Application of oryza sativa leaf whitening gene OsLCD2 in regulation and control of oryza sativa leaf color traits

Abstract

The invention belongs to the field of plant genetic engineering, particularly relates to application of a rice leaf whitening gene OsLCD2 in regulation and control of rice leaf color traits, and further provides a low-temperature sensitive rice leaf whitening gene oslcd2. The OsLCD2 gene and the oslcd2 gene provided by the invention have application prospects in purity identification of hybrid F1-generation hybrid in rice breeding and breed conservation purity identification of sterile line selfing breeding, and have important significance in improving the seed purity and reducing the production risk of hybrid rice.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the application of a rice leaf albino gene OsLCD2 in regulating rice leaf color traits. Background Art

[0002] Ribosomes are the basic components of the translation system of all living cells. Similar to fungi and bacteria, plant chloroplasts have 70S ribosomes, which are composed of 30S small subunits and 50S large subunits (Schmid LM, et al. Chloroplastribosome biogenesis factors. Plant Cell Physiol. 2024, 65: 516-536). The 30S small subunit is composed of 25 plastid ribosomal proteins (PRP) and 16S rRNA, of which 13 PRP proteins are encoded by the nuclear genome and the remaining 12 are encoded by the plastid genome; the 50S large subunit contains 23S, 5S and 4.5S rRNA and 33 PRP proteins, of which 9 PRP proteins are encoded by the nuclear genome and the remaining 24 are encoded by the plastid genome (Yamaguchi K, Subramanian AR. The plastid ribosomal proteins. Identification of all the proteins in the 50S subunit of an organelle ribosome (chloroplast). J Biol Chem. 2000, 275: 28466-28482; Yamaguchi K, et al. The plastid ribosomal proteins. Identification of all the proteins in the 30S subunit of an organelle ribosome (chloroplast). J Biol Chem. 2000, 275: 28466-28482; Yamaguchi K, et al. The plastid ribosomal proteins. Identification of all the proteins in the 30S subunit of an organelle ribosome (chloroplast). J Biol Chem. 2000, 275: 28455-28465; Stoppel R, Meurer J. The cutting crew-ribonucleases are key players in the control of plastid gene expression. J Exp Bot. 2012, 63: 1663-1673.).Based on protein two-dimensional electrophoresis and yeast two-hybrid techniques, RP proteins such as PRPS1-5 and PRPS10 that constitute 30S have been identified and cloned from plants (Yamaguchi K, Subramanian AR. Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit. Eur J Biochem. 2003, 270: 190-205; Xiong HB, et al. AtNusG, a chloroplast nucleoid protein of bacterial origin linking chloroplast transcriptional and translationalmachineries, is required for proper chloroplast gene expression in Arabidopsisthaliana. Nucleic Acids Res. 2022, 50: 6715-6734). Studies have shown that the new leaves of the single-base substitution mutant rps5 of the Arabidopsis PRPS5 gene are light yellow, and the plant growth and development are significantly delayed and weak. The results indicate that this type of protein is closely related to chloroplast production and development (Zhang J, et al. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis. J Exp Bot. 2016, 67: 2731-2744). Using gene editing technology, two mutants us10c-1 (single base T insertion) and us10c2 (single base substitution leading to amino acid substitution) were created for the Arabidopsis PRPS10 gene. Studies have shown that both mutants exhibit homozygous albinism and lethality, while heterozygous mutants exhibit albinism and spotted leaves (Sun X, et al. The uS10c-BPG2 module mediates ribosomal RNA processing in chloroplastnucleoids. Nucleic Acids Res. 2024, 52: 7893-7909). However, there have been no reports on the temperature sensitivity of the PRPS10 gene.

