01±4.35 mg / 100g, an average chlorophyll b content of 7.43±0.61 mg / 100g, an average total chlorophyll content of 34.73±6.58 mg / 100g, an average xanthophyll content of 4.08±1.12 mg / 100g, an average carotene content of 4.53±0.89 mg / 100g, and an average carotenoid content of 8.56±1.32 mg / 100g.

[0064] Both significance and correlation analyses showed that the molecular marker BcSG1 was closely related to the chlorophyll and carotenoid content in non-heading Chinese cabbage leaves. The correlation coefficients between this marker and total chlorophyll content, chlorophyll a content, chlorophyll b content, and carotenoid content were 0.90, 0.93, 0.83, and 0.93, respectively. However, the correlation between this marker and xanthophyll and carotene was low, with correlation coefficients of 0.61 and 0.70, respectively. Therefore, this molecular marker can only identify the total chlorophyll content, chlorophyll a content, chlorophyll b content, and carotenoid content in non-heading Chinese cabbage leaves; it cannot identify the levels of xanthophyll and carotene. Figure 2 ).

[0065] The above description is merely a preferred embodiment of the present invention. It should be emphasized that anyone skilled in the art can make various improvements and adjustments without departing from the core principles of the invention. These improvements and adjustments based on the present invention should also be considered within the scope of protection of the present invention. The essence of the present invention is not limited to the specific embodiments described above, but lies in the core concepts it reveals. When understanding and applying the present invention, those skilled in the art can make necessary modifications and optimizations according to actual circumstances to adapt to different application scenarios or solve specific problems. This flexibility and adaptability is also one of the important characteristics of the present invention.

Claims

1. The application of InDel molecular markers closely linked to the pigment content trait of green leaves in non-heading Chinese cabbage in assisted breeding of the pigment content trait of green leaves in non-heading Chinese cabbage, characterized in that, The InDel molecular marker nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4; the application is as follows: (1) If the InDel molecular marker nucleotide sequence is the fragment shown in SEQ ID NO.3, the non-heading Chinese cabbage to be tested is determined to be a high pigment content variety; (2) If the InDel molecular marker nucleotide sequence is the fragment shown in SEQ ID NO.4, the non-heading Chinese cabbage to be tested is determined to be a low pigment content variety; (3) If the InDel molecular marker nucleotide sequence is the fragment shown in SEQ ID NO.3 and SEQ ID NO.4, the non-heading Chinese cabbage to be tested is determined to be an intermediate pigment content variety; The green leaf-related pigments are total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids; The non-heading Chinese cabbage is selected from at least one of the following 24 core germplasms of non-heading Chinese cabbage, or from at least one of the non-heading Chinese cabbage varieties bred using the aforementioned 24 core germplasms of non-heading Chinese cabbage as parents.

2. The application of the primer pair for amplifying the InDel molecular marker described in claim 1 in assisted breeding of the green leaf-related pigment content trait in non-heading Chinese cabbage is characterized by, The primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.2: SEQ ID No.1: GCTATATTTTTTAAAGCCCAC; SEQ ID No.2: AAGGATGAAGAGGTGTGAGTG; The green leaf-related pigments are total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids; The non-heading Chinese cabbage is selected from at least one of the following 24 core germplasms of non-heading Chinese cabbage, or from at least one of the non-heading Chinese cabbage varieties bred using the aforementioned 24 core germplasms of non-heading Chinese cabbage as parents.

3. The application of the kit for detecting the InDel molecular marker described in claim 1 in assisted breeding of non-heading Chinese cabbage with green leaf-related pigment content; the kit comprising primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2; the green leaf-related pigments being total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids; The non-heading Chinese cabbage is selected from at least one of the following 24 core germplasms of non-heading Chinese cabbage, or from at least one of the non-heading Chinese cabbage varieties bred using the aforementioned 24 core germplasms of non-heading Chinese cabbage as parents.

4. A method for assisted breeding of non-heading Chinese cabbage with green leaf pigment content, characterized in that, The method includes the following steps: (1) Extract total genomic DNA from the non-heading Chinese cabbage to be tested; (2) The total genomic DNA of the non-heading Chinese cabbage to be tested was amplified by PCR using the InDel molecular marker primers shown in SEQ ID NO.1 and SEQ ID NO.2; (3) Perform electrophoresis and / or sequencing on the PCR amplification products, and determine the content of green leaf-related pigments in non-heading Chinese cabbage based on the length differences of the amplified fragments and / or the sequencing results of the PCR products. The green leaf-related pigments are total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids; The non-heading Chinese cabbage is selected from at least one of the following 24 core germplasms of non-heading Chinese cabbage, or from at least one of the non-heading Chinese cabbage varieties bred using the aforementioned 24 core germplasms of non-heading Chinese cabbage as parents. If the amplification product contains only a 192 bp band and / or only the fragment with the sequence shown in SEQ ID NO.3, the tested non-heading Chinese cabbage is a high-pigment-content variety; if it contains only a 179 bp band and / or only the fragment with the sequence shown in SEQ ID NO.4, the tested non-heading Chinese cabbage is a low-pigment-content variety; if it contains both 192 bp and 179 bp bands and / or simultaneously includes the InDel molecular marker shown in SEQ ID NO.3 and SEQ ID NO.4, the tested non-heading Chinese cabbage is a heterozygous individual and is an intermediate-pigment-content variety.

5. The method according to claim 4, characterized in that, The PCR amplification reaction system in step (2) is as follows: the total PCR amplification system is 10 μl, including 0.5 μl each of forward and reverse primers, 5 μl of Taq enzyme, 1 μl of DNA (50 ng / μl), and 3 μl of ddH2O; the PCR amplification reaction program in step (2) is: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 53℃ annealing for 15 s, 72℃ extension for 20 s, 30 cycles; 72℃ extension for 5 min, and storage at 10℃.

6. The method according to claim 4, characterized in that, The specific operation of electrophoresis in step (3) is as follows: load 5 μl onto a 2% agarose gel, electrophores at a constant voltage of 120 V for 40 min, turn off the power, remove the gel, and perform UV color development.

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