Linkage Molecular Markers of Major QTL for Resistance to White-backed Planthopper in Rice and Their Application

Abstract

The present invention discloses a molecular marker linked to a major QTL regulating rice whiteback whitefly resistance. The QTL is located on rice chromosome 1, with a genetic distance of 30.88-34.32 cM and a physical distance of 7203302-8007653 bp. Molecular markers are then set on both sides of the QTL, and the molecular markers are used to screen rice varieties resistant to whiteback whiteflies, thereby improving the efficiency of screening for ideal rice plant types.

Description

Technical Field

[0001] The invention relates to the technical field of rice whitebacked whitefly molecular markers, and more particularly to a molecular marker linked to a major effect QTL of rice whitebacked whitefly resistance and an application thereof. Background Art

[0002] Rice is one of the world's most important food crops, and its safe production is of great significance to ensuring food security. However, rice production is threatened by various pests and diseases all year round. Among them, the white-backed planthopper is widely distributed in East Asia, South Asia, and Southeast Asia, and is one of the main pests of rice. As a large-scale migratory pest, its occurrence is sudden and explosive, which increases the difficulty of controlling the white-backed planthopper. The white-backed planthopper is a piercing-sucking pest that sucks the phloem sap of rice stalks through its mouth needle, resulting in slowed plant growth, yellowing of leaves, and in severe cases, the entire plant withering and death. In addition, the white-backed planthopper is also the vector of southern rice black streaked dwarf disease. SRBSDV is a viral disease with no drug treatment and is known as "rice cancer". It seriously threatens rice production in East Asia and Southeast Asia.

[0003] Currently, chemical pesticides are the primary control measure for white-backed planthoppers. However, excessive use not only increases production costs, pollutes the environment, and produces pesticide residues, but also kills natural enemies, disrupts ecological balance, and leads to the development of pesticide resistance in pests, causing resurgence. Therefore, breeding and promoting resistant varieties is considered the most economical and effective approach to controlling white-backed planthoppers. Identifying sources of resistance and genes responsible for white-backed planthopper resistance is a prerequisite and foundation for breeding resistant varieties. To date, several genes or QTLs for white-backed planthopper resistance have been reported. However, only qWL6 and qWBPH11 have been finely mapped; no map-based cloning of white-backed planthopper resistance genes has been reported. The mechanisms of white-backed planthopper resistance in rice remain largely unknown.

[0004] Therefore, developing a new QTL-linked molecular marker for rice resistance to white-backed planthoppers and further applying it to breed superior rice varieties is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a major QTL-linked molecular marker for rice resistance to white-backed planthoppers, which can be used to breed rice resistant to white-backed planthoppers, thereby improving rice breeding efficiency.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Molecular markers linked to the major QTL regulating resistance to white-backed whitefly in rice, the rice molecular markers include IndelWbph-1 and Indel Wbph-2;

[0008] Among them, the primer pair for the molecular marker Indel Wbph-1 is:

[0009] Upstream primer: 5′-AGGAGACCGCCGTTTTTGAT-3′, SEQ ID NO. 1;

[0010] Downstream primer: 5′-AAGCGTTTCGTTTCGGTTGG-3′, SEQ ID NO. 2;

[0011] The primer pair for the molecular marker Indel Wbph-2 is:

[0012] Upstream primer: 5′-CTATGGCTACCGCGACGTAC-3′, SEQ ID NO. 3;

[0013] Downstream primer: 5'-GCTGCTCACGTCGACGAC-3', SEQ ID NO.4.

[0014] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the aforementioned QTL-linked molecular marker for regulating rice whitebacked whitefly resistance in breeding whitebacked planthopper-resistant rice varieties.

[0015] As an inventive concept identical to the above technical solution, the present invention also seeks protection for a method for breeding white-backed planthopper-resistant rice varieties, the process comprising:

[0016] Extract rice DNA, perform PCR amplification on the DNA using the molecular marker primer pair described in claim 1, perform electrophoresis detection on the amplified product, and analyze the resistance of rice to white-backed planthopper by banding pattern.

[0017] Preferably, the reaction system for PCR amplification is: 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of DNA template, 6 μL of mix enzyme, and 1 μL of ddH2O; the reaction procedure for PCR amplification is:

[0018] Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, amplification for 38 cycles; and final extension at 72°C for 10 min.

[0019] As an inventive concept identical to the above technical solution, the present invention also seeks protection for a kit for breeding white-backed planthopper-resistant rice varieties, comprising the primer pair of the molecular marker.

[0020] Through the above technical solution, it can be seen that compared with the existing technology, the present invention locates the main effect QTL that regulates rice resistance to white-backed planthoppers, which is located on rice chromosome 1, with a genetic distance of 30.88-34.32 cM and a physical distance of 7203302-8007653 bp. The QTL site is used to obtain two pairs of molecular markers tightly linked to it. These molecular markers can be used to predict rice resistance to white-backed planthoppers and accelerate the breeding of ideal rice plant types. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 Shown is a flowchart for constructing genetic materials used in the process of mapping the major QTL regulating resistance to white-backed planthopper in rice;

[0023] Figure 2 Shown is the frequency distribution of white-backed planthopper resistance in the RIL population; Nekken represents the rice variety Reyan 2, and HZ represents the rice variety Huazhan;

[0024] Figure 3 Shown is the location of the major QTL qWBPH1.1 regulating resistance to white-backed planthopper in rice on chromosome 1;

[0025] Figure 4 Shown are electrophoretic patterns generated by amplification of the primer pair for the molecular marker IndelWbph-1 in the parental rice, its F1 generation, and RIL population; wherein, 1 is Huazhan, 2 is Reyan 2, 3 is the F1 offspring of the Reyan 2 / Huazhan hybrid, and 4-12 are rice lines with higher resistance in the RIL population of the Reyan 2 / Huazhan hybrid combination;

[0026] Figure 5 Shown are electrophoretic patterns generated by amplification of the primer pair for the molecular marker IndelWbph-2 in the parental rice, its F1 generation, and RIL population; 1 is Huazhan, 2 is Reyan 2, 3 is the F1 offspring of the Reyan 2 / Huazhan hybrid (with resistance biased towards the male parent Huazhan), and 4-12 are rice lines with higher resistance in the RIL population of the Reyan 2 / Huazhan hybrid combination;

[0027] Figure 6Shown are electropherograms generated by amplifying the molecular marker IndelWbph-1 using the primer pair in the parental lines Huazhan and Nipponbare, their F1 offspring, and the BC3F1 generation with Nipponbare as the recurrent parent. Figure 1 shows Huazhan, 2 shows Nipponbare, 3 shows the F1 offspring of the Huazhan / Nipponbare hybrid, and 4-10 show electropherograms generated by amplifying the BC3F1 generation with Nipponbare as the recurrent parent.

[0028] Figure 7 Shown are electropherograms generated by amplifying the molecular marker IndelWbph-2 using the primer pair from the parental lines Huazhan and Nipponbare, their F1 generations, and the BC3F1 generation with Nipponbare as the recurrent parent. Figure 1 represents Huazhan, 2 represents Nipponbare, 3 represents the F1 hybrid offspring of Huazhan / Nipponbare, and 4-10 represent electropherograms generated by amplifying the BC3F1 generation with Nipponbare as the recurrent parent. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Example 1 Mapping of the major QTL regulating resistance to white-backed planthopper in rice

[0031] 1. Acquisition of Experimental Materials

[0032] Reyan 2 was used as the donor parent and Huazhan rice was used as the recipient parent for hybridization. The single seed method (i.e., the F1 was bagged and seeded individually until the phenotype of the offspring lines did not segregate) was used to obtain 120 stable genetic lines (F12, all lines were phenotypically stable), which constituted the recombinant inbred line (RIL) population. Figure 1 .

[0033] Sixty seeds each of the parent and each strain (F12) were selected, surface disinfected, soaked for two days, wrapped in a damp towel, and placed in a 37°C incubator for 48 hours to accelerate germination. Seeds that showed consistent whitening were then sown. Thirty days later, 24 seedlings of the parent and each strain with similar growth conditions were selected and transplanted. All rice materials were planted in the experimental fields of the College of Biochemistry, Zhejiang Normal University, Jinhua, Zhejiang Province, and managed as usual.

[0034] 2. Determination of resistance data of white-backed planthopper

[0035] The international standard seedling screening method (Standard Seed-box Screening Test, SSST method) was used to identify rice resistance to white-backed planthoppers.

[0036] The results are as follows Figure 2 As shown, the resistance data of white-backed planthopper showed a continuous normal distribution with a wide range, and there were many super-parent individuals (level 1, high resistance), showing the genetic characteristics of quantitative traits.

[0037] 3. QTL Mapping Analysis

[0038] Using a genetic map constructed using a large number of SNP and Indel markers developed earlier in the laboratory, quantitative trait loci (QTL) interval mapping was performed for resistance to white-backed planthoppers in rice. Using R-QTL professional software, the relationships between markers and quantitative trait phenotypic values ​​across the entire genome were analyzed. QTLs were individually located on linkage groups and their genetic effects were estimated. If a molecular marker with a LOD > 2.5 was detected, a QTL was considered to exist between the two markers corresponding to the highest LOD value.

[0039] Finally, a major QTL for resistance to white-backed planthoppers was found between the Indel Wbph-1 and Indel Wbph-2 markers on chromosome 1 in the entire chromosome set of rice Huazhan. The LOD value was as high as 3.32, the genetic distance was 30.88-34.32 cM, and the physical distance was 7203302-8007653 bp. It was named qWBPH1.1 ( Figure 3 ).

