A molecular marker primer set and detection method for identifying chickpea kabuli and desi types

Abstract

The application provides a molecular marker primer set and a detection method for identifying chickpea Kabuli type and Desi type, and belongs to the technical field of biotechnology. Flower color and seed coat color are main classification characteristics of Kabuli type and Desi type chickpea. The molecular marker CAKD1 primer set for identifying Kabuli type and Desi type chickpea provided by the application uses two pairs of primers to respectively perform PCR amplification reaction on DNA of Kabuli type and Desi type chickpea, different site bands are shown, and the amplification band modes are complementary, and the results are verified with each other, so that the two types of chickpea can be accurately distinguished. The specific primer set and the matching amplification system provided by the application can effectively distinguish and identify Kabuli type and Desi type chickpea varieties, can conveniently, accurately and efficiently identify the difference of the two types at specific sites, and thus reliable identification of the variety types can be realized.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and molecular marker technology, specifically to a molecular marker primer set for identifying Kabuli and Desi types of chickpeas, and also to reagents and methods thereof. Background Technology

[0002] Chickpeas (Cicer arietinum L., 2n = 16) are annual herbs belonging to the genus Cicera in the legume family (Fabaceae). They are the third most widely cultivated legume crop globally, originating in West Asia and the Near East, and are grown in 49 countries and regions worldwide. Based on differences in plant morphology, flower color, and seed appearance, chickpeas are mainly divided into two types: Kabuli and Desi. Kabuli chickpeas have larger, plump seeds with a light yellow or beige seed coat, and typically white flowers. Desi chickpeas have relatively smaller seeds with a brown, reddish-brown, or black, wrinkled seed coat, and usually pink or purplish-red flowers.

[0003] Chickpeas are highly nutritious, rich in high-quality protein, essential amino acids, fatty acids, vitamins, carbohydrates, and various minerals. They are an important source of high-quality protein in arid and semi-arid regions and play an irreplaceable role in food security in developing countries. According to the latest statistics from the Food and Agriculture Organization of the United Nations (FAO), the global chickpea planting area has reached 15.3562 million hectares, with an annual output of 18.095 million tons. India is the world's largest producer and consumer of chickpeas, accounting for approximately 71.2% of global production. Pakistan, Turkey, Australia, and Ethiopia are also important producers, jointly driving the development of the global chickpea industry and market cooperation. In China, chickpeas are mainly grown in Xinjiang, Qinghai, Gansu, and Yunnan, with Xinjiang being the main chickpea growing area and a core export destination.

[0004] Flower color and seed coat color are core phenotypic characteristics that distinguish Kabuli and Desi type chickpeas. Traditional methods for identifying chickpea resource types mainly rely on phenotypic observation and lack systematic and standardized molecular-level detection and identification criteria. Researchers typically differentiate between the two types of chickpeas based solely on phenotypic characteristics such as plant morphology, flower color, seed size, and seed coat color. However, this method is highly subjective and dependent on the experience of the researchers, making it difficult to guarantee the scientific validity and accuracy of the identification results. This seriously affects the identification, effective protection, and sustainable utilization of chickpea germplasm resources. Therefore, there is an urgent need to develop an efficient and accurate identification technique, which has significant theoretical and practical value for the assessment of Kabuli and Desi type chickpea germplasm resources, gene function research, variety breeding, and new germplasm creation.

[0005] Molecular markers are genetic markers based on the gene level, capable of scientifically and accurately detecting genetic variations at the DNA level, effectively avoiding interference from factors such as tissue / organ type and environmental conditions. Due to their significant advantages such as simplicity, speed, ease of operation, and insensitivity to external environmental influences, molecular marker technology has been widely applied in fields such as crop variety identification and germplasm resource screening and evaluation, providing an effective and precise approach to address the shortcomings of traditional phenotypic identification. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a molecular marker primer set for identifying Kabuli and Desi types of chickpeas; a second objective of the present invention is to provide a reagent containing the aforementioned molecular marker primer set for identifying Kabuli and Desi types of chickpeas; and a third objective of the present invention is to provide a method for identifying Kabuli and Desi types of chickpeas using the aforementioned primer set.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. A set of molecular marker primers for identifying Kabuli and Desi types of chickpeas, the primer set comprising primer sets P1, P2, P3, P4, P5, and P6, wherein the nucleotide sequences of primer set P1 are shown in SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequences of primer set P2 are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of primer set P3 are shown in SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequences of primer set P4 are shown in SEQ ID NO.7 and SEQ ID NO.8; the nucleotide sequences of primer set P5 are shown in SEQ ID NO.9 and SEQ ID NO.10; and the nucleotide sequences of primer set P6 are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0008] 2. The reagent containing the molecular marker primer set for identifying the Kabuli and Desi types of chickpeas, the reagent further comprising Taq PCR Master Mix and ddH2O.

[0009] Preferably, the reagent further includes a DNA extraction solution, which is 2% (w / v) CTAB, 1.4 mol / L NaCl, 25 mmol / L EDTA, and 100 mmol / L Tris-HCl.

