Method for producing and identifying rice haploid

Abstract

The invention discloses a method for producing and identifying rice haploids, and belongs to the technical field of biology. The method comprises the following steps: (1) carrying out cross pollination by taking a dominant male sterile line as a female parent and a haploid induction line carrying a red fluorescent protein gene mCherry as a male parent to obtain hybrid seeds; (2) after the hybrid seeds germinate, screening fluorescent-free candidate haploid seeds by observing red fluorescence of roots and bud parts; and (3) culturing the candidate haploid seeds to obtain seedlings, and identifying haploid plants by adopting a flow cytometry. The dominant male sterile line is used as a hybrid female parent, so that the pollination and hybridization efficiency is improved, and the manual workload and time are greatly reduced; the haploid induction line is combined with a fluorescence labeling technology, the ploidy is determined by screening non-fluorescence seeds and then carrying out flow cytometry background determination, haploid seedlings can be screened out in the early stage, the screening efficiency is greatly improved, and meanwhile, the time and the workload of field planting are saved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for producing and identifying rice haploids. Background Technology

[0002] Haploid breeding is one of the most effective crop breeding methods to date. Through the hybridization and recombination of the genomes of two or more varieties, it can produce an extremely rich variety of superior gene combinations, which rapidly become homozygous through chromosome doubling. Currently, there are three main pathways for haploid production: anther (microspore) culture, haploid induction line induction, and chromosome disappearance induced by distant hybridization. Haploid breeding of maize based on haploid induction lines is the most successful haploid breeding system. Anther (microspore) culture technology has also been applied in rice, wheat, and rapeseed, but its widespread application has been limited by genotypic differences.

[0003] Rice is one of the most important crops for breeding in southern China and plays a vital role in our food security. Although successful reports of rice haploid breeding date back to the 1970s, its application is still affected by genotypic differences, especially in indica rice, where its application is very limited. In rice, the technique of obtaining haploids through anther culture is relatively mature, but this process is time-consuming, complex, and highly susceptible to genotypic differences. Most indica rice materials exhibit low callus rates and even lower differentiation rates during anther culture, resulting in a high proportion of albino seedlings. Therefore, only a few breeding units have applied this technique to date. Recently, some progress has been made in cultivating haploid inducible lines through OsMATL gene editing, but its practical application faces challenges. First, rice is hermaphroditic, requiring artificial emasculation before hybridization. This step is not only labor-intensive but also prone to self-pollination contamination (incomplete or improper emasculation leading to self-pollination). Secondly, there is currently no marker system for visually distinguishing between hybrid and haploid seeds, and the shape of seeds obtained after emasculation and demasking differs significantly from that of normal seeds, making them difficult to differentiate by appearance alone. Thirdly, using... Osmatl When mutants are used for male-parent hybridization, the hybridization seed set rate is low (only about 10-20%), and the hybrid seeds typically contain only 0.5%-5% haploids (the highest reported percentage is 12.4%, but haploid induction varies greatly depending on the plant background). Obtaining a sufficient number of haploid seeds requires extensive hybridization. Therefore, this technology is almost impossible to directly apply to rice breeding because the cost of using this approach to cultivate doubled haploid systems is too high. Rice OsmatlThe seed set rate of mutants used for paternal hybridization is 20%, the average proportion of haploids in hybrid seeds is 5%, and the efficiency of obtaining DH plants from haploid seedlings through artificial doubling is 20%. Therefore, obtaining one DH plant requires emasculating 1 / (0.2*0.05*0.2) = 500 florets. Assuming that a hybrid combination needs to obtain at least 200 DH lines to have a high probability of selecting a new variety that meets the breeding objectives, then 100,000 florets need to be artificially emasculated, which would require a skilled technician working for at least 40 days (including emasculation, pollination, and hybridization). Furthermore, since it is impossible to visually identify whether hybrid seeds are haploid, molecular identification is required for the 4,000 seedlings produced from these seeds, which is not only very costly but also difficult to complete in a short period. Therefore, there is an urgent need to combine the haploid induction system with other genetic systems to innovate emasculation hybridization and haploid identification techniques, thereby significantly improving haploid production efficiency and reducing production costs. Summary of the Invention

[0004] To address the aforementioned technical challenges, this application organically combines dominant male-sterile lines, haploid induction lines, and fluorescent labeling technology to construct an efficient haploid production and identification technology system, pioneering a new path for haploid rice breeding.

