CN112662800B - Molecular marker primers for rice cold tolerance main QTL CTS12 and its marker method and application - Google Patents

Abstract

A molecular marker primer of a rice cold-resistant major QTL CTS12 and a marking method and application thereof discover a molecular marker M5 closely linked with the rice cold-resistant major QTL CTS12, and utilize a primer corresponding to the molecular marker to carry out PCR amplification and detect an amplification product; the invention also provides application of the molecular marker or the molecular marker identification method in positioning and cloning of cold-resistant CTS12 of rice. The method can accelerate the cloning of the cold-resistant QTL CTS12, can be applied to auxiliary selective breeding, is convenient for timely cross breeding and accelerates the breeding process.

Description

Molecular marker primers and marker methods for rice cold tolerance major QTL CTS12 application

technical field

The invention relates to the field of molecular biology, in particular to a molecular marker of rice seedling stage cold tolerance main QTL CTS12 and its identification method and application.

Background technique

Rice is a temperature-loving crop and is extremely sensitive to temperature. The optimum temperature for its growth is between 15°C and 33°C. Japonica rice and indica rice will suffer chilling injury when the temperature is below 15°C and 18°C respectively. For a long time, chilling injury has been one of the key problems to be solved urgently in my country's rice production. In addition, temperature is also an important environmental factor affecting rice quality. Research on rice chilling damage can promote the expansion of rice planting area, improve the economic benefits of rice production areas, and also help to ensure the quality of rice grains.

When rice chilling injury occurs, the permeability of the outer cell membrane and the plastid membrane of the organelle increases, the content of active oxygen in the cell increases, and photosynthesis and respiration decrease, resulting in a decrease in plant metabolism, slow growth, yellowing or withering of leaves. These physiological symptoms are Mapping of QTL for cold tolerance provides the phenotypic basis. In 1996, several QTLs for rice seedling low temperature tolerance were first reported, and with the rapid development of biotechnology, rice cold tolerance QTLs were continuously discovered (Redona ED, MDMappingquantitative trait loci for seedling vigor in rice using RFLPs. Theoretical and applied genetics, 92: 395–402 (1996)). So far, more than 250 low-temperature-related QTLs have been mapped on 12 rice chromosomes, but only 12 QTLs have been fine-mapped, and 7 genes have been cloned and confirmed to be related to low-temperature resistance through functional identification, including germination qLTG3-1 at the seedling stage, COLD1, qCTS-9 and GSTZ2 at the seedling stage, LTG1 at the vegetative stage, Ctb1 and CTB4a at the booting stage. qLTG3-1 is located on chromosome 3 and encodes a glycine-rich conserved domain. High expression at low temperature can increase the germination rate of rice seeds (Fujino, K. et al. Molecular identification of a major quantitative trait locus, qLTG3–1, controlling low-temperature germinability in rice. Proceedings of the National Academy of Sciences 105, 12623-12628, doi: 10.1073/pnas.0805303105 (2008)). COLD1 encodes a G protein signaling regulator, localized in the plasma membrane and endoplasmic reticulum (ER), and can interact with the α subunit RGA1 of G protein to activate Ca2 ⁺ channels, thereby sensing low temperature and increasing the GTP of G protein Enzyme (GTPase) activity enhances cold tolerance of rice (Ma, Y. et al. COLD1 confers chilling tolerance in rice. Cell 160, 1209-1221, doi:10.1016/j.cell.2015.01.046(2015)). An InDel marker was detected in the promoter region of the qCTS-9 gene between the parents Lijiang Xintuan Heigu (LTH) and Huangshanzhan 2 (SHZ-2), which was significantly related to the low temperature tolerance phenotype at the seedling stage. Transgenic plants overexpressing qCTS-9 have enhanced cold tolerance (Zhao, J. et al. A novel functional gene associated with cold tolerance at the seedling stage in rice. Plant BiotechnolJournal 15, 1141-1148, doi:10.1111/pbi.12704 (2017 )). OsGSTZ2 encodes glutathione transferase, the 99th amino acid mutation (I99V) plays an important role in improving rice seedling low temperature tolerance (Kim,SI,Andaya,VC&Tai,THCold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathionetransferase isoenzyme GSTZ2. Biochem Journal 435,373-380, doi:10.1042/BJ20101610(2011)). LTG1 encodes casein kinase I, and a single amino acid substitution (I357K) in its coding region affects growth rate, heading date, and yield in rice at low temperatures (Lu, G. et al. Rice LTG1 is involved inadaptive growth and fitness under low ambient temperature . The Plant Journal 78, 468-480, doi: 10.1111/tpj.12487 (2014)). The E3 ubiquitin ligase subunit Skp1 interacts with the F-box domain protein encoded by Ctb1, participates in the low temperature signal transduction of the ubiquitin-proteasome pathway, and affects the low temperature tolerance of rice at the booting stage (Saito, K., Hayano- Saito, Y., Kuroki, M. & Sato, Y. Map-based cloning of the ricecold tolerance gene Ctb1. Plant Science 179, 97-102, doi:10.1016/j.plantsci.2010.04.004(2010)). CTB4a encodes a conserved leucine-rich repeat receptor-like kinase that interacts with the β subunit of ATP synthase, AtpB. The enhanced ATP synthase activity under cold stress conditions improves the cold tolerance of rice at the filling stage; at the same time, the haplotypes of 119 rice varieties were analyzed and found that the differences in the expression levels of SNP-2536, SNP-2511 and SNP-1930 determine the Variety tolerance to low temperature (Zhang, Z. et al. Natural variation in CTB4a enhances rice adaptation to cold habitats. Nature Communication 8, 14788, doi: 10.1038/ncomms14788 (2017)).