[0010] 3. The method for identifying Kabuli and Desi types of chickpeas using the primer set described above involves using primer set P1, P2, P3, P4, P5, and P6 as detection primers, and performing PCR amplification using the DNA of the chickpea material to be tested as a template. The amplification products are then detected. If the target band obtained by amplification is 486 bp, the chickpea material is determined to be of the Kabuli type; if the target band obtained by amplification is 701 bp, the chickpea material is determined to be of the Desi type.

[0011] Preferably, the DNA of the chickpea material to be tested is extracted using the CTAB method.

[0012] Preferably, the DNA extraction material is selected from 0.5 g of young leaves of plants 7-10 days after emergence.

[0013] Preferably, the extraction solution used in this invention is 2% (w / v) CTAB, 1.4 mol / L NaCl solution, 25 mmol / L EDTA, and 100 mmol / L Tris-HCl.

[0014] Preferably, the PCR amplification conditions of the present invention are: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 56℃ annealing for 25 s, 72℃ extension for 25 s, for 35 cycles; 72℃ extension for 5 min.

[0015] The beneficial effects of this invention are as follows: This invention provides a set of molecular marker primers for identifying Kabuli and Desi types of chickpeas. These primers can be efficiently applied to the identification of Kabuli and Desi types of chickpeas, and are applicable to scenarios such as chickpea germplasm screening and type identification, significantly improving the efficiency of chickpea parental screening and breeding. PCR amplification using specific primers allows for convenient and accurate identification of structural variations and differences in related sites between the two types of chickpeas, achieving rapid and accurate identification of Kabuli and Desi types. This effectively solves the technical pain points of traditional phenotypic identification methods, such as high subjectivity, low accuracy, and low efficiency, filling the technical gap in precise molecular-level identification of the two types of chickpeas. It provides reliable molecular technical support for chickpea germplasm resource protection, gene function research, variety breeding, and quality improvement.

[0016] The two primer pairs provided in this patent for identifying Kabuli and Desi types of chickpeas also have the following advantages: Results showed that primer set P5 (486 bp) had high amplification sensitivity and high sample detection rate; primer set P6 (701 bp) had better specificity but limited sensitivity, and some samples experienced amplification deletion.

[0017] High specificity: Extensive primer screening yielded species-specific primer sets P5 and P6, which can be efficiently applied to the identification of Kabuli and Desi type chickpeas. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 To identify CaTT8, a gene associated with flower color, for genome-wide association analysis of chickpeas; Figure 2 The sequence alignment results of the coding region of the Ca_09761 (TT8) gene in Kabuli-type chickpea material PY1Ca04G02100 (tt8) and Desi-type material ICC4958; Figure 3 Correlation analysis between Kabuli and Desi type chickpeas and the functional molecular marker CaTT8_InDel (Structure variation, SV); Figure 4 The results of PCR amplification of chickpea material Pinying 1 (PY1) and ICCV89330 primer sets P1, P2, P3, P4, P5 and P6.

[0019] Figure 5 Results of PCR amplification of primer sets P5 and P6 for 24 chickpea materials. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Example 1

[0022] A significant association signal related to flower color was detected on the short arm of chromosome 4 of chickpea using genome-wide association analysis (GWAS). Figure 1 The signal is anchored to the CaTT8 gene, which encodes a basic helical-loop-helical (bHLH) transcription factor responsible for regulating flower and seed color. Its function is homologous to the Mendelian A gene (PsbHLH) in peas. Sequence alignment results of the coding regions of the Ca_09761 (TT8) gene in the Kabuli-type chickpea material Pinying 1 (PY1) Ca04G02100 (tt8) and the Desi-type material ICC4958 are shown below. Figure 2 Correlation analysis of TT8 gene structure () Figure 3Based on the previously assembled T2T genome of the Kabuli-type chickpea material PY1, and utilizing the published genome of the Desi-type chickpea material ICC4958, six pairs of specific primers were designed to construct a molecular marker system capable of efficiently identifying the two chickpea types. The primer set is P5, and the optional P6 is as follows: Table 1. Names and sequences of detection primer pairs

[0023] Amplification was performed using the six primer pairs P1, P2, P3, P4, P5, and P6 listed in Table 1. The results are as follows: Figure 4 As shown in the figure, P1 and P2 had low amplification efficiency and failed to effectively amplify the target band; although P3 and P4 yielded amplification products, the band size did not match the expectations, and were judged as non-specific amplifications. In contrast, P5 and P6 had better amplification effects, with single and highly specific bands, and the product size was correct. Further combining P5 and P6 can effectively improve the accuracy of identification.

[0024] Twenty-six test materials were selected, as detailed in Table 2. Among them, PY1 was of the Kabuli type and ICCV89330 was of the Desi type.