[0005] This invention provides a method for producing and identifying rice haploids, comprising the following steps: (1) Using a dominant male-sterile line as the female parent and a haploid inducible line carrying the red fluorescent protein gene mCherry as the male parent, hybrid pollination was carried out to obtain hybrid seeds; (2) After the hybrid seeds germinate, candidate haploid seeds without fluorescence are screened by observing the red fluorescence of the roots and buds.

[0006] Preferably, the method for creating the dominant male-sterile line in step (1) is as follows: using three dominant male-sterile rice varieties as the female parent and Wuxiangjing 7375, Meixiangzhan 2, or Indica rice DR610 as the recurrent parent, dominant male-sterile materials with the same genetic background as the recurrent parent are obtained through continuous backcrossing and selection.

[0007] More preferably, the method for cultivating the haploid inducible line in step (1) includes: (a) The rice OsMATL gene was edited using CRISPR-Cas9 technology to obtain OsMATL gene mutants; (b) The constitutively expressed red fluorescent protein gene mCherry was introduced into the mutant by transgenic technology, and lines with single-copy insertion and stable expression of the fluorescent protein gene were screened to obtain haploid inducible lines.

[0008] More preferably, the rice mentioned in step (a) is Xidao No. 1.

[0009] More preferably, the target site sequence of the CRISPR-Cas9 technology in step (a) is GTCCGGTGGCTGCGCAACAA.

[0010] More preferably, the OsMATL gene mutant described in step (a) is a vector-free edited pure line, and the encoded OsMATL protein sequence is changed from SEQ ID NO.9 to SEQ ID NO.10.

[0011] More preferably, the method further includes step (3) identifying haploid plants by flow cytometry of seedlings obtained from candidate haploid seed culture.

[0012] This invention also provides the application of the above method in the production and identification of rice haploids.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention organically combines dominant male-sterile lines, haploid induction lines, and fluorescent labeling technology to construct a highly efficient haploid production and identification technology system. Dominant male-sterile lines, used as the female parent in hybridization, improve pollination and hybridization efficiency, significantly reducing manual labor and time. The haploid induction lines, combined with fluorescent labeling technology, determine ploidy by screening for non-fluorescent seeds and then performing flow cytometry background measurement. Haploid seedlings can be screened at an early stage, with greatly improved screening efficiency, while saving time and field planting workload. Attached Figure Description

[0014] Figure 1 The editing sites and sequence changes in the OsMATL gene-edited seedlings in Example 1 are shown, where A is... OsMATL A schematic diagram of the gene structure and the changes in gene sequence at the editing site. B is a sequence comparison diagram of wild-type OsMATL protein and edited mutant OsMATL protein.

[0015] Figure 2 The images show the haploid identification results in the hybrid offspring of Example 1. In the images, A represents photographs of fluorescent and non-fluorescent seeds under white light and excitation light in the hybrid offspring, B represents the peak shapes of diploid and haploid seedlings identified by flow cytometry, and C represents the hybridization results of the induction line with Jingeng7B DGMS, Wuxianggeng DGMS, Meixiangzhan DGMS, and DR610 DGMS. Detailed Implementation

[0016] Example 1 1. Creation of different types of dominant male-sterile lines Using tristigious male sterile rice (SMDGMS, see Huang Xianbo, Tian Zhihong, Deng Zeqin, et al. Preliminary identification of tristigious nuclear male sterility gene in rice [J]. Acta Agronomica Sinica, 2008, 34(010):1865-1868.DOI:10.3724 / SP.J.1006.2008.01865.) as the female parent, and Jinjing 7B (an excellent maintainer line bred by Hubble), Wuxiangjing 7375, Meixiangzhan 2, and indica rice DR610 (see National Rice Data Center, DR610, parental origin: female parent R538 / R91, male parent 10-3084) were used as recurrent parents. Through continuous backcrossing, the plant phenotype was observed in each backcross generation. The sterile plants that most resembled the recurrent parents were selected for further backcrossing. Finally, new dominant male sterile materials that were consistent with the recurrent parents were obtained and numbered as (Jingeng7B DGMS; Wuxianggeng DGMS; Meixiangzhan DGMS; DR610DGMS).

