A molecular marker for enhancing rice salt tolerance gene OsHAK11, its detection and identification method, and its application.

Abstract

This invention discloses a gene for improving salt tolerance in rice. OsHAK11 This research on molecular markers, their detection and identification methods, and their applications belongs to the field of plant molecular breeding technology. The molecular marker consists of three primers: 52390-WF1 (SEQ ID NO. 6), 52390-MF1 (SEQ ID NO. 7), and 52390-R1 (SEQ ID NO. 8). It can rapidly and accurately distinguish between different types of plants through PCR amplification and electrophoretic analysis. OsHAK11 The invention identifies the wild-type, heterozygous, and homozygous mutant types of genes. It also provides a method for genotyping using this marker and its application in backcross breeding for selecting salt-tolerant rice lines. This marker system has advantages such as accurate detection, simple operation, and high breeding efficiency, making it suitable for large-scale application and significant for accelerating the breeding of salt-tolerant rice varieties and improving rice production capacity in saline-alkali land.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, and specifically relates to a gene for improving salt tolerance in rice. OsHAK11 Molecular markers and their detection and identification methods and applications. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is a major global food crop, crucial for ensuring global food security. It is projected that by 2050, food production will need to increase by approximately 50% to meet the demands of population growth. However, rice growth is often severely constrained by abiotic stresses such as salt stress. Globally, saline-alkali land covers 945 million hectares, accounting for about 20% of agricultural land. China, as a major country with saline-alkali land, ranks third in the world in terms of saline-alkali land area, mainly concentrated in the Northeast, Northwest, North China, and coastal areas, severely limiting the expansion of arable land resources. Utilizing the hydroponic characteristics of rice for saline-alkali land improvement is one of the effective ways to expand arable land area and increase food production capacity.

[0003] Current rice salt-tolerant breeding methods largely rely on chromosome 1. Saltol The genetic resources of QTL loci such as qSKC-1 are relatively limited. With the development of marker-assisted selection (MAS) and gene editing technologies, cloning salt-tolerant genes and developing molecular markers linked to them has become crucial for accelerating the breeding of salt-tolerant rice. Potassium transporters (HAK family) play an important role in plant K⁺ uptake and salt stress response. The rice genome contains 27 HAK family members. HAK Genes, among which OsHAK9 and OsHAK21 It has been reported to participate in salt tolerance regulation. OsHAK18 It participates in regulating rice plant height, tiller number, panicle length, seed setting rate, number of grains per panicle, yield, and grain-to-weed ratio. OsHAK26 It plays an important role in maintaining rice pollen development and fertility, and OsHAK11 Its function in salt stress and its application in molecular marker-assisted breeding have not been reported. Summary of the Invention

[0004] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0005] This invention provides a method for identifying rice mutants. oshak11 The genotype primer set consists of a forward primer as shown in SEQ ID NO. 6, a mutant-specific forward primer as shown in SEQ ID NO. 7, and a reverse primer as shown in SEQ ID NO. 8.

[0006] The present invention also provides a method for identifying rice mutants. oshak11A genotype detection kit containing the aforementioned primer set.

[0007] This invention also provides a method for identifying rice mutants. oshak11 The genotyping method includes the following steps:

[0008] S1. Extract genomic DNA from the rice plants to be tested;

[0009] S2. Using the genomic DNA as a template, perform PCR amplification using the primer set;

[0010] S3. Detect PCR amplification products and determine the appropriate amplification method based on the band size. OsHAK11 Genotype: If only a 177 bp band is present, it is wild-type; if both 177 bp and 171 bp bands are present, it is heterozygous; if only a 171 bp band is present, it is homozygous mutant.

[0011] Furthermore, the PCR amplification conditions are as follows: pre-denaturation temperature of 94 degrees Celsius for 5 minutes, denaturation temperature of 94 degrees Celsius for 30 seconds, annealing temperature of 55 degrees Celsius for 30 seconds, extension temperature of 72 degrees Celsius for 30 seconds, 35 cycles, and a final extension temperature of 72 degrees Celsius for 5 minutes.

[0012] This invention also provides a method for breeding salt-tolerant rice varieties, comprising the following steps:

[0013] (a) Those with salt-tolerant phenotypes oshak11 The mutant was used as a donor parent and hybridized with a target rice parent to obtain hybrid offspring;

[0014] (b) In the hybrid offspring, the method for identifying rice mutants is used. oshak11 Genotyping methods for screening OsHAK11 A single plant with a heterozygous genotype;

[0015] (c) Perform genetic background analysis on the selected individual plants, select individual plants with high genetic background similarity to the target rice parent, and thus breed rice lines with improved salt tolerance and agronomic traits close to the target rice parent.