[0025] Table 2. Information on 26 chickpea materials

[0026] The Kabuli and Desi types of chickpeas were identified using the designed P5 and P6 primers, and the specific steps are as follows: (1) Extraction of DNA from chickpea material: Select 3-5 plump seeds for sowing and cultivate in a greenhouse. 7-10 days after emergence, cut 0.5 g of young leaves from the plant and place them in a 2 mL centrifuge tube with added steel balls. Quickly place the tube in liquid nitrogen for 30 s to cool, and grind it in a grinder at 1000 r / 25 s for 2-3 s to grind the young leaves into powder. Add 800 μL of CTAB (hexadecyltrimethylammonium bromide) to the centrifuge tube and heat it in a 65℃ oven for 50 min, shaking once every 10 min. Then add an equal volume of chloroform-isoamyl alcohol (24:1) and shake to mix. Centrifuge at 10000 r / min for 10 min at room temperature, transfer 600 μL of supernatant to a 1.5 mL centrifuge tube, add an equal volume of isopropanol at 4℃, gently invert the tube, and place it in a -20℃ refrigerator to precipitate DNA for 20 min. Centrifuge at 10000 r / min for 10 min, discard the supernatant, and add 75% Wash the DNA with 600 μL of ethanol solution, centrifuge at 10000 r / min for 3 min, collect the precipitate by centrifugation, repeat twice, and then air dry the DNA; add 20-50 μL of ddH2O to dissolve the DNA and store it in a refrigerator at 4℃. (2) DNA quality detection: The extracted DNA was tested for quality. 4 μL of DNA solution was electrophoresed in an agarose gel for 30 min and photographed using a gel imaging system. (3) PCR amplification reaction: The amplification product was obtained by using a 10 μL reaction system; the primer for PCR amplification reaction was a primer, and the reaction program was as follows: 95℃, pre-denaturation for 3 min; 94℃, 25 s, 56℃, 25 s, 72℃, 25 s, for 35 cycles; 72℃, extension for 5 min. (4) Detection of PCR amplification products: Electrophoresis was performed using an electrophoresis apparatus and its matching electrophoresis tank, 2% agarose gel, at 180 volts for 30 minutes, and the results were recorded by taking pictures using a gel imaging system. Figure 5 ); (5) Result determination: When the target band obtained by amplification is 486 bp, the chickpea material is determined to be of the Kabuli type; when the target band obtained by amplification is 701 bp, the chickpea material is determined to be of the Desi type.

[0027] The amplification results showed that 15 samples (PY1, S_2, S_3, S_6, S_7, S_8, S_9, S_10, S_11, S_17, S_18, S_19, S_21, S_22, and S_23) amplified to 486 bp, indicating that these materials are of the Kabuli type. 11 samples (89330, S_1, S_4, S_5, S_12, S_13, S_14, S_15, S_16, S_20, and S_24) amplified to 701 bp, indicating that they are of the Desi type.

[0028] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A molecular marker primer set for identifying Kabuli and Desi types of chickpeas, characterized in that: The primer set includes primer sets P1, P2, P3, P4, P5, and P6. The nucleotide sequences of primer set P1 are shown in SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequences of primer set P2 are shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleotide sequences of primer set P3 are shown in SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequences of primer set P4 are shown in SEQ ID NO.7 and SEQ ID NO.8; the nucleotide sequences of primer set P5 are shown in SEQ ID NO.9 and SEQ ID NO.10; and the nucleotide sequences of primer set P6 are shown in SEQ ID NO.11 and SEQ ID NO.

12.

2. A reagent containing the molecular marker primer set for identifying Kabuli and Desi types of chickpeas as described in claim 1, characterized in that: The reagents also include Taq PCR Master Mix and ddH2O.

3. The reagent according to claim 2, characterized in that: The reagents also include a DNA extraction solution, which is 2% (w / v) CTAB, 1.4 mol / L NaCl, 25 mmol / L EDTA, and 100 mmol / L Tris-HCl.

4. A method for identifying Kabuli and Desi types of chickpeas using the primer set described in claim 1, characterized in that: Using primer set P1, P2, P3, P4, P5, and P6 as detection primers, PCR amplification was performed using the DNA of the chickpea material to be tested as a template. The amplification products were then detected. If the target band obtained by amplification was 486 bp, the chickpea material was determined to be of the Kabuli type; if the target band obtained by amplification was 701 bp, the chickpea material was determined to be of the Desi type.

5. The method according to claim 4, characterized in that: DNA from the chickpea material to be tested was extracted using the CTAB method.

6. The method according to claim 5, characterized in that: The DNA extraction material was selected from 0.5 g of young leaves from plants 7-10 days after emergence.

7. The method according to claim 6, characterized in that: The extraction solution used was 2% (w / v) CTAB, 1.4 mol / L NaCl solution, 25 mmol / L EDTA, and 100 mmol / L Tris-HCl.

8. The method according to claim 4, characterized in that: The PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 94℃, 25 s, 56℃, 25 s, 72℃, 25 s, for 35 cycles; 72℃ extension for 5 min.

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