Due to the mining of cold tolerance QTL, we can deepen our knowledge and understanding of the rice cold tolerance regulatory network, and at the same time, the molecular markers obtained can also be used for assisted selection breeding. Since the marker-assisted selection (Marker-Assisted Selection, MAS) method is the selection of the genotype of the variety, it is not affected by the environment, so the breeding application of MAS is helpful for the cold-tolerant genetic modification of rice varieties, and will effectively improve the breeding efficiency. efficiency.

Contents of the invention

The present invention provides a molecular marker of rice cold tolerance main QTL CTS12 and its identification method and application. By detecting the molecular marker closely linked with rice cold tolerance main QTL CTS12, it can be determined whether the cold tolerance CTS12 locus is introduced into the breeding line In this process, the selection efficiency of this trait can be improved and the breeding progress can be accelerated.

In order to achieve the above object, the technical solution of the present invention is: a molecular marker of rice cold tolerance main effect QTL CTS12, the molecular marker is M5, and its corresponding primers are respectively:

Upstream primer: 5'—TATCCCCAAGGAAATAAACT—3'

Downstream primer: 5'—AGAGTATGATGCGATGTCCC—3'.

The method for obtaining the closely linked molecular markers of the rice cold tolerance major QTL CTS12 comprises the following steps:

Using the cold-tolerant Guangxi common wild rice DP15 as the donor parent and the cold-sensitive rice variety 9311 as the recurrent parent, the chromosome segment substitution lines (chromosome segment substitution lines, CSSLs) were constructed, through continuous backcrossing, selfing and directional selection, in BC ₅ In the F2 generation, a stable cold _- tolerant chromosome segment substitution line DC90 was obtained; then the substitution line _DC90 was crossed with 9311 to obtain F1 hybrids, and F2 segregation populations _were obtained after selfing, and the F2 segregation populations _were genotyped by molecular markers Identification and identification of cold tolerance phenotype, and 5 exchanged individual plants (W1, W2, W3, W4, W5) were obtained. The molecular marker M5 was obtained based on genetic linkage analysis of materials 9311, DC90, NC, W1, W2, W3, W4, and W5.