[0016] Furthermore, step (a) is followed by a backcrossing step: the hybrid offspring are backcrossed at least once with the target rice parent to obtain backcross offspring, and steps (b) and (c) are performed on the backcross offspring.

[0017] Furthermore, the genetic background analysis is performed using polymorphic molecular markers distributed on the rice genome for assisted selection, including SSR markers, SNP markers, or InDel markers.

[0018] Furthermore, the single plant with a high genetic background similarity to the target rice parent refers to a single plant with a genotypic similarity greater than 88%. Beneficial effects

[0019] (1) The molecular marker developed in this invention consists of three specific primers (SEQ ID NO.6–8), which can quickly distinguish between wild type (177 bp), heterozygous type (177 bp / 171 bp double band) and homozygous mutant type (171 bp) by PCR and electrophoresis, and is suitable for large-scale germplasm screening.

[0020] (2) The molecular markers developed in this invention and oshak11 Co-segregation of mutation sites can be directly used for early genotypic selection during backcrossing and conversion, significantly shortening the breeding cycle.

[0021] (3) This invention is applicable to rice materials with different genetic backgrounds and can be used in conjunction with multiple molecular markers such as SSR and SNP to achieve synchronous selection of genotype and genetic background, and accelerate the breeding of superior salt-tolerant varieties. Attached Figure Description

[0022] Picture 1 for OsHAK11 Schematic diagram of gene structure and target sequence elements of CRISPR / Cas-OsHAK11 vector.

[0023] Picture 2 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. oshak11 Target sequencing peak diagram.

[0024] Picture 3 Salt tolerance test for the OsHAK11 mutant. A represents ZH11 and... oshak11 Plant growth status before and after salt treatment, bar = 5cm; B represents ZH11 and oshak11 Survival rates before and after salt treatment were statistically analyzed. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01). One-way ANOVA was used for statistical analysis.

[0025] Picture 4 A schematic diagram of competitive typing primer design for target mutation sites.

[0026] Picture 5 To validate the results of T-clone detection using molecular markers.

[0027] Picture 6 This is a schematic diagram of the backcrossing and transformation route using molecular markers. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments. Example 1

[0029] This embodiment provides rice. OsHAK11 The functions and applications of genes include the following:

[0030] 1. Rice OsHAK11 Functional verification of genes

[0031] To clarify OsHAK11 To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice.

[0032] This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. This invention selects the nucleotide sequence from base 2362 to base 2384 of the gene coding region, starting from the start codon ATG (as shown in SEQ ID NO. 4), as the target region for CRISPR / Cas9 gene editing (see...). Picture 1 ).

[0033] (1) OsHAK11 Construction of CRISPR / Cas9 gene editing vector

[0034] The gene editing vector of this invention is pEGCas9Pubi-B-OsHAK11, and the base vector of this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows:

[0035] i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsHAK11 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequence of the target region selected in this invention is shown in SEQ ID NO.4.

[0036] ii) Amplify the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. Then, the primer pairs were ligated with the binary vector pEGCas9Pubi-B (see Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi-OsHAK11. The recombinant vector pEGCas9Pubi-OsHAK11 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific steps were as described in the reference "Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327."

[0037] iii) Sequencing verification.

[0038] A positive clone with correct sequencing results is a successfully constructed pEGCas9Pubi-OsHAK11 gene editing vector.

[0039] (2) Agrobacterium-mediated genetic transformation of rice

[0040] The pEGCas9Pubi-OsHAK11 gene editing vector successfully constructed above was transformed into Agrobacterium EHA105 via heat shock. After identification by PCR, the bacterial culture was stored at -80 °C with glycerol.

[0041] Freshly peeled embryos of the hybrid rice variety Zhonghua 11, approximately 1.5 mm in size, were used as recipient materials. The peeled rice embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension and left for no more than 1 hour. Approximately 100 embryos were placed in each centrifuge tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43°C for 2 minutes, followed by an ice bath for 1 minute. The remaining washings at the bottom of the tubes were aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then left to stand in the dark for 8 minutes.

[0042] Next, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove any excess infection solution. Place all embryos with their scutes facing upwards and co-culture at 23°C in the dark for 3 days.

[0043] After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28°C for 7-14 days. During this process, be careful to remove any sprouts that grow on the embryos.

[0044] After the recovery culture was completed, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for 3 rounds of selection culture, with each round lasting 2 weeks. Then, they were transferred to a selection medium containing 2 mg / L Bialaphos for 2 rounds of selection culture, with each round lasting 2 weeks.