[0017] 2. Cultivation of haploid inducible lines expressing red fluorescent markers Using gene editing technology to target and knock out the japonica rice variety Xidao No. 1 OsMATL Using the haploid inducible line Osmatl-XD as the parent, the constitutively expressed red fluorescent protein gene mCherry was introduced via transgenic technology. Analysis of the fluorescent protein expression segregation characteristics in the self-crossed progeny yielded the haploid inducible line Osmatl-XD-mCherry, which exhibits a single copy insertion and stable expression of the fluorescent protein gene. Through hybridization with indica rice DR610 and subsequent self-crossing of the progeny, an inducible line with an indica rice background, Osmatl-DR610-mCherry, was obtained.

[0018] The specific operating method is as follows: (1) Target design: Based on the target site design principles of CRISPR-Cas9 technology, one gRNA target site was designed on the OsMATL gene sequence using an online design website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), with the guide RNA sequence being GTCCGGTGGCTGCGCAACAA. A gRNA sequence primer with a linker was synthesized.

[0019] U6AF: GCCGGTCCGGTGGCTGCGCAACAA (SEQ ID NO. 1); U6AR: AAACTTGTTGCGCAGCCACCGGAC (SEQ ID NO. 2).

[0020] (2) Construction of gene editing vectors: The vector pHun4c12s was digested with Bsa I enzyme, and the digestion product was recovered to obtain a linearized vector. Primer annealing: Add 10 µL each of the gRNA and its reverse complementary sequence to a PCR tube. Perform the following program in a PCR instrument: 95℃, 5 min; 4℃, 1 h.

[0021] gRNA was ligated into the gene editing vector pHun4c12s. The ligated vector was then transformed into competent E. coli cells, plated, and incubated overnight at 37°C. Single colonies were picked and cultured, and PCR was performed to confirm whether the target fragment had been ligated into the vector. Plasmids with correct sequencing results were extracted and sequenced. Plasmids with correct sequencing results were electroporated into Agrobacterium tumefaciens EHA105. The PCR identification and sequencing primer sequences are as follows: ZT F: GGGAACCCTGTGGTTGACAT (SEQ ID NO.3); ZT R: GCTGGTTGGGTCCGTTAGA (SEQ ID NO. 4).

[0022] The PCR system and procedure are as follows: Table 1

[0023] Table 2

[0024] (3) Transgenic: Mature embryo callus tissue of rice Xidao No. 1 was used as explant; Agrobacterium tumefaciens carrying the OsMATL gene editing vector was used for infection and transformation; transgenic plants were obtained through resistance selection, tissue differentiation, rooting and hardening. Primers MATL F / R were designed on both sides of the OsMATL gene editing site. DNA was extracted from the leaves of the transgenic plants, amplified by PCR, sequenced, and compared with the reference genome sequence to identify plants homozygous for OsMATL gene editing.

[0025] MATL F: GCGGTAAGTTCCTGGTGC (SEQ ID NO.5); MATL R: TGGATGCGGAGGTAGTCG (SEQ ID NO. 6).

[0026] The PCR system and procedure are as follows: Table 3

[0027] Table 4

[0028] (4) Screening for skeletonless editing pure lines: Leaves were taken from homozygous OsMATL gene-edited plants, and DNA was extracted. The vector backbone was identified using hygromycin primers, and plants without the vector backbone were ultimately screened out as homozygous for OsMATL gene editing. The hygromycin primer sequences are as follows: HYG-F: AGAAGAAGATGTTGGCGACCT (SEQ ID NO.7); HYG-R: GTCCTGCGGGTAAATAGCT (SEQ ID NO. 8).

