Molecular marker for identifying boron poisoning resistance of citrus, primer and application

Abstract

The invention belongs to the technical field of molecular markers for boron poison resistance of citrus varieties, and relates to a molecular marker for identifying boron poison resistance of citrus, a primer and application of the molecular marker, the nucleotide sequence of the molecular marker is shown as SEQ ID No.5, if a citrus genome contains a DNA fragment, the citrus is a boron poison resistance citrus variety or a candidate is a boron poison resistance citrus variety; the nucleotide sequence of the PCR detection primer for identifying the boron poisoning resistance of the citrus is as shown in SEQ ID No.6-7; the PCR detection primer is used for identifying the boron poison resistance of the citrus, if a PCR product containing 442 bp can be specifically amplified, the citrus variety is diagnosed as a boron poison resistant citrus variety, and if only a PCR product containing 231 bp is specifically amplified, the citrus variety is a boron poison sensitive variety. By utilizing the developed molecular marker, resistance and susceptible allelic mutation of the 3-KAT promoter can be distinguished at high throughput and low cost, marker-assisted selection in boron poison resistance breeding is facilitated, and a 3-KAT resistant material can be accurately and efficiently utilized to breed a citrus boron poison resistance variety.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology for boron resistance in citrus varieties, and relates to a molecular marker, primer, and application for identifying boron resistance in citrus. Background Technology

[0002] Boron is an essential micronutrient for plant growth, development, and reproduction, playing a crucial role in sexual reproduction and the formation of yield and quality. Citrus is my country's largest fruit tree, with a planting area of ​​44.93 million mu (approximately 3.65 million hectares) and a yield of 64.33 million tons (accounting for 32.5% of the global total) in 2022. Given the high fertilization efficiency of boron application to soil, boron supply through citrus orchard soil is widely used as an official guideline in various regions. However, the optimal concentration range for boron in citrus growth is very narrow, leading to widespread boron toxicity in citrus-producing areas worldwide (Papadakis et al. 2004; Martínez-Cuenca et al. 2015; Shah et al. 2017; Simón-Grao et al. 2018; Ferreira et al. 2020; Aparicio-Durán et al. 2021; Yang et al. 2023). Boron toxicity not only has a wide range of effects on the physiological functions of citrus roots and leaves, but may also increase the risk of fruit granulation, which has become an urgent problem to be solved in citrus production.

[0003] Solutions to boron toxicity in plants mainly include: ① Boron flushing for mitigation – however, this method is costly and difficult to implement on a large scale. ② Planting boron-tolerant varieties. To prevent and control boron toxicity, breeding and planting boron-tolerant varieties is the most economical and effective measure. Therefore, developing molecular markers for boron toxicity and screening boron-tolerant resources are crucial for effectively controlling boron toxicity in citrus and ensuring the sustainable development of my country's citrus industry.

[0004] Previous studies have shown that plant tolerance to boron toxicity mainly depends on their ability to redistribute and / or translocate free boron in plastids. However, the tolerance to boron toxicity in plants such as citrus has not shown a reliable correlation with the boron content in leaves / roots, indicating that citrus plants possess a distinctly different mechanism for boron tolerance (Huang et al. 2014, 2022; Huang, 2017). However, current research on the localization of genes related to boron toxicity in citrus is limited, and there is a lack of molecular markers that can be used to breed boron-tolerant citrus varieties, severely restricting the breeding of such varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker, primers, and applications for identifying boron toxicity resistance in citrus fruits. The developed molecular marker enables high-throughput, low-cost differentiation. 3-KATThe resistance and susceptibility allelic mutations of the promoter facilitate marker-assisted selection in boron toxicity resistance breeding.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a molecular marker for identifying boron resistance in citrus, the nucleotide sequence of which is shown in SEQ ID No. 5. If the citrus genome contains the DNA fragment of SEQ ID NO. 5, the citrus is or is a candidate for a boron-resistant citrus variety.

[0007] Secondly, this invention provides a PCR detection primer for identifying boron resistance in citrus fruits. The PCR detection primer is used to amplify the aforementioned molecular marker, and its nucleotide sequence is as follows: BS-P2-F: 5'-TGGGTGAATTTGATAATTTGAA-3' BS-P2-R: 5'-TTGAACGGGTGATGATGACG-3'.

[0008] A PCR product containing 442 bp was specifically amplified for boron-resistant citrus varieties, and a PCR product containing 231 bp was specifically amplified for boron-sensitive citrus varieties.

[0009] This invention also provides a PCR detection method for identifying boron resistance in citrus using the aforementioned PCR detection primers, comprising the following steps: (1) Extracting total DNA from the vegetative organs of citrus fruits; (2) Using the total DNA extracted in step (1) as a template, PCR amplification was performed using the PCR detection primers BS-P2-F / BS-P2-R to obtain the PCR amplification product; (3) Observe the PCR results. If a PCR product containing 442 bp can be specifically amplified, it is diagnosed as a boron-resistant citrus variety. If only a PCR product containing 231 bp is specifically amplified, it is a boron-sensitive variety.

[0010] The specific method is as follows: (1) Total DNA was extracted from vegetative organs such as citrus leaves using the CTAB method. (2) Using the total DNA extracted in step (1) as a template, PCR amplification was performed using the appropriate primers BS-P2-F / BS-P2-R (SEQ ID No. 6, SEQ ID No. 7). The amplification conditions were as follows: The PCR reaction system was 20 µL, including 10 µL of PCR Mix (2× Es Taq MasterMix (Dye)), 0.5 µL each of 10 µM BS-P2-F and 10 µM BS-P2-R, 1 µL of 50-100 ng total DNA template, and sterile double-distilled water to make up to 20 µL.

[0011] The PCR reaction program was as follows: 98 °C pre-denaturation for 3 min, 98 °C denaturation for 20 s, 56 °C annealing for 30 s, 72 °C extension for 40 s, 35 cycles, and a final extension at 72 °C for 10 min.

[0012] (3) Take 7 μL of the amplification product and perform 1% agarose gel electrophoresis. Observe the size of the amplification product with a gel imaging instrument to determine the result. If a PCR product of 442 bp can be specifically amplified, it can be diagnosed as a boron-resistant citrus variety (highly resistant or moderately resistant). If a PCR product of 231 bp is specifically amplified, it is a boron-sensitive variety.

[0013] Finally, this invention provides a breeding method for boron-resistant citrus varieties. According to the PCR detection method described above, citrus varieties are subjected to PCR detection to determine the mutation type based on the size of the target fragment. If the 3-KAT promoter insertion site of the citrus variety has a homozygous or heterozygous insertion, it is selected as a parent for breeding. The insertion site is a molecular marker for identifying boron resistance in citrus, and its nucleotide sequence is shown in SEQ ID No. 5.

[0014] The present invention has the following advantages: This invention utilizes DNA mutation sites on the upstream chromosomal segment of 3-KAT to develop a set of specific molecular markers at Indel sites present in both resistant and susceptible materials. The insertion site associated with these markers is approximately 360 bp upstream of the 3-KAT promoter associated with boron toxicity, and the insertion fragment is 211 bp in size. The development and utilization of these markers have significant theoretical and practical value for marker-assisted breeding of citrus varieties highly resistant to boron toxicity.

[0015] The developed molecular markers can be used to distinguish between resistant and susceptible allelic mutations of the 3-KAT promoter at high throughput and low cost, which facilitates marker-assisted selection in boron toxicity resistance breeding and helps to accurately and efficiently use 3KAT resistant materials to breed boron toxicity resistant citrus varieties. Attached Figure Description

[0016] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] Figure 1The results of 3-KAT promoter sequence alignment from different citrus types in the Citrus Genome Database (partial).

[0018] Figure 2 Cluster analysis of 3-KAT promoter sequences from different citrus types in the Citrus Genome Database.

[0019] Figure 3 The image shows the PCR detection results of the resistance molecular marker primers for Fuju and sour grapefruit.

[0020] Figure 4 Symptoms of boron toxicity in different citrus varieties.

[0021] Figure 5 This is a graph showing the PCR detection results of the molecular marker primers of this invention in the identification of boron resistance in citrus varieties. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0023] Example 1: Citrus Boron Toxicity Response Alleles 3-KAT Acquisition of promoters The boron-tolerant variety 'Fukuju' ( Citrus reticµLate cv. Fujü) and the boron-sensitive variety 'Sour Pomelo' ( C. grandis Using *Sour pummelo* cv. as material, 0.5 g of young leaves were collected from each material, and total genomic DNA was extracted using the CTAB method. The total genomic DNA from both materials was used as a template. 3-KAT Promoter-specific primers were used to amplify the full-length promoter sequence using high-fidelity PCR.

[0024] The primer pair is: Pro3KAT-f: 5'-CAGTATGAAGTTACCCTTGGGAGTG-3' (SEQ ID No.1) Pro3KAT-r: 5'-TTATCCCTTGGTGTTGGTGCTAT-3' (SEQ ID No.2) The PCR reaction system was a 50 µL TaKaRa (Code DRR100A) system: TaKaRa Ex Taq (5 U / µL), 0.25 µL 10×Ex Taq Buffer 5 µL, dNTP Mixture 4 µL, template DNA 1 µL (100 ng), Pro3KAT-f (10 uM) 1 µL, Pro3KAT-r (10 uM) 1 µL, and sterile double-distilled water was added to make up to 50 µL.

[0025] The PCR amplification conditions were: 98 °C pre-denaturation for 3 min, 98 °C denaturation for 20 s, 60 °C annealing for 20 s, 72 °C extension for 1 min 30 s, 34 cycles, and a final extension at 72 °C for 10 min.

[0026] PCR products from the two materials were analyzed by gel sequencing, respectively. The boron-tolerant variety 'Fukuju'... 3-KAT The full-length promoter sequence is 1852 bp (nucleotide sequence shown in SEQ ID No. 3), while the boron-sensitive variety 'Sour Pomelo'... 3-KAT The full-length promoter sequence is 1533 bp (nucleotide sequence as shown in SEQ ID No. 4).

[0027] Sequence alignment revealed that, compared to the boron-sensitive variety 'Sour Pomelo', the boron-tolerant variety 'Fujuju' has a 211 bp insertion at approximately 360 bp upstream of the 3-KAT promoter (as shown in SEQ ID No. 5). This sequence contains multiple transcription factor binding sites, including CAAT-box, MYB, CTAG-motif, GT1-motif, and TATA-motif.

[0028] Further analysis of information from the Citrus Genome Database (https: / / www.citrusgenomedb.org / ) revealed that the inserted sequence is evolutionarily conserved. Figure 1 ), and it exhibits two types of insertion / deletion in different citrus varieties ( Figure 2 ).

[0029] Using this inserted sequence as the target site for PCR primer design, two specific molecular marker primers (BS-P2-F and BS-P2-R) were designed on the flanking side, with sequences shown in SEQ ID No. 6 and SEQ ID No. 7.

[0030] BS-P2-F: 5'-TGGGTGAATTTGATAATTTGAA-3' (SEQ ID No. 6) BS-P2-R: 5'-TTGAACGGGTGATGATGACG-3' (SEQ ID No.7) Using the marker pair as primers and genomic DNA from 'sour grapefruit' and 'Fuju' as templates, PCR amplification was performed using 2× Es Taq MasterMix (Dye).

[0031] The PCR reaction system is 20 µL, consisting of 10 µL PCR Mix (2× Es Taq MasterMix (Dye)), 0.5 µL each of 10 µM BS-P2-F and 10 µM BS-P2-R, 1 µL of 50-100 ng total DNA template, and sterile double-distilled water to a final volume of 20 µL.

[0032] The PCR reaction program was as follows: 98 °C pre-denaturation for 3 min, 98 °C denaturation for 20 s, 56 °C annealing for 30 s, 72 °C extension for 40 s, 35 cycles, and a final extension at 72 °C for 10 min.

[0033] The amplification products, as detected by agarose gel electrophoresis, showed distinct specific bands of 442 bp and 231 bp, respectively. Figure 3 ).

[0034] Example 3: Application of molecular markers in identifying boron toxicity resistance in citrus varieties Thirty-two representative citrus varieties (Table 1) were collected and grafted onto one-year-old trifoliate orange rootstocks. The grafts were then transplanted into ceramic pots containing 6L of river sand and placed in a glass chamber under natural light. The pots were watered with 500 mL of Hoagland's nutrient solution (Huang Jinghao, 2017) every one day. Once the scions reached a height of 15 cm, seedlings with uniform growth were selected for boron toxicity treatment. Every one day, 500 mL of Hoagland's nutrient solution containing different boron concentrations [10 μM (control) and 400 μM (boron toxicity)] was applied. After 16 weeks of treatment, boron toxicity symptoms were observed in mature leaves at the base of the plant (approximately 1 / 4 of the plant height). Simultaneously, young leaves at the top were collected, and genomic DNA was extracted using the CTAB method and PCR was performed according to the method in Example 2.

[0035] Table 1. Results of boron toxicity resistance detection and PCR amplification of grafted seedlings of different citrus varieties.

[0036] The test results are shown in Table 1. Figure 4 and Figure 5Boron toxicity treatment caused mottled yellowing between the veins on the basal older leaves of sensitive varieties, starting from the leaf tips. Necrosis then appeared at the chlorotic areas of the tips, gradually spreading towards the leaf base, eventually leading to leaf senescence and abscission. In moderately resistant varieties, only slight, scattered yellowing appeared between the veins on the basal older leaves, and the leaves did not fall off. In contrast, in highly resistant varieties treated for the same duration, the older leaves remained dark green. Figure 4 ).Depend on Figure 5 It is evident that the 3-KAT promoter insertion site in citrus varieties exhibits three types: homozygous insertion, homozygous deletion, and heterozygous. Resistant (highly resistant and moderately resistant) varieties show homozygous or heterozygous insertions, while boron-sensitive varieties show homozygous deletions. Selecting genotypes with 3-KAT promoter fragment insertions during variety breeding can accelerate the selection of boron-resistant citrus varieties.

Claims

1. A molecular marker for identifying boron toxicity resistance in citrus fruits, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID No.

5. If the citrus genome contains the DNA fragment of SEQ ID NO. 5, the citrus is or is a candidate for a boron-resistant citrus variety.

2. A PCR detection primer for identifying boron toxicity resistance in citrus fruits, characterized in that, The PCR detection primers are used to amplify the molecular marker described in claim 1, and their nucleotide sequence is as follows: BS-P2-F: 5'-TGGGTGAATTTGATAATTTGAA-3' BS-P2-R: 5'-TTGAACGGGTGATGATGACG-3'; A PCR product containing 442 bp was specifically amplified for boron-resistant citrus varieties, and a PCR product containing 231 bp was specifically amplified for boron-sensitive citrus varieties.

3. A PCR detection method for identifying boron resistance in citrus using the PCR detection primers described in claim 2, characterized in that: Includes the following steps: (1) Extracting total DNA from the vegetative organs of citrus fruits; (2) Using the total DNA extracted in step (1) as a template, PCR amplification was performed using the PCR detection primers BS-P2-F / BS-P2-R to obtain the PCR amplification product; (3) Observe the PCR results. If a PCR product containing 442 bp can be specifically amplified, it is diagnosed as a boron-resistant citrus variety. If only a PCR product containing 231 bp is specifically amplified, it is a boron-sensitive variety.

4. The PCR detection method for identifying boron resistance in citrus according to claim 3, characterized in that: The PCR amplification conditions described in step (2) are as follows: the PCR reaction system is 20 µL, including 10 µL PCR Mix, 0.5 µL each of 10 µM BS-P2-F and 10 µMBS-P2-R, 50-100 ng total DNA template, and sterile double-distilled water to make up to 20 µL; the amplification program is 98 °C pre-denaturation for 3 min, 98 °C denaturation for 20 s, 56 °C annealing for 30 s, 72 °C extension for 40 s, 35 cycles, and a final extension at 72 °C for 10 min.

5. A breeding method for boron-resistant citrus varieties, characterized in that, According to the PCR detection method described in claim 3, PCR detection is performed on citrus varieties to determine the mutation type based on the size of the target fragment; if the 3-KAT promoter insertion site of the citrus variety has a homozygous or heterozygous type, it is selected as a parent for breeding; the insertion site is a molecular marker for identifying boron toxicity resistance in citrus, and its nucleotide sequence is shown in SEQ ID No. 5.

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