The application of the molecular marker of rice seedling stage cold tolerance main QTL CTS12 in the location and cloning of rice cold tolerance CTS12 comprises the following steps:

Using the whole genome DNA of the rice material to be identified as a template, and using the primers corresponding to the molecular marker M5 to perform PCR amplification, if an amplified fragment of 227bp is amplified, it indicates the existence of the main QTL CTS12 for cold tolerance at the seedling stage.

The reaction system of the PCR is as follows:

The PCR amplification reaction conditions are: 94°C for 2min, 98°C for 10s, 60°C for 30s, 68°C for 30s, 35 cycles of amplification, and finally 68°C for 1min.

The DNA amplification products were detected by agarose gel electrophoresis, and the amplified DNA bands were recorded by color development.

The beneficial effects of the present invention are:

(1) Through the discovery of closely linked molecular markers of rice cold-tolerant main QTL CTS12, the fine mapping and cloning of the cold-tolerant QTLCTS12 can be accelerated, and it can be applied to assisted selection breeding to facilitate timely cross-breeding and speed up the breeding process;

(2) Established a molecular marker identification method for rice cold-tolerant main QTL CTS12, which can accurately and quickly identify cold-tolerant single plants with CTS12 loci, improve the selection efficiency of this trait, and speed up the breeding progress;

(3) A molecular marker of rice cold tolerance main effect QTL CTS12 was discovered, and the location of the main effect gene locus located by the present invention is clear and easy to identify. By detecting the molecular markers linked to the gene locus, the seedling cold resistance of rice plants can be predicted, which can be used for genotype detection of rice varieties or lines to determine whether the variety or line has cold resistance at seedling stage, and then Rapid selection of cold-tolerant varieties or lines for rice breeding. It has enriched people's knowledge and understanding of the cold-tolerant regulatory network of rice, and also increased the diversity of cold-tolerant gene sources for rice breeding applications, providing more choices for cultivating different types of cold-tolerant rice varieties.

Description of drawings

Figure 1 is the genetic background of the chromosome segment substitution line DC90.

Figure 2 is the low temperature stress phenotype of DC90 seedling stage, wherein: A in Figure 2 is the phenotype of 9311 and DC90 before cold treatment; B in Figure 2 is the phenotype of 9311 and DC90 after cold treatment at 8-10°C for 5 days; C in 2 is the 9311 and DC90 phenotypes after 5 days of cold treatment and recovery of 7 days under normal conditions.

Fig. 3 is the identification of cold stress phenotype of a single plant of F ₃ generation at seedling stage.

Figure 4 is a preliminary map of the rice CTS12 gene on chromosome 12.

Fig. 5 is a partial amplified band pattern amplified by the molecular marker primer M5 for 45 different rice varieties.

detailed description

The technical solutions of the present invention will be further described below in conjunction with examples, but the implementation of the present invention is not limited to the following examples.

Example 1

Screening and mapping of molecular markers

(1) Construction of the cold-tolerant chromosomal segment substitution line DC90 and analysis of the cold-resistant phenotype of the replaced segment

(1) Using the cold-tolerant Guangxi common wild rice DP15 as the donor parent and the cold-sensitive rice variety 9311 as the recurrent parent to construct chromosome segment substitution lines (chromosome segment substitution lines, CSSLs), through continuous backcrossing, selfing and directional selection , in the _second generation of BC ₅ F, DC90 and NC, two chromosome segment replacement lines with similar genetic backgrounds, were obtained. Most of the genetic background of DC90 is the same as that of the recurrent parent 9311, only the fragment replacement of DP15 on chromosome 3 and chromosome 12, the length of which is 10Mb and 20Mb respectively. However, NC only has a fragment replacement of DP15 on chromosome 3, which is the same as the replacement region of chromosome 3 in DC90.

(2) The cold tolerance phenotypes of DC90 and NC were identified, and it was found that DC90 had the ability to tolerate cold stress, while NC showed cold sensitivity. Combined with the genetic background of DC90 and NC, it is deduced that the cold tolerance of DC90 is controlled by the fragment of DP15 on chromosome 12, which is located in the 20Mb region between the molecular markers M3 and M12, so it has acquired cold resistance in seedling stage The replacement line fragments (Figure 1).

(3) Construction and genetic analysis of the F ₂ segregation population of DC90/9311

(1) Use the cold-tolerant chromosome segment substitution line DC90 to cross with the recipient parent 9311 to obtain F ₁ hybrids, plant the F ₁ hybrids, and self _- cross to obtain F ₂ segregation populations. Type identification, and 5 important exchanged individual plants were obtained for genetic analysis.

( ₂ ) Genomic DNA from leaves of each individual plant of the parent and F2 populations was extracted by the CTAB (cetyltrimethylamine bromide) method.

(3) The sequence information of DP15 and 9311 genes was compared, and more than 50 STS markers were designed in the M3 and M12 segments for further fine-tuning. Among them, InDel molecules with polymorphisms are marked with M4, M5, M6, M7, M8, M9, M10, and M11.

(4) Using molecular markers to screen the F ₃ population and fine-map the CTS-12 gene

According to the QTL mapping results, use the obtained polymorphic molecular markers M4, M5, M6, M7, M8, M9, M10, M11 to identify the genotype of the target region _of each individual in the _F2 population, obtained from the F3 population A series of exchange-type individual plants were collected, and the genotype and phenotype of each individual plant were detected to investigate the co-segregation of markers and resistance phenotypes.

The above linkage analysis and preliminary positioning process, the DNA extraction method and PCR reaction conditions adopted are respectively:

A. Extract the DNA of each individual plant of the donor parent, the recipient parent and the F2 segregation population with the conventional CTAB method _;

B. The reaction system of PCR is as follows:

PCR reaction conditions: 94°C for 2min, 98°C for 10s, 60°C for 30s, 68°C for 30s, 35 cycles of amplification, and finally 68°C for 1min.

The DNA amplification products were detected by agarose gel electrophoresis, and the amplified DNA bands were recorded by color development.

It can be seen from the above process that the molecular markers linked to the cold-tolerant main effect QTL CTS12 can be effectively carried out molecular marker-assisted selection breeding of the cold-tolerant QTL CTS12 by using the above method steps; Positioning and map bit cloning.

results and analysis

Through genotype and corresponding cold-tolerant phenotype identification of recombinant single plant (Figure 4), combined with genotype and phenotype genetic linkage analysis: W5 is cold-sensitive, so qCTS12 should be located on the left side of molecular marker M6. W4 showed cold tolerance, so qCTS12 should be located on the right side of M4. In summary, qCTS12 should be located in the vicinity of the molecular marker M5. Compared with the previous mapping results, qCTS12 belongs to a new QTL locus for cold tolerance in rice seedling stage.

Example 2

Validation of Molecular Markers

1. Materials and methods

1.1 Materials

45 rice varieties from different origins

(1) extracting rice variety DNA with conventional CTAB method;

(2) The reaction system of PCR is as follows:

PCR reaction conditions: 94°C for 2min, 98°C for 10s, 60°C for 30s, 68°C for 30s, 35 cycles of amplification, and finally 68°C for 1min.

The DNA amplification products were detected by agarose gel electrophoresis, and the amplified DNA bands were recorded by color development ( FIG. 5 ).

The detection results of 45 rice varieties using molecular marker M5 are shown in Table 1. Table 1 shows the identification results of the cold stress phenotypes and molecular marker M5 genotypes of 45 different rice varieties at the seedling stage.

Table 1 Detection results of molecular marker M5 on 45 rice varieties

Note: In Table 1, A represents the genotype of 9311, B represents the genotype of DC90, 1 represents alive, and 0 represents dead.

As can be seen from the results in Table 1, the use of molecular marker M5 primers for amplification, if a 227bp amplified fragment (genotype B) is amplified, it indicates the existence of the cold-resistant main effect QTL CTS12, if the 194bp amplified fragment can be amplified The amplified fragment (genotype A) indicates that there is an amplified fragment of the parent 9311. Most varieties containing genotype B are cold tolerant; whereas most varieties containing genotype A are cold intolerant.

The detection of rice varieties based on the above-mentioned molecular markers can verify the existence of the cold-tolerant main QTL CTS12, and the selection of cold-tolerant rice varieties can be carried out, thereby speeding up the breeding progress.

sequence listing

<110> Guangxi University

<120> Molecular marker primers and marker methods and applications of rice cold tolerance main QTL CTS12

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tatccccaag gaaataaact 20

<210> 2

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agagtatgat gcgatgtccc 20

<210> 3

<211> 227

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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atccccaagg aaataaactt acaataatct cctcttagtc cattcactct ctcctgttat 60

atttatgtag ttgtagtctg tgtctgtcat cagtaaatgg ccaccgtcaa taaatacacg 120

aagtaacagc agtgggactt aaaaatattt gttcttaaag cagataacag tgtaagctta 180

agtgatgaac aaaagaaagc taaatcaggg acatcgcatc atactct 227

Claims (5)

1. Rice cold-resistant major QTLCTS12A closely linked molecular marker M5 characterized by: the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2.

2. Cold-resistant major QTL of rice as claimed in claim 1CTS12The marking method of the closely linked molecular marker M5 is characterized by comprising the following steps:

chromosome fragment replacement line population constructed by using cold-resistant Guangxi common wild rice DP15 as a donor parent and using a cold-sensitive rice variety 9311 as a recurrent parent (C: (C))chromosome segment substitution lines，CSSLs) by continuous backcrossing, selfing and directional selection at BC ₅ F ₂ Obtaining a cold-resistant chromosome fragment substitution line DC90 with stable inheritance; then the substitution line DC90 and 9311 are hybridized to obtain F ₁ Hybridizing to obtain F ₂ Segregating the population, F ₂ The segregating population obtains 5 crossover individuals W1, W2, W3, W4 and W5 through the genotype identification and the cold-resistant phenotype identification of the molecular marker, and the molecular marker M5 is obtained based on the genetic linkage analysis of 9311, DC90, NC, W1, W2, W3, W4 and W5.

3. Cold-resistant major QTL of rice as claimed in claim 1CTS12Identification of closely linked molecular marker M5The method is characterized in that: the method comprises the following steps:

(1) Using the whole genome DNA of rice material to be identified as a template, carrying out PCR amplification by using the primer of claim 1, and if an amplified fragment of 261 bp is amplified, indicating that the cold-resistant major QTL in the seedling stage existsCTS12(ii) present;

(2) The reaction system of the PCR is as follows:

upstream primer (10. Mu.M) 0.3. Mu.L

Downstream primer (10. Mu.M) 0.3. Mu.L

dNTPs (2 mM) 2.0 μL

DNA template (100 ng/. Mu.L) 1.0. Mu.L

KOD FX DNA Polymerase (1.0 unit/μL) 0.2 μL

2× PCR Buffer for KOD FX 5.0 μL

ddH ₂ O 1.2 μL

(3) The PCR amplification reaction conditions are as follows: amplifying at 94 deg.C for 2min,98 deg.C for 10s,60 deg.C for 30s,68 deg.C for 30s for 35 cycles, finally extending at 68 deg.C for 1min,

(4) The DNA amplification products were detected by agarose gel electrophoresis and the amplified DNA bands were recorded by color development.

4. Cold-resistant major QTL of rice as claimed in claim 1CTS12Closely linked molecular marker M5 in rice cold resistanceCTS12And cloning.

5. Rice cold-resistant major QTL of claim 3CTS12The application of the closely linked molecular marker M5 in breeding rice cold-resistant varieties.

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