[0029] The PCR system and procedure are as follows: Table 5

[0030] Table 6

[0031] The carrier-free skeleton obtained by identification OsMATL In homozygous gene-edited plants, an A atom was inserted at the gene-editing site, leading to subsequent changes in the protein sequence. Figure 1 ).

[0032] Figure 1 The wild-type OsMATL protein sequence (SEQ ID NO.9) is as follows: MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKMLGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTD DATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLRNKGMAPIIDIFMAASSDLVDIHAAV MFQSLHSDGDYLRIQDNTLHGDAATVDAATRDNMRALVGIGERMLAQRVSRVNVETGRYVEVPGAGSNADALRGFARQLSEERRARLGRRNACGGGGEGEPSGVACKR* The sequence of the mutant OsMATL protein (SEQ ID NO.10) is as follows: MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKMLGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTDD ATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLRKQGDGAHHRHLHGGQLRPRRHPRRRH VPVAAQRRRLPPHPGQHAPRRRRHGGRRHQGQHAGARRDRRADAGAAGVEGQRRDRQVRRGARRRQQRRRAEGLROAALRGEEGEARSAKRLRRRRRRRAQRRGVQAL* (5) Constructing the UBI:mCherry vector Using the 163-mCherry vector as a template, primers with adapters were designed to clone the mCherry gene fragment. The primer sequences are as follows: 1390 mcherry F: GTTACTTCTGCACTAGGTACCatggtgagcaagggcgaggag (SEQ IDNO.11); 1390 mcherry R: TCTTAGAATTCCCGGGGATCCttacttgtacagctcgtcca (SEQ ID NO. 12).

[0033] The PCR system and procedure are as follows: Table 7

[0034] Table 8

[0035] PCR products were detected using a 1% agarose gel electrophoresis, and the mCherry gene fragment was recovered using a gel extraction kit (EasyCare). The pCUBi 1390 vector plasmid was digested with KpnI and BamHI in the following manner: Table 9

[0036] The enzyme digestion time was 4 h and the incubation temperature was 37℃. The enzyme digestion products were detected by 1% agarose gel, and the pCUBi 1390 vector enzyme digestion products were recovered by a gel recovery kit (EasySpeed) and the concentration was determined.

[0037] The pCUBi 1390 vector digestion product was ligated with the mCherry clone fragment using a homologous recombinase and then transformed into E. coli. The ligation system is as follows: Table 10

[0038] Transformation and validation of *E. coli*: The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing kanamycin (50 mg / L), and incubated at 37°C for 16 h. Single colonies were picked, plasmids were extracted by shaking, and the correctness of the inserted fragment was verified by Sanger sequencing.

[0039] (6) Transgenic: Seeds of the skeletonless edited pure line were induced to form callus. Agrobacterium carrying the UBI:mCherry vector was used to infect the callus tissue, and transgenic seedlings were obtained. By analyzing the fluorescence protein expression segregation characteristics of the self-pollinated progeny, a haploid inducible line with single-copy insertion and stable expression of the fluorescence protein gene was obtained. Osmatl -XD-mCherry.

[0040] (7) Creation of haploid induction lines with indica rice background haploid induction system Osmatl XD-mCherry (as male parent) was crossed with indica rice DR610 (as female parent). Seeds from the hybrids germinated, and seedlings exhibiting red fluorescence were selected for self-pollination. After maturity, individual plants were harvested, and each packet of seeds was tested for fluorescence and... OsMATL Based on the editing situation, select all those with red fluorescence and OsMATL By editing homozygous single plants, an inducer line with an indica rice background was obtained. Osmatl -DR610-mCherry.

[0041] 3. Hybrid production of haploid seeds Using different types of dominant male-sterile lines as maternal parents, and haploid induction lines carrying mCherry fluorescent protein... Osmatl -DR610-mCherry was used as the male parent, and cross-pollination was completed by clipping the bark.

