High quercetin 7-O-beta-D-glucuronic acid content molecular marker related to resistance to root knot nematode disease and application

Abstract

The invention provides a high quercetin 7-O-beta-D-glucuronic acid content molecular marker related to resistance to root knot nematode disease and application, and belongs to the technical field of molecular biology, an SV molecular marker is Chr142657774del1565 and is used for predicting the high or low content of quercetin 7-O-beta-D-glucuronic acid in cassava; the primer group for detecting the SV molecular marker comprises an upstream primer with the sequence of 5 '-ATGATCCGAGATCCAATG-3', a downstream primer with the sequence of 5 '-ATGATCCGAGATCCAATG-3', and a The sequence of the downstream primer is 5 '-GTTACCCAAATGTGATGC-3'; preparing the kit by utilizing the primer group; the cassava is detected by using the primer group or the kit so as to predict whether the content of quercetin 7-O-beta-D-glucuronic acid in the cassava is high or low. The SV molecular marker disclosed by the invention can accurately and efficiently predict the high or low content of quercetin 7-O-beta-D-glucuronic acid in the cassava seedling stage, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and relates to a molecular marker method for breeding related to the content of metabolites associated with resistance to root-knot nematode disease, and a breeding technique for screening high-quercetin materials using an SV molecular marker related to the content of quercetin 7-O-β-D-glucuronide in cassava as a key index. Specifically, it relates to a molecular marker of quercetin 7-O-β-D-glucuronide content related to resistance to root-knot nematode disease and its application, which solves the problems of long breeding time and high breeding cost for new cassava varieties with high quercetin 7-O-β-D-glucuronide content. Background Art

[0002] Root-knot nematode disease is a disease caused by root-knot nematodes, which mainly harms the roots of plants and seriously affects their growth and yield. Root-knot nematodes mainly damage the root systems of plants, resulting in the formation of tumor-like root knots of different sizes on lateral roots and fibrous roots. For plants parasitized by root-knot nematodes, the above-ground part grows weakly, the plants are short, the leaves turn yellow and curl, and in severe cases, the leaves wither and fall off, resulting in a decrease in yield. Flavonoids may play an important role in the anti-nematode response of plants. The prevention and control of root-knot nematode disease is difficult and costly, and traditional prevention and control methods are difficult to fundamentally solve the problem.

[0003] Flavonoids play an important role in the anti-nematode response of plants. Quercetin is a natural flavonoid with various biological activities such as antioxidant, anti-inflammatory and antibacterial. Existing studies have shown that quercetin and its derivatives have been proven to have an inhibitory effect on root-knot nematodes. Quercetin 7-O-β-D-glucuronide is a metabolite of quercetin, and materials with high content of it usually show stronger antioxidant activity and the ability to resist root-knot nematode stress, and are suitable for planting in areas with high incidence of root-knot nematodes to improve crop resistance.

[0004] In traditional cassava breeding, breeders mainly select individual plants with excellent traits, fix the excellent traits through hybridization or backcrossing, and then carry out the breeding of excellent seedlings through asexual reproduction. At present, to obtain a new cassava variety with high quercetin 7-O-β-D-glucuronide content, it is necessary to wait for the plants to reach adulthood and then investigate the quercetin 7-O-β-D-glucuronide content level in their roots to screen excellent plants. Due to the lack of molecular markers significantly associated with the content of quercetin 7-O-β-D-glucuronide, this breeding method has a long breeding time, high cost and low efficiency. Summary of the Invention

[0005] In view of the above problems, the present invention provides a molecular marker related to the content of quercetin 7-O-β-D-glucuronic acid associated with resistance to root-knot nematode disease and its application.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A molecular marker related to the content of quercetin 7-O-β-D-glucuronic acid associated with resistance to root-knot nematode disease, wherein the molecular marker is an SV molecular marker, and the SV molecular marker is a large fragment of bases 2657774 to 2659338 on chromosome 14 of the cassava genome, with a sequence length of 1565 bp, named Chr14_2657774_del_1565, and the sequence is as shown in SEQ ID NO: 1; The content of quercetin 7-O-β-D-glucuronic acid in cassava carrying the SV molecular marker is significantly higher (i.e., greater than) that in cassava lacking the SV molecular marker (i.e., the large fragment deletion occurring at bases 2657774 to 2659338 on chromosome 14). That is, the content of quercetin 7-O-β-D-glucuronic acid in cassava carrying this SV molecular marker is relatively high, and it is cassava with a high content of quercetin 7-O-β-D-glucuronic acid; The content of quercetin 7-O-β-D-glucuronic acid in cassava lacking this SV molecular marker is relatively low, and it is cassava with a low content of quercetin 7-O-β-D-glucuronic acid.

[0007] An application of a reagent for detecting the above molecular marker related to the content of quercetin 7-O-β-D-glucuronic acid associated with resistance to root-knot nematode disease, wherein the application is to detect whether the cassava carries the SV molecular marker through the reagent to predict whether the content of quercetin 7-O-β-D-glucuronic acid in the cassava is high or low.

[0008] A primer set for detecting the above molecular marker related to the content of quercetin 7-O-β-D-glucuronic acid associated with resistance to root-knot nematode disease, wherein the primer set is a set of primers designed using the above SV molecular marker, that is, the primers are designed according to nucleotides 2657774 to 2659338 on chromosome 14 of the cassava genome, including: The upstream primer sequence is 5'-ATGATCCGAGATCCAATG-3'; The downstream primer sequence is 5'-GTTACCCAAATGTGATGC-3'.

[0009] A kit including the above primer set.

[0010] Furthermore, the kit includes reagents for PCR amplification.

[0011] Furthermore, the kit includes: 2× Rapid Taq Master Mix and ddH2O.

[0012] A method for identifying high or low quercetin 7-O-β-D-glucuronide content in cassava. The method uses the above primer set with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, sequence the PCR amplification product, and determine whether the SV molecular marker is carried to predict high or low quercetin 7-O-β-D-glucuronide content in cassava. Alternatively, use the above kit with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, sequence the PCR amplification product, and determine whether the SV molecular marker is carried to predict high or low quercetin 7-O-β-D-glucuronide content in cassava. When the nucleotide sequence of the PCR amplification product is as shown in SEQ ID NO: 1, with a length of 1565 bp, this sequence is the DNA fragment represented by the SV molecular marker, that is, the sequence within the 1-1565 coordinate range from the 5' end of the SV molecular marker, then the cassava carries this SV molecular marker, and this cassava has a relatively high quercetin 7-O-β-D-glucuronide content and is a cassava with high quercetin 7-O-β-D-glucuronide content. Otherwise, the cassava lacks this SV molecular marker, that is, a large fragment deletion occurred at the 2657774-2659338 bases on chromosome 14 in the sample genome, and there is no amplification product in the PCR amplification, then this cassava has a relatively low quercetin 7-O-β-D-glucuronide content and is a cassava with low quercetin 7-O-β-D-glucuronide content.

[0013] Furthermore, the PCR amplification system includes: 12.5 μL of 2× Rapid Taq Master Mix, 1 μL of the upstream primer with a concentration of 10 μM, 1 μL of the downstream primer with a concentration of 10 μM, 1 μL of cassava genomic DNA, and 9.5 μL of ddH2O. The PCR amplification reaction program is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 5 min.

[0014] A method for identifying high or low quercetin 7-O-β-D-glucuronide content in cassava. The method uses the above primer set with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, perform electrophoresis detection on the PCR amplification product, and interpret the electrophoresis detection result to determine whether the SV molecular marker is carried to predict the quercetin 7-O-β-D-glucuronide content in cassava. Alternatively, using the above kit with cassava genomic DNA as a template, perform PCR amplification on the SV molecular marker, subject the PCR amplification product to electrophoresis detection, and interpret the electrophoresis detection result to determine whether the SV molecular marker is carried, so as to predict whether the quercetin 7-O-β-D-glucuronide content in cassava is high or low; When only a 1565bp band appears in the electrophoresis detection result, the cassava carries this SV molecular marker, and the quercetin 7-O-β-D-glucuronide content of this cassava is relatively high, being a cassava with a high quercetin 7-O-β-D-glucuronide content; Otherwise, when there is no band in the electrophoresis detection result, the cassava lacks this SV molecular marker, and the quercetin 7-O-β-D-glucuronide content of this cassava is relatively low, being a cassava with a low quercetin 7-O-β-D-glucuronide content.

[0015] Furthermore, the PCR amplification system includes: 12.5 μL of 2× Rapid Taq Master Mix, 1 μL of the upstream primer with a concentration of 10 μM, 1 μL of the downstream primer with a concentration of 10 μM, 1 μL of cassava genomic DNA, and 9.5 μL of ddH2O; The PCR amplification reaction program is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 5 min.

[0016] The beneficial effects of the molecular marker related to the high quercetin 7-O-β-D-glucuronide content and its application in the present invention related to resistance to Meloidogyne incognita are as follows: A molecular marker related to the high quercetin 7-O-β-D-glucuronide content in the present invention related to resistance to Meloidogyne incognita solves the problems of long breeding time, high breeding cost, and low efficiency in the breeding of new cassava varieties related to the quercetin 7-O-β-D-glucuronide content index in the prior art; this SV molecular marker occurs at the 2657774th base of chromosome 14 (Chr14) of the cassava genome, and the sequence deletion length is 1565 bp, named Chr14_2657774_del_1565; this SV molecular marker can accurately and efficiently predict whether the quercetin 7-O-β-D-glucuronide content in the fibrous roots and tuberous roots of cassava is high or low, greatly improving the efficiency of breeding for cassava with a high quercetin 7-O-β-D-glucuronide content resistant to Meloidogyne incognita and reducing costs; Using this SV molecular marker, it is possible to accurately and efficiently predict whether the quercetin 7-O-β-D-glucuronide content in the fibrous roots and tuberous roots of cassava is high or low by whether the leaf DNA carries the target molecular marker at the seedling stage of cassava. Excellent plants with high content can be quickly screened out at the seedling stage of cassava, greatly improving the selection efficiency of cassava breeding, and can efficiently assist the breeding selection of cassava, with extremely high economic value; The present invention has developed a molecular marker related to the content of quercetin 7-O-β-D-glucuronide associated with resistance to root-knot nematodes, and established a method for accurately and rapidly identifying high or low quercetin 7-O-β-D-glucuronide content in cassava, providing a more effective theoretical and practical basis for molecular marker-assisted breeding of new cassava varieties with specific quercetin 7-O-β-D-glucuronide content levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the Manhattan plot of GWAS signals for the trait data of the relative content of quercetin 7-O-β-D-glucuronide in 337 cassava samples in Example 1 of the present invention, which is located by genome-wide chromosome association analysis; Figure 2 is the T-test result of the relative content level of quercetin 7-O-β-D-glucuronide in 207 cassava samples in Example 5 of the present invention; among them, REF represents cassava carrying the SV molecular marker, n = 113 represents the sample size of 113; SV represents cassava lacking the SV molecular marker, n = 94 represents the sample size of 94. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments for those skilled in the art to understand.

[0019] Example 1 Obtaining a molecular marker related to the content of quercetin 7-O-β-D-glucuronide associated with resistance to root-knot nematodes Collect 337 fresh leaf samples of cassava planted in Danzhou City, Hainan Province, China (109.5° east longitude, 19.5° north latitude), extract genomic DNA for whole-genome sequencing. Align the high-quality sequencing data after quality control to the cassava reference genome, and perform genome-wide association analysis (GWAS) using the obtained cassava whole-genome SV molecular marker map and the phenotypic data of the relative content of quercetin 7-O-β-D-glucuronide in cassava germplasm resources. The results are as Figure 1 shown. It can be seen that there are significant association signals (indicated by the arrow) with the content level of quercetin 7-O-β-D-glucuronide in the cassava genome, including 1 SV molecular marker (Chr14_2657774_del_1565).