[0045] The resistant callus was transferred to propagation medium and cultured in the dark at 28°C for 2 weeks. The propagated resistant callus was then transferred to induction medium and cultured in the dark at 28°C for 2 weeks. Finally, it was transferred to differentiation medium and cultured under light at 25°C and 5000 lx for 2 weeks.

[0046] After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested after 3-4 months.

[0047] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0048] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0049] This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0050] PCR was performed using 5 μL of Biomiga 2×PCR premix (containing Mg2+, Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer), 1 μL of primers (containing 0.5 μL each of forward and reverse primers), 1 μL of template DNA, and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were analyzed by 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after color development with formaldehyde and NaOH.

[0051] For mutants oshak11 Nucleotide sequence alignment analysis revealed that ( Picture 2 Compared to the unedited wild type (WT), the mutated OsHAK11 The mutant nucleotide sequence is shown in SEQ ID NO. 5, which describes the deletion of a T base at position 2368, starting from the start codon ATG. This deletion causes a frameshift in the amino acid sequence, leading to premature termination of translation. Example 2

[0052] This embodiment describes the mutant obtained in Example 1. oshak11 Phenotypic analysis was performed, as follows:

[0053] 1. Mutant oshak11 Salt tolerance assessment

[0054] The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0055] The rice seeds to be tested are oshak11 - L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0056] (1) For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48h.

[0057] (2) After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0058] (3) After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0059] (4) After completing step 3, place the 96-well plate (with germinated seeds on it) on a plastic box containing Yoshida rice culture solution and immerse the germinated seeds in the culture solution. Culture in alternating light and dark for 3 weeks to obtain rice seedlings that have grown to the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture solution needs to be replaced every 7 days.

[0060] (5) After completing step 4, place the 96-well plate (on which rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and completely immerse the roots in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and then replace it with 150 mM NaCl Yoshida rice culture solution. The high salt stress was carried out for 11 days under alternating light and dark culture (during the high salt stress period, the Yoshida rice culture solution was replaced every 2 days).

[0061] (6) After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and recover for 7 days under alternating light and dark culture.

[0062] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0063] See the growth status of rice seedlings before treatment. Picture 3 The growth status after treatment A is shown in Figure 1. Picture 3 B. Survival rate statistics are shown in [the original text]. Picture 3 C in the middle.

[0064] The results showed that before salt treatment, oshak11 Slightly shorter than ZH11; freshwater control ZH11 and mutant oshak11 The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... oshak11 The survival rate was 91.66%, and statistical analysis results showed that... oshak11 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... oshak11 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference. Example 3

[0065] This embodiment provides a method for identifying mutants. oshak11 The co-separation molecular markers and their applications are described in the following process:

[0066] 1. Development of co-separated molecular markers

[0067] In this invention, primers were designed using Primer 5.0 software to target mutation sites in sterile lines, and molecular markers were developed ( Picture 4 The assay contains three primers: 52390-WF1 (SEQ ID NO. 6), 52390-MF1 (SEQ ID NO. 7), and 52390-R1 (SEQ ID NO. 8). By combining PCR with agarose gel electrophoresis and non-denaturing polyacrylamide gel electrophoresis (PAGE) or agarose gel electrophoresis, the genotype of the mutant can be identified based on the presence and size of the amplified bands.

[0068] like Picture 5 As shown, molecular markers can specifically detect rice OsHAK11 Homozygous mutants and mutant genes in rice materials derived from them oshak11 And can distinguish between wild type at the same time. OsHAK11 Genes and mutants oshak11 Genes; targeting mutated genes oshak11 A 171 bp band was amplified in the middle, while in the wild type... OsHAK11 The gene amplified into a 177 bp band:

[0069] 2. Application of co-separated molecular markers

[0070] Theoretically, 52390-WF1 / 52390-MF1 / 52390-R1 in OsHAK11 / OsHAK11 A 177 bp band can be amplified in homozygous wild-type (AA) DNA. oshak11 / oshak11 A 171 bp band can be amplified in the DNA of the homozygous mutant material (aa), while... OsHAK11 / oshak11In hybrid (Aa) materials, the corresponding two bands can be amplified simultaneously. The T-cloning verification results of the 52390-WF1 / 52390-MF1 / 52390-R1 molecular markers are as follows... Picture 5 As shown, the results indicate that the designed functional molecular markers achieved the expected detection results for the segregating population. OsHAK11 / OsHAK11 Homozygous wild type (AA) OsHAK11 / oshak11 Heterozygous (Aa) and oshak11 / oshak11 The homozygous mutant material (aa) amplified bands of corresponding sizes, which can be used as... OsHAK11 An ideal marker for allele detection. Example 4

[0071] This embodiment performs OsHAK11 Transgenic experiments using mutant genes, using mutants oshak11 Hybridization, backcrossing, and self-pollination with recipients exhibiting excellent agronomic traits, such as R3261, were performed, with molecular markers used throughout the process. OsHAK11 Genetic and genetic prospect selection ultimately yields a restorer line with a homozygous mutant gene in the R3261 background. The technical route for hybridization and conversion is as follows: Picture 6 As shown, the specific implementation steps are as follows:

[0072] 1. Using the recipient parent, such as R3261, as the paternal and maternal mutants. oshak11 F1 was obtained through hybridization.