[0042] 4. Preliminary differentiation between diploids and candidate haploids was achieved through fluorescence observation. The hybrid seeds were dehulled, sterilized with 20% sodium hypochlorite for 10 minutes, and soaked in water at 37°C for 10 hours. The seeds were then placed in petri dishes with moistened filter paper at the bottom and cultured until roots and shoots emerged. Germinated seeds were observed under a fluorescence microscope for red fluorescence, paying particular attention to the fluorescence at the roots and shoots. Seeds without fluorescence were selected and cultured further. Using this method, we screened for non-fluorescent seeds from hybrids of Osmatl-DR610-mCherry with different types of dominant male-sterile lines. We obtained 588, 804, 433, and 5269 seeds from hybrids with Jinjing 7B, Wuxiangjing 7375, Meixiangzhan, and DR610 background DGMS, respectively. Of these, 14, 247, 86, and 212 seeds without fluorescence were selected, respectively. Further flow cytometry analysis yielded 4, 5, 2, and 9 haploid seedlings (Table 11). Comparisons of fluorescence observation between haploid and diploid plants in these progeny, flow cytometry peak patterns, and overall plant phenotypes are shown below. Figure 2 As shown, all haploid plants were significantly shorter and sterile compared to diploid plants in the same background, consistent with the characteristics of haploids. Figure 2 C).

[0043] Table 11. Statistics on fluorescence and haploid counts in hybrid seeds. 5. Flow cytometry was used to determine the ploidy of candidate plants. Cut 1cm lengths of freshly grown rice (10 days old) and place them in a culture dish. Add 200µL of nuclear extraction buffer from the Kono ploidy analysis kit (catalog number KRP2301-250T). Fix one end of the leaf with forceps and quickly and evenly mince each tissue vertically downwards with a scalpel. Add 200µL of cell staining solution (mainly DAPI) and mix well. Filter the mixture through a 40μm nylon mesh into a 2ml round-bottom centrifuge tube. Then, perform flow cytometry analysis on a BECKMAN COULTER cytoFLEX A00-1-1102 flow cytometer. Flow cytometry parameters are set as follows: excitation wavelength UV 355nm or 405nm, detection channel DAPI channel (emission wavelength 450±50nm), flow rate controlled at 30-40μL / min, and at least 5000 cell nuclei detected per sample. Haploid or diploid can be determined based on the peak chromatogram. Figure 2 B).

[0044] OsMATL genome sequence (SEQ ID NO.13):

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for producing and identifying rice haploids, characterized in that, Includes the following steps: (1) Using a dominant male-sterile line as the female parent and a haploid inducible line carrying the red fluorescent protein gene mCherry as the male parent, hybrid pollination was carried out to obtain hybrid seeds; (2) After the hybrid seeds germinate, candidate haploid seeds without fluorescence are screened by observing the red fluorescence of the roots and buds.

2. The method according to claim 1, characterized in that, The method for creating the dominant male-sterile line described in step (1) is as follows: using three dominant male-sterile rice varieties as the female parent and Wuxiangjing 7375, Meixiangzhan 2, or Indica rice DR610 as the recurrent parent, dominant male-sterile materials with the same genetic background as the recurrent parent are obtained through continuous backcrossing and selection.

3. The method according to claim 2, characterized in that, The method for cultivating the haploid inducible line in step (1) includes: (a) The rice OsMATL gene was edited using CRISPR-Cas9 technology to obtain OsMATL gene mutants; (b) The constitutively expressed red fluorescent protein gene mCherry was introduced into the mutant by transgenic technology, and lines with single-copy insertion and stable expression of the fluorescent protein gene were screened to obtain haploid inducible lines.

4. The method according to claim 3, characterized in that, The rice mentioned in step (a) is Xidao No.

1.

5. The method according to claim 4, characterized in that, The target site sequence of the CRISPR-Cas9 technology described in step (a) is GTCCGGTGGCTGCGCAACAA.

6. The method according to claim 5, characterized in that, The OsMATL gene mutant described in step (a) is a vector-free edited pure line, and the encoded OsMATL protein sequence is changed from SEQ ID NO.9 to SEQ ID NO.

10.

7. The method according to claim 6, characterized in that, It also includes step (3) identifying haploid plants by flow cytometry of seedlings obtained from candidate haploid seeds.

8. The application of the method according to any one of claims 1 to 7 in the production and identification of rice haploids.

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