[0040] Example 2 Molecular marker-assisted selection

[0041] Molecular markers Indel Wbph-1 and IndelWbph-2 were set up upstream and downstream of QTL locus qWBPH1.1, and primers were designed.

[0042] The primer pair for the molecular marker Indel Wbph-1 is:

[0043] Upstream primer: 5′-AGGAGACCGCCGTTTTTGAT-3′, SEQ ID NO. 1;

[0044] Downstream primer: 5'-AAGCGTTTCGTTTCGGTTGG-3', SEQ ID NO.2

[0045] The primer pair for the molecular marker Indel Wbph-2 is:

[0046] Upstream primer: 5′-CTATGGCTACCGCGACGTAC-3′, SEQ ID NO. 3;

[0047] Downstream primer: 5'-GCTGCTCACGTCGACGAC-3', SEQ ID NO.4.

[0048] The rice leaves of parental rice varieties Reyan 2, Huazhan, their F1 generation and RIL population were taken to extract genomic DNA, and the genomic DNA was amplified by PCR using the above molecular markers.

[0049] PCR reaction system: upstream primer (10 μmol) 1 μL, downstream primer (10 μmol) 1 μL, DNA template (>100 ng / μL) 2 μL, mix enzyme (Qingke Biotechnology, 2× Taq MasterMix) 6 μL, ddH2O 1 μL;

[0050] The reaction procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, amplification for 38 cycles; and final extension at 72°C for 10 min.

[0051] The PCR amplification products were detected by 4% agarose gel electrophoresis. Some of the results are shown in the figure. Figure 4-Figure 5 shown.

[0052] The band patterns of the electrophoresis detection bands were analyzed. If the bands tended towards the parent Huazhan, it indicated that the rice line had good resistance to white-backed planthoppers. If they tended towards Reyan 2, it indicated that the resistance was poor.

[0053] The resistance of the tested rice lines to white-backed planthoppers was compared with the results predicted by banding analysis, which showed that the predicted results were consistent with the actual test results.

[0054] Example 3 Application of QTLs Related to Rice White-backed Planthopper Resistance in Rice Breeding

[0055] The less resistant rice variety, Nipponbare, was hybridized with Huazhan, the female parent, to obtain the corresponding F1 line. Backcrosses were then performed using Nipponbare as the recurrent parent, resulting in the BC3F1 generation. DNA from individual plants in the BC3F1 generation was extracted and amplified using PCR using primers targeting Indel Wbph-1 and Indel Wbph-2, followed by electrophoresis.

[0056] The band patterns of electrophoresis detection bands were analyzed. If the bands tended to be similar to those of its parent, Huazhan, it indicated that the rice strain had good resistance to white-backed planthopper. Figure 6 and Figure 7 By using this method for screening and targeted selection, rice with strong resistance to white-backed planthoppers and retaining the excellent traits of Nipponbare can be obtained, greatly improving breeding efficiency.

[0057] In summary, the major QTL for regulating resistance to white-backed planthoppers in rice disclosed herein can effectively accelerate the process of optimizing rice varieties. During molecular-assisted rice breeding, rice with greater resistance to white-backed planthoppers can be cultivated while simultaneously optimizing rice quality and yield. This method is simple, safe, and effective, contributing to improving the economic value of rice varieties while balancing economic and ecological benefits, making it suitable for large-scale application.

[0058] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0059] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for breeding white-backed planthopper-resistant rice varieties, characterized in that the process include: Rice DNA was extracted and PCR amplified using molecularly labeled primers. The amplified products were then analyzed by electrophoresis. Banding patterns were used to analyze the rice's resistance to white-backed planthoppers. If the bands resembled those of its parent, Huazhan, the rice line had good resistance to white-backed planthoppers. If the bands resembled those of Reyan 2, the resistance was poor. Molecular markers include IndelWbph-1 and IndelWbph-2; Among them, the primer pair for the molecular marker IndelWbph-1 is: Upstream primer: 5′-AGGAGACCGCCGTTTTTGAT-3′, SEQ ID NO. 1; Downstream primer: 5′-AAGCGTTTCGTTTCGGTTGG-3′, SEQ ID NO. 2; The primer pair for the molecular marker IndelWbph-2 is: Upstream primer: 5′-CTATGGCTACCGCGACGTAC-3′, SEQ ID NO. 3; Downstream primer: 5'-GCTGCTCACGTCGACGAC-3', SEQ ID NO.

4.

2. The method for breeding a white-backed planthopper-resistant rice variety according to claim 1, characterized in that: The reaction system for PCR amplification is: 1 μL upstream primer, 1 μL downstream primer, 2 μL DNA template, 6 μL mix enzyme, 1 μL ddH2O; the reaction procedure for PCR amplification is: Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, amplification for 38 cycles; and final extension at 72°C for 10 min.

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