[0020] The molecular marker related to the resistance to root-knot nematode disease with high quercetin 7-O-β-D-glucuronide content is a large fragment of bases 2657774 to 2659338 on chromosome 14 of the cassava genome, with a sequence length of 1565 bp, named Chr14_2657774_del_1565, and the sequence is as shown in SEQ ID NO: 1. Using this SV molecular marker to detect cassava breeding materials, the quercetin 7-O-β-D-glucuronide content of cassava carrying the SV molecular marker is significantly higher than that of cassava lacking the SV molecular marker (i.e., the large fragment deletion occurring at bases 2657774 to 2659338 on chromosome 14).

[0021] Therefore, using this SV molecular marker, it is possible to accurately and efficiently predict whether the quercetin 7-O-β-D-glucuronide content in the fibrous roots and tuberous roots of cassava is high or low by detecting whether the leaf DNA carries the target molecular marker at the seedling stage of cassava. Excellent plants with high content can be quickly screened out at the seedling stage of cassava, greatly improving the selection efficiency of cassava breeding, and can efficiently assist the breeding selection of cassava, with extremely high economic value.

[0022] Example 2 Primer set and kit for identifying high or low quercetin 7-O-β-D-glucuronide content Using the characteristics of the molecular marker related to the resistance to root-knot nematode disease with high quercetin 7-O-β-D-glucuronide content in Example 1 on the cassava genome, a set of primers was designed (that is, the primers were designed according to nucleotides 2657774 to 2659338 on chromosome 14 of the cassava genome), specifically as follows: The upstream primer sequence is 5'-ATGATCCGAGATCCAATG-3'; The downstream primer sequence is 5'-GTTACCCAAATGTGATGC-3'.

[0023] When designing the primers, in order to enhance the applicability and sensitivity of the primers, the designed primer length is between 18 and 25 bp, and the primers do not interfere with each other. The above 2 primers can be obtained by artificial synthesis.

[0024] This example also provides a kit for identifying high or low quercetin 7-O-β-D-glucuronide content in cassava. The kit includes the above primer set.

[0025] The kit also includes conventional reagents for PCR amplification, such as 2× Rapid Taq Master Mix and ddH2O.

[0026] The primer set designed by the present invention has strong specificity and can accurately amplify the sequence carrying the SV molecular marker of the present invention.

[0027] Using the primer set or kit of the present invention, the high or low content of quercetin 7-O-β-D-glucuronide in cassava can be accurately and efficiently predicted at the seedling stage of cassava.

[0028] Example 3 Method for identifying high or low content of quercetin 7-O-β-D-glucuronide in cassava This example provides a method for identifying high or low content of quercetin 7-O-β-D-glucuronide in cassava. The specific method includes the following steps: S1. Extract the genomic DNA of the cassava to be tested (at the seedling stage of cassava) as the PCR template; S2. Using the primer set in Example 2, with the cassava genomic DNA as the template, perform PCR amplification on the SV molecular marker; The reaction system for PCR amplification is: 12.5 μL of 2× Rapid Taq Master Mix, 1 μL of the upstream primer with a concentration of 10 μM, 1 μL of the downstream primer with a concentration of 10 μM, 1 μL of cassava genomic DNA, and 9.5 μL of ddH2O. Among them, Mix (amplification buffer) is purchased from Novoprotein Scientific Inc., and the primer set is synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.

[0029] The reaction program for PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 5 min.

[0030] S3. Sequence the obtained PCR amplification product to determine whether it carries the SV molecular marker fragment.