[0003] In hybrid rice breeding, especially two-line hybrid breeding, since sterile lines, such as thermosensitive two-line sterile lines, are usually fertile at low temperatures but sterile at high temperatures, the fertility conversion of such sterile lines is affected by the sterility threshold temperature (usually the sterility threshold temperature of sterile lines is required to be lower than 23.5°C). However, in the two-line rice seed production process, due to the complexity and variability of natural temperature, sterile lines often self-pollinate during seed production, thus affecting seed production purity and even leading to seed production failure. Leaf color is an important morphological marker of crops and can be used to simplify the breeding of improved varieties and improve the efficiency of hybrid production. Rice leaf color markers have the advantages of being intuitive, reliable, simple and easy to use in identifying true and false hybrids. They have been applied to rice two-line breeding, such as the light green leaf high-quality fragrant sterile line Huangxiangzhan S (Eshendao 20210099) bred by Huanggang Academy of Agricultural Sciences and other units (Cai Xingxing et al. Breeding of high-quality fragrant rice two-line sterile line Huangxiangzhan S. Hybrid Rice, 2024, 39: 70-72). Huangliangyou 913 (Eshendao 20210017) was bred using this sterile line; Hunan Taohuayuan Agricultural Science and Technology Co., Ltd. and Hunan Zhihe Seed Technology Co., Ltd. K Liangyou 22 (National Approved Rice No. 20196004) was bred from the yellow-leaf thermosensitive sterile line KT27S of rice, realizing the full mechanized seed production of hybrid rice with direct seeding and drone-assisted pollination (Zhang Qing et al. Fully mechanized seed production technology of hybrid rice based on leaf color-marked sterile line. Hybrid Rice, 2021, 36: 25-28); Yunnan Academy of Agricultural Sciences Institute of Grain Crops and Zhejiang University Institute of Nuclear Agriculture used the two-line sterile line NHR111S with albino-green leaf color markers and the restorer line Zhejiang Fu 26 to breed Yunfuyou 26 (Dian Te (Honghe) Approved Rice No. 2008010) ) (Deng Anfeng et al. Yunnan Province's first high-yield hybrid rice combination "Yunfuyou 26" prepared using a marker sterile line. Seed, 2008, 27: 94-96); Hefei Fenghai Marker Rice Research Institute used the sterile line Luzhou 86A carrying albino marker to breed rice varieties such as Luyou 855 (Anhui Pinshen 07010606), Luyou 827 (Anhui Pinshen 07010602) and Luyou 136 (Anhui Pinshen 05010474); Liuzhou Agricultural Science Research Institute used the light green leaf two-line sterile line Qian S and the restorer line Liuhui 1552 to select Qian Liangyou 1552 (Qin Ruide et al. Breeding and cultivation technology of a new hybrid rice combination Qianliangyou 1552. Agricultural Science and Technology Communications, 2019, 7: 303-304); The Rice Research Institute of Guangxi Academy of Agricultural Sciences used the purple-red leaf marker sterile line Xianhong A and the restorer lines Gui 981 and Gui 826 to breed Xianhongyou 981 (Guishen Rice No. 2020046) and Xianhongyou 826 (Guishen Rice No. 2020149) (Deng Guofu et al. Breeding and application of rice purple-red leaf marker sterile line Xianhong A. Chinese Rice, 2008, 5: 26-28). Summary of the invention

[0004] In view of the need for rice breeding screening through leaf color morphological markers in the prior art, the present invention provides the application of rice leaf albino gene OsLCD2 in regulating rice leaf color traits. The specific technical scheme is as follows:

[0005] In a first aspect, the present invention provides an application of a rice leaf albino gene OsLCD2 in regulating rice leaf color traits, and the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1.

[0006] In a second aspect, the present invention provides an application of a rice leaf albino gene OsLCD2 in molecular breeding of rice leaf color traits, wherein the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1.

[0007] In a third aspect, the present invention provides the use of the rice leaf albino gene OsLCD2 in the purity identification of hybrid rice F1 generation hybrids and / or the purity identification of sterile line self-pollinated seeds, and its genome sequence is shown in SEQ ID NO.1.

[0008] In a fourth aspect, the present invention provides a low-temperature-sensitive rice leaf albino gene oslcd2, the nucleotide sequence of the oslcd2 gene is shown in SEQ ID NO.2.

[0009] In a fifth aspect, the present invention provides the use of the above-mentioned oslcd2 gene in the molecular breeding of rice leaf color traits.