[0073] 2. Use F1 as the female parent and the recipient parent, such as R3261, to backcross and obtain BC1F1.

[0074] 3. Plant BC1F1 and detect it using primer pairs with primer sequences such as SEQ ID No. 6-8. OsHAK11 Genotype, selection OsHAK11 Heterozygous genotype, meaning the PCR amplification product shows both 171 bp and 177 bp bands.

[0075] 4. Use a set of genotypes (e.g., 200) in oshak11 Polymorphism exists between mutants and recurrent parents, and molecular markers (including but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, and SCAR type markers) are evenly distributed. Genetic background identification is performed on the single plants selected in step 3, and plants with high genotypic similarity to recurrent parents (e.g., greater than 88% similarity, or 2% selection rate) are selected.

[0076] 5. Use the plants selected in step 4 and the recipient parent, such as R3261, to backcross and obtain BC2F1.

[0077] 6. Plant BC2F1, repeat steps 3 and 4, and select... OsHAK11 Plants with heterozygous genotypes and high genetic background reversion rates (e.g., greater than 98%, or a selection rate of 2%) were collected from cross BC2F2.

[0078] 7. Plant BC2F2, repeat steps 3 and 4, and select... OsHAK11 The plant with the highest genotypic heterozygosity and the highest homozygous genetic background was collected from the cross BC2F3. Segregation occurred in the progeny of BC2F3. OsHAK11 homozygous strain, i.e., under the background of R3261 OsHAK11 Gene restoration line.

[0079] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for identifying a rice mutant oshak11 genotype, characterized in that, The primer set consists of a forward primer with the sequence as shown in SEQ ID NO. 6, a mutant-specific forward primer with the sequence as shown in SEQ ID NO. 7, and a reverse primer with the sequence as shown in SEQ ID NO.

8.

2. A kit for identifying a rice mutant oshak11 a kit for detecting a genotype, characterized by, The kit comprises the primer set of claim 1.

3. A method of identifying a rice mutant oshak11 genotype, characterized in that The method comprises the following steps: S1, extracting genomic DNA of the rice plant to be tested; S2, using the primer set of claim 1 to perform PCR amplification with the genomic DNA as a template; S3, detecting the PCR amplification product, and determining the genotype according to the band size of the amplification product: if only a 177 bp band appears, it is wild type; if 177 bp and 171 bp bands appear at the same time, it is heterozygous type; if only a 171 bp band appears, it is homozygous mutant type.

4. The method of claim 3, wherein, The PCR amplification conditions are: pre-denaturation temperature of 94 degrees Celsius for 5 minutes; denaturation temperature of 94 degrees Celsius for 30 seconds, annealing temperature of 55 degrees Celsius for 30 seconds, extension temperature of 72 degrees Celsius for 30 seconds, and cycling for 35 times; and finally extension temperature of 72 degrees Celsius for 5 minutes.

5. A method for breeding a salt-tolerant rice variety, characterized by, The method comprises the following steps: (a) obtaining a mutant having a salt tolerance phenotype as a donor parent, wherein the donor parent is a rice plant; oshak11 (b) crossing the mutant with a target rice parent to obtain hybrid offspring, wherein the target rice parent is a rice plant; (b) screening a single plant with a heterozygous genotype in the hybrid offspring by using the method of claim 3; (c) performing genetic background analysis on the screened single plant, and selecting a single plant with a genetic background similar to the target rice parent genotype with a similarity of more than 88%, thereby breeding a rice line with improved salt tolerance and agronomic traits close to the target rice parent.

6. The method of claim 5, wherein, After step (a), a backcross step is further included: performing at least one backcross between the hybrid offspring and the target rice parent to obtain a backcross offspring, and steps (b) and (c) are performed in the backcross offspring.

7. The method of claim 5, wherein, The genetic background analysis is assisted selection using polymorphic molecular markers distributed on the rice genome, and the polymorphic molecular markers include SSR markers, SNP markers or InDel markers.

Images