[0031] When the nucleotide sequence of the PCR amplification product is as shown in SEQ ID NO: 1, with a length of 1565 bp, this sequence is the DNA fragment represented by the SV molecular marker, that is, the sequence within the 1-1565 coordinate range from the 5' end of the SV molecular marker, then the cassava to be tested carries this SV molecular marker, and the content of quercetin 7-O-β-D-glucuronide in this cassava to be tested is relatively high, and it is a cassava with a high content of quercetin 7-O-β-D-glucuronide.

[0032] Otherwise, the cassava to be tested lacks this SV molecular marker, that is, a large fragment deletion occurs at the 2657774-2659338 bases of chromosome 14 in the sample genome, and there is no amplification product in PCR amplification, then the content of quercetin 7-O-β-D-glucuronide in this cassava to be tested is relatively low, and it is a cassava with a low content of quercetin 7-O-β-D-glucuronide.

[0033] Alternatively, perform electrophoresis detection on the PCR amplification product and interpret the electrophoresis detection result; When only a 1565bp band appears in the electrophoresis detection result, the cassava to be tested carries this SV molecular marker, and the content of quercetin 7-O-β-D-glucuronide in this cassava to be tested is relatively high, being a cassava with a high content of quercetin 7-O-β-D-glucuronide; Otherwise, when there is no band in the electrophoresis detection result, the cassava to be tested lacks this SV molecular marker, and the content of quercetin 7-O-β-D-glucuronide in this cassava to be tested is relatively low, being a cassava with a low content of quercetin 7-O-β-D-glucuronide.

[0034] Among them, the relative content of quercetin 7-O-β-D-glucuronide in cassava with a high content of quercetin 7-O-β-D-glucuronide is generally ≥10416; The relative content of quercetin 7-O-β-D-glucuronide in cassava with a low content of quercetin 7-O-β-D-glucuronide is generally <10416.

[0035] Example 4 Detection and verification of SV molecular markers in cassava To verify the practicality of this SV molecular marker, in the cassava planting area of Danzhou City, Hainan Province, China (109.5° east longitude, 19.5° north latitude), several cassava plants were randomly selected (excluding 337 cassava plants used for the development of SV molecular markers), and the method in Example 3 was used to determine whether they carried the SV molecular marker by PCR amplification, and to measure the relative content of quercetin 7-O-β-D-glucuronide in these cassava samples. The sequencing results are shown in Tables 1 - 2 below.

[0036] Table 1 Whether 20 cassava plants carry the SV molecular marker and the relative content of quercetin 7-O-β-D-glucuronide

[0037] Table 2 Statistical table of whether 20 cassava plants carry the SV molecular marker and the relative content of quercetin 7-O-β-D-glucuronide

[0038] It can be seen from Tables 1 to 2 that the method of the present invention can identify that the relative content of quercetin 7-O-β-D-glucuronide in cassava carrying this SV molecular marker is relatively high, being a cassava with a high content of quercetin 7-O-β-D-glucuronide; the relative content of quercetin 7-O-β-D-glucuronide in cassava lacking the SV molecular marker is relatively low, being a cassava with a low content of quercetin 7-O-β-D-glucuronide, indicating that the method of the present invention can be used to identify whether the content of quercetin 7-O-β-D-glucuronide in cassava is high or low.

[0039] Example 5 Detection of SV molecular markers in cassava Furthermore, the method in Example 3 was used to genotype 207 cassava samples, and the results are asFigure 2 As shown, among them, 113 samples carry the SV molecular marker, and 94 samples lack the SV molecular marker. The relative content levels of quercetin 7-O-β-D-glucuronide in these samples were detected, and a T-test was performed. The results showed that the P value was 0.000001, that is, the relative content of quercetin 7-O-β-D-glucuronide in cassava carrying the SV molecular marker was higher than that in cassava lacking the SV molecular marker, and there was a significant difference.

[0040] Other parts not described in detail are all prior art. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Those of ordinary skill in the art can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A molecular marker related to the content of quercetin 7-O-β-D-glucuronide associated with resistance to root-knot nematode disease, characterized in that, The molecular marker is an SV molecular marker, and the sequence of the SV molecular marker is shown as SEQ ID NO: 1; The content of quercetin 7-O-β-D-glucuronide in cassava carrying the SV molecular marker is greater than that in cassava lacking the SV molecular marker.

2. An application of a reagent for detecting the molecular marker related to the content of quercetin 7-O-β-D-glucuronide associated with resistance to root-knot nematode disease according to claim 1, characterized in that, The application is to detect whether the cassava carries the SV molecular marker through the reagent to predict whether the content of quercetin 7-O-β-D-glucuronide in the cassava is high or low.

3. A primer set for detecting the molecular marker related to the content of quercetin 7-O-β-D-glucuronide associated with resistance to root-knot nematode disease according to claim 1, characterized in that, The primer set includes: The upstream primer sequence is 5'-ATGATCCGAGATCCAATG-3'; The downstream primer sequence is 5'-GTTACCCAAATGTGATGC-3'.

4. A kit comprising the primer set according to claim 3.

5. The kit according to claim 4, characterized in that, The kit includes reagents for PCR amplification.

6. The kit according to claim 5, characterized in that, The kit includes: 2× Rapid TaqMaster Mix and ddH2O.

7. A method for identifying high or low content of quercetin 7-O-β-D-glucuronide in cassava, characterized in that, The method is to use the primer set described in claim 3 with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, sequence the PCR amplification product, and determine whether the SV molecular marker is carried to predict whether the content of quercetin 7-O-β-D-glucuronide in the cassava is high or low; Alternatively, use the kit described in any one of claims 4-6 with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, sequence the PCR amplification product, and determine whether the SV molecular marker is carried to predict whether the content of quercetin 7-O-β-D-glucuronide in the cassava is high or low; When the length of the PCR amplification product is 1565bp, the cassava is a cassava with a high content of quercetin 7-O-β-D-glucuronide; If there is no amplification product in the PCR amplification, the cassava is a cassava with a low content of quercetin 7-O-β-D-glucuronide.

8. The method for identifying the content of quercetin 7-O-β-D-glucuronide in cassava according to claim 7, characterized in that, The PCR amplification system includes: 2× Rapid Taq Master Mix, the upstream primer, the downstream primer, cassava genomic DNA and ddH2O; The reaction procedure of PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min.

9. A method for identifying high or low content of quercetin 7-O-β-D-glucuronide in cassava, characterized in that, The method is to use the primer set described in claim 3 with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, perform electrophoresis detection on the PCR amplification product, and interpret the electrophoresis detection result to determine whether the SV molecular marker is carried to predict whether the content of quercetin 7-O-β-D-glucuronide in the cassava is high or low; Alternatively, use the kit described in any one of claims 4-6 with cassava genomic DNA as a template to perform PCR amplification on the SV molecular marker, perform electrophoresis detection on the PCR amplification product, and interpret the electrophoresis detection result to determine whether the SV molecular marker is carried to predict whether the content of quercetin 7-O-β-D-glucuronide in the cassava is high or low; When only a 1565bp band appears in the electrophoresis detection result, the cassava carries the SV molecular marker, and the cassava is a cassava with a high content of quercetin 7-O-β-D-glucuronide; When there is no band in the electrophoresis detection result, the cassava lacks this SV molecular marker, and this cassava is a cassava with low quercetin 7-O-β-D-glucuronide content.

10. The method for identifying the content of quercetin 7-O-β-D-glucuronide in cassava according to claim 9, characterized in that, The system for PCR amplification includes: 2× Rapid Taq Master Mix, the upstream primer, the downstream primer, cassava genomic DNA, and ddH2O; The reaction program for PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, for 35 cycles; 72°C for 5 min.

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