[0010] In a sixth aspect, the present invention provides the use of the above-mentioned oslcd2 gene in the purity identification of hybrid rice F1 generation hybrids and / or the purity identification of sterile line self-pollinated seeds.

[0011] The rice low temperature sensitive leaf albino gene oslcd2 of the present invention is obtained by screening from the offspring of the EMS mutant library of Xiushui 134. The phenotype of the mutant oslcd2 is that the leaves show albino characteristics under low temperature, and it is a recessive trait. Further artificial temperature control experiments show that the mutant leaves show obvious albino characteristics at 22°C. The present invention uses a map-based cloning method to preliminarily locate the oslcd2 gene in an approximately 198kb interval between RM14525 and RM14531 on the short arm of chromosome 3, which contains 36 genes. These genes are further cloned by PCR and sequenced for comparative analysis, and it is determined that the plastid ribosomal protein gene PRPS10 is the candidate gene of OsLCD2. Then, a genetic complementation expression vector is constructed and introduced into the mutant oslcd2, and the transgenic offspring show normal leaf color, thereby confirming that the low temperature sensitive albino leaf characteristics of the mutant oslcd2 are caused by a single base substitution of the OsLCD2 gene.

[0012] In a seventh aspect, the present invention provides an application of OsLCD2 protein in regulating leaf color traits of rice, and the amino acid sequence of OsLCD2 protein is shown in SEQ ID NO.3.

[0013] In an eighth aspect, the present invention provides a method for removing false hybrids in hybrid breeding, comprising the following steps:

[0014] (1) constructing rice leaf albino gene OsLCD2 mutant plants in which the gene was disrupted, knocked out, or silenced; the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1;

[0015] (2) hybridizing and backcrossing the mutant plants constructed in step (1) with rice maintainer lines and sterile line materials to cultivate three-line and / or two-line sterile lines with low-temperature-sensitive leaf albino traits, and producing seeds to obtain F1 hybrids;

[0016] The hybrid F1 generation obtained from seed production was identified at the seedling stage. Plants with albino leaves were identified as pseudo-hybrids of the sterile line self-pollination and were removed.

[0017] Furthermore, in step (1), a mutant plant containing the above-mentioned low-temperature-sensitive rice leaf albino trait gene oslcd2 is constructed.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The OsLCD2 gene and the oslcd2 gene provided by the present invention have application prospects in the hybrid purity identification of the hybrid F1 generation of rice breeding and the purity identification of sterile line self-pollination and seed preservation, which is of great significance for improving seed purity and reducing the risk of hybrid rice production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the phenotype and chlorophyll content of the mutant oslcd2 and its wild-type control at different temperatures; A is the seedling phenotype under different temperature conditions; B is the statistical graph of leaf chlorophyll content under different temperature conditions.

[0021] Figure 2 The results of the positional cloning of the OsLCD2 gene; A is the linkage molecular marker of the OsLCD2 gene; B is the BAC clone of the fine positioning interval; C is the structure of the OsLCD2 gene; D is the sequencing analysis of the mutation site of the OsLCD2 gene; E is the field phenotype of the complementary transgenic offspring; F is the determination of the total chlorophyll content of the transgenic offspring.

[0022] Figure 3This is an analysis diagram of the original plants of the three-line sterile line Xiushui 134A carrying the leaf albino gene oslcd2; wherein, A is a plant photo of the three-line sterile line Xiushui 134A carrying the albino leaf gene oslcd2, and B is the chlorophyll content analysis result of the three-line sterile line Xiushui 134A carrying the albino leaf gene oslcd2. DETAILED DESCRIPTION

[0023] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0026] In the following examples, the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1, the amino acid sequence of the OsLCD2 protein is shown in SEQ ID NO.3, the nucleotide sequence of the oslcd2 gene is shown in SEQ ID NO.2, and the amino acid sequence of the oslcd2 protein is shown in SEQ ID NO.4.

[0027] SEQ ID NO.1:

[0028] ATGGCCGTCTCCACCTCCGCCTCCTCCACCCTