Major QTL for regulating and controlling cold tolerance of rice in seedling stage, molecular marker and application

Abstract

The invention discloses a major QTL for regulating and controlling the cold tolerance of rice in the seedling stage, a molecular marker and application. Belongs to the technical field of rice breeding and molecular biology. The invention provides the major QTL for regulating and controlling the cold resistance of the rice in the seedling stage and the molecular marker thereof, the molecular marker is applied to breeding of the rice with strong cold resistance in the seedling stage, rice offspring can be screened, and the breeding efficiency of the excellent cold-resistant rice is greatly improved while the cost is saved.

Description

Technical Field

[0001] The present invention relates to the technical field of rice breeding and molecular biology, and more particularly to a major QTL for regulating cold tolerance at the seedling stage of rice, molecular markers linked thereto, and applications thereof. Background Art

[0002] As one of the three major food crops in the world, rice is the most important food crop in China, and its yield is directly related to national food security and social stability. However, as a typical thermophilic crop, rice is often highly sensitive to low temperature stress. The seedling stage is a critical stage in the growth and development of rice. If low temperature stress occurs during this period, it will significantly inhibit the growth rate of rice, and in severe cases, it may even cause seedling death, resulting in a significant reduction in rice yield. Therefore, in-depth exploration of QTLs and major genes related to cold tolerance at the seedling stage of rice, so as to analyze the genetic regulatory network of cold tolerance in rice, will provide a theoretical basis and excellent germplasm resources for the improvement and breeding of cold-tolerant rice.

[0003] So far, certain research progress has been made on the genetic mechanism of cold tolerance at the seedling stage of rice by predecessors. Li et al. detected 12 QTLs related to seedling survival rate through genome-wide association studies, among which qSR9 is related to the stem length growth rate under cold stress. Liu et al. used the RILs population constructed from japonica rice variety 'Zhonghua 11' and cold-tolerant indica rice 'IR26' to map a major QTL CTB4a on chromosome 4. This locus may contain the coding gene of carotenoid hydroxylase and maintain the stability of chloroplasts under low temperature. Maiko et al. used the F2 population constructed from cold-tolerant wild rice 'W1943' and cold-sensitive indica rice variety 'Guangluai 4' to detect a total of 3 cold tolerance QTLs at the seedling stage on chromosomes 3, 10, and 11. Among them, the LOD value of the qCtss11 locus is as high as 17.8, which can explain about 40% of the phenotypic variation. Wang et al. used the F2 population constructed from cold-tolerant variety 'Kunmingxiaobaigu' and cold-sensitive variety 'Nipponbare' as experimental materials, combined with BSA-seq and linkage analysis, and detected the OsMAPK3 gene on chromosome 8. This gene regulates the expression of downstream cold-responsive genes by phosphorylating the transcription factor OsbZIP46, inducing the cold stress response pathway in rice.

[0004] At present, the research on the genetic regulatory mechanism of cold tolerance at the seedling stage of rice is still not deep enough, especially the key genes involved in the low temperature stress response and their interaction networks have not been fully elucidated. Therefore, how to precisely map the major QTLs for cold tolerance at the seedling stage of rice and screen the molecular markers linked thereto has become an urgent problem 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 for regulating cold tolerance at the seedling stage of rice, molecular markers thereof, and applications.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A major QTL for regulating cold tolerance at the seedling stage of rice, the major QTL is located on chromosome 3 of rice, named qCTS-3, with a genetic distance of 11.37 - 21.63 cM and a physical distance of 2,652,824 - 5,046,260 bp.

[0008] The molecular markers of the above-mentioned major QTL for regulating cold tolerance at the seedling stage of rice, the molecular markers include two pairs of closely linked molecular markers Indel cts-1 and Indel cts-2; wherein,

[0009] The primer pair of the molecular marker Indel cts-1 is:

[0010] Upstream primer cts-1-F: 5'-ATTGGTTGTGGGTAGGCTGA-3', SEQ ID NO.1;

[0011] Downstream primer cts-1-R: 5'-TTGCGATGACTTGGGTTGAA-3', SEQ ID NO.2;

[0012] The primer pair of the molecular marker Indel cts-2 is:

[0013] Upstream primer cts-2-F: 5'-GGATGCTTTCGGTTATGGCC-3', SEQ ID NO.3;

[0014] Downstream primer cts-2-R: 5'-AGGATAGTGCAGGTTGTGGT-3', SEQ ID NO.4.

[0015] The application of the above-mentioned major QTL for regulating cold tolerance at the seedling stage of rice in selecting and breeding rice with strong cold tolerance at the seedling stage.

[0016] The application of the above-mentioned molecular markers in selecting and breeding rice with strong cold tolerance at the seedling stage.

[0017] A method for breeding rice with strong cold tolerance at the seedling stage, the process is:

[0018] Extract rice DNA, use the primer pairs of the above-mentioned molecular markers Indel cts-1 and Indel cts-2 to perform PCR amplification on the DNA, perform electrophoresis detection on the amplification products, and analyze the cold tolerance of rice through band patterns.

[0019] Furthermore,

[0020] The reaction system for PCR amplification is as follows: 10 μmol·L -1 0.75 μL of the upstream primer, 10 μmol·L -1 0.75 μL of the downstream primer, 1 μL of the DNA template, 12.5 μL of Mix enzyme, and 10 μL of ddH2O.

[0021] Furthermore,

[0022] The reaction program for PCR amplification is as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 5 s, 35 cycles of amplification, and finally final extension at 72°C for 1 min.

[0023] The present invention also simultaneously provides a kit for breeding rice with strong cold tolerance at the seedling stage, including the primer pairs of the above-mentioned molecular markers Indel cts-1 and Indel cts-2.

[0024] It can be seen from the above technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] The present invention uses a population of 120 recombinant inbred lines obtained by continuous self-crossing of the F1 generation of a cross between indica rice variety Huazhan as the male parent and japonica rice variety Reyan as the female parent. Using the dense genetic map constructed for this population, QTL mapping analysis is performed on the data, and a QTL with a LOD value as high as 5.20 is detected. The molecular marker-assisted breeding technology can effectively solve the problem of incomplete understanding of related genes. By constructing a genetic linkage map and quantitative trait locus (QTL) analysis, molecular markers tightly linked to the major QTL related to cold tolerance at the seedling stage can be effectively found. Using these markers, the rice offspring can be screened, which not only saves costs but also greatly improves the breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0027] Figure 1 It is a flowchart for constructing the genetic materials used in the process of mapping the major QTL for regulating cold tolerance at the seedling stage in Embodiment 1 of the present invention;

[0028] Figure 2This is the determination result of cold tolerance at the seedling stage of the RILs population in Example 1 of the present invention. Note: 'HZ' and 'Nekken2' marked in the figure represent the survival rates of these two varieties. At the actual survival rate, there are other strains;

[0029] Figure 3 This is the position of the major QTL qCTS-3 regulating cold tolerance at the seedling stage of rice on chromosome 3 in Example 1 of the present invention;

[0030] Figure 4 This is the electrophoresis pattern generated by the primer pair of the molecular marker Indel cts-1 in the parents, their F1 generation, and the RILs population in Example 2 of the present invention. Among them, 1 is Huazhan, 2 is Reyan, 3 is the F1 generation of the Huazhan / Reyan hybrid, and 4-10 are the RILs population of the Huazhan / Reyan hybrid combination;

[0031] Figure 5 This is the electrophoresis pattern generated by the primer pair of the molecular marker Indel cts-2 in the parents, their F1 generation, and the RILs population in Example 2 of the present invention. Among them, 1 is Huazhan, 2 is Reyan, 3 is the F1 generation of the Huazhan / Reyan hybrid, and 4-10 are the RILs population of the Huazhan / Reyan hybrid combination;

[0032] Figure 6 This is the electrophoresis pattern generated by the primer pair of the molecular marker Indel cts-1 in the parents and their BC3F1 generation in Example 3 of the present invention. Among them, 1 is 9311, 2 is Reyan, 3 is the F1 generation of the 9311 / Reyan hybrid, and 4-10 are the BC3F1 population of the 9311 / Reyan hybrid;

[0033] Figure 7 This is the electrophoresis pattern generated by the primer pair of the molecular marker Indel cts-2 in the parents and their BC3F1 generation in Example 3 of the present invention. Among them, 1 is 9311, 2 is Reyan, 3 is the F1 generation of the 9311 / Reyan hybrid, and 4-10 are the BC3F1 population of the 9311 / Reyan hybrid. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] The reagents required for the present invention are conventional experimental reagents and are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be elaborated here one by one.

[0036] Example 1

[0037] Mapping of Major QTL Controlling Cold Tolerance at the Seedling Stage in Rice

[0038] 1. Obtaining of Experimental Materials

[0039] Using Huazhan as the donor parent and the local rice variety Reyanshan as the recipient parent, RILs were constructed by hybridization. The single-seed descent method (i.e., bagging and single-plant seeding treatment for F1 until the phenotypes of the offspring lines did not segregate) was used, and finally 120 stable genetic lines (F 12 , with stable phenotypes for all lines) were obtained, as Figure 1 .

[0040] Select 100 seeds each of the parent and each line (F 12 ). After surface sterilization, soak the seeds for 2 days, then wrap them with a moist towel and place them in an incubator at 37°C for 48 h of germination. After that, select the seeds with consistent radicle emergence and transfer them to a 96-well hydroponic plate device for cultivation at 32°C.

[0041] 2. Determination of Cold Tolerance at the Seedling Stage in Rice

[0042] When the rice grew to the three-leaf stage, it was treated at 4°C for 4 days and then transferred to an incubator at 32°C for 5 days of recovery. Three biological replicates were set up to measure the seedling survival rates of Huazhan, Reyanshan and each line.

[0043] The results are as Figure 2 shown. The data of the seedling survival rate of rice showed a normal distribution and a wide range, with many transgressive individuals, showing the genetic characteristics of quantitative traits.

[0044] 3. QTL Mapping Analysis

[0045] Using the genetic map constructed with a large number of SNP and Indel markers developed in the early stage of the laboratory, quantitative trait locus (QTL) interval mapping was performed on the cold tolerance at the seedling stage in rice. The relationship between the markers in the whole genome and the phenotypic values of quantitative traits was analyzed by the professional software R-QTL. The QTLs were mapped to the corresponding positions on the linkage groups one by one, and their genetic effects were estimated. If a molecular marker with LOD > 2.5 was detected, it was considered that there was one QTL between the two markers corresponding to the highest LOD value.

[0046] Finally, in the whole genome, we found a major QTL between the Indel cts-1 marker and the Indel cts-2 marker located on chromosome 3. The LOD value of cold tolerance at the seedling stage was as high as 5.20. Its genetic distance was 11.37 - 21.63 cM, and the physical distance was 2652824 - 5046260 bp, and it was named qCTS-3, as Figure 3 shown.

[0047] Example 2

[0048] Molecular marker-assisted selection

[0049] Molecular markers Indel cts-1 and Indel cts-2 were respectively set upstream and downstream of the QTL locus qCTS-3, and primers were designed.

[0050] The primer pair for molecular marker Indel cts-1 is:

[0051] Forward primer: 5’-ATTGGTTGTGGGTAGGCTGA-3’, SEQ ID NO.1;

[0052] Reverse primer: 5’-TTGCGATGACTTGGGTTGAA-3’, SEQ ID NO.2;

[0053] The primer pair for molecular marker Indel cts-2 is:

[0054] Forward primer: 5’-GGATGCTTTCGGTTATGGCC-3’, SEQ ID NO.3;

[0055] Reverse primer: 5’-AGGATAGTGCAGGTTGTGGT-3’, SEQ ID NO.4.

[0056] The detection method is as follows:

[0057] Extract the genomic DNA of the rice leaves to be tested, and perform PCR amplification on its genomic DNA using the above molecular markers.

[0058] PCR reaction system: 10 μmol·L -1 Forward primer 0.75 μL, 10 μmol·L -1 Reverse primer 0.75 μL, DNA template 1 μL, Mix enzyme 12.5 μL, ddH2O 10 μL.

[0059] The reaction program is: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 20 s, annealing at 57 °C for 30 s, extension at 72 °C for 5 s, 35 cycles of amplification, and finally final extension at 72 °C for 1 min.

[0060] The PCR amplification products were detected by 4% agarose gel electrophoresis, and the band patterns of the electrophoresis detection were analyzed. Among them, if the bands of Indel cts-1 and Indel cts-2 both showed a tendency towards the parent Reyan (Indel cts-1: 165 bp; Indel cts-2: 209 bp), it indicated that the cold tolerance of this rice line at the seedling stage was relatively strong. If both showed a tendency towards Huazhan (Indel cts-1: 138 bp; Indel cts-2: 185 bp), it indicated that the cold tolerance at the seedling stage was relatively poor.

[0061] Specifically:

[0062] Seven rice lines with stable and excellent cold tolerance traits (the survival rate after cold stress at the seedling stage was 0.83 - 0.96) were selected from the parents Reyan, Huazhan, their F1 generation, and the RILs population of the Huazhan / Reyan hybrid combination. The DNA of these lines was extracted, and then PCR amplification was performed using the primers of Indel cts-1 and Indel cts-2.

[0063] The results were as Figure 4 、 Figure 5 shown. The bands of the seven lines in the RILs population of the Huazhan / Reyan hybrid combination tended towards Reyan, indicating that the excellent traits of strong cold tolerance at the seedling stage were retained. Comparing the results predicted by band pattern analysis with the cold tolerance of the tested rice lines at the seedling stage showed that the predicted results were consistent with the actual detection results.

[0064] Example 3

[0065] Application of QTL related to cold tolerance at the rice seedling stage in rice breeding

[0066] The molecular markers set in Example 2 can be applied to rice molecular assisted breeding. The specific implementation method is as follows: Other rice varieties with poor cold tolerance at the seedling stage, such as 9311, were crossed with Reyan to obtain the corresponding F1. Backcrossing was carried out with 9311 as the recurrent parent until the BC3F1 generation. The DNA of some individual plants in the BC3F1 generation was extracted, and then PCR amplification was performed using the primers of Indel cts-1 and Indel cts-2. The PCR amplification products were detected by 4% agarose gel electrophoresis, and the band patterns of the electrophoresis detection were analyzed. Among them, if the bands of Indel cts-1 and Indel cts-2 both showed a tendency towards the parent Reyan (Indel cts-1: 165 bp; Indel cts-2: 209 bp), it indicated that the cold tolerance of this rice line at the seedling stage was relatively strong. By using this method for screening and directional selection, rice with strong cold tolerance at the seedling stage and retaining the excellent traits of 9311 can be obtained, greatly improving the breeding efficiency.

[0067] In this laboratory, the rice variety 9311 with poor cold tolerance at the seedling stage was backcrossed with Reyan, and directional selection was carried out by screening using the above method. Seven different rice progeny lines with strong cold tolerance at the seedling stage and retaining the excellent traits of 9311 were obtained. Through Figure 6 and Figure 7 It can be seen that the bands of the seven selected rice lines tend to be Reyan, indicating that they retain the excellent trait of strong cold tolerance at the seedling stage. 30 plants of each line were planted to measure the survival rate after cold stress, and the survival rate after cold stress at the seedling stage was 0.80 - 0.93.

[0068] In summary, the major QTL for regulating cold tolerance at the rice seedling stage of the present invention can effectively increase the cold tolerance of rice, effectively improve the yield and quality of rice during the breeding process, and at the same time accelerate the process of optimizing rice varieties. This method is simple, easy to implement, safe and effective, beneficial to improving the economic value of rice varieties, taking into account economic and ecological benefits, and suitable for large-scale popularization and application.

[0069] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0070] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A major QTL regulating cold tolerance at the seedling stage of rice, characterized in that, The major-effect QTL is located on chromosome 3 of rice, named qCTS-3, with a genetic distance of 11.37 - 21.63 cM and a physical distance of 2652824 - 5046260 bp.

2. The molecular marker for the major QTL regulating cold tolerance at the seedling stage of rice according to claim 1, characterized in that, The molecular markers include two pairs of closely linked molecular markers Indel cts-1 and Indel cts-2; among them, The primer pair of the molecular marker Indel cts-1 is: Forward primer cts-1-F: 5’-ATTGGTTGTGGGTAGGCTGA-3’, SEQ ID NO.1; Reverse primer cts-1-R: 5’-TTGCGATGACTTGGGTTGAA-3’, SEQ ID NO.2; The primer pair of the molecular marker Indel cts-2 is: Forward primer cts-2-F: 5’-GGATGCTTTCGGTTATGGCC-3’, SEQ ID NO.3; Reverse primer cts-2-R: 5’-AGGATAGTGCAGGTTGTGGT-3’, SEQ ID NO.

4.

3. Application of the major-effect QTL for regulating cold tolerance at the seedling stage of rice as claimed in claim 1 in selecting strong cold-tolerant rice at the seedling stage.

4. Application of the molecular marker as claimed in claim 2 in selecting strong cold-tolerant rice at the seedling stage.

5. A breeding method for cold-tolerant rice at the seedling stage, characterized in that, The process is: Extract rice DNA, perform PCR amplification on the DNA using the primer pairs of the molecular markers Indel cts-1 and Indel cts-2 as claimed in claim 2, subject the amplification products to electrophoresis detection, and analyze the cold tolerance of rice through band patterns.

6. The method as claimed in claim 5, wherein, The reaction system for PCR amplification is: 10 μmol·L -1 0.75 μL of the upstream primer, 10 μmol·L -1 0.75 μL of the downstream primer, 1 μL of the DNA template, 12.5 μL of Mix enzyme, and 10 μL of ddH2O.

7. The method as claimed in claim 5, wherein, The reaction program of PCR amplification is: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 5 s, 35 cycles of amplification, and finally final extension at 72°C for 1 min.

8. A kit for breeding rice with strong cold tolerance at the seedling stage, characterized in that, It includes the primer pairs of the molecular markers Indel cts-1 and Indel cts-2 as claimed in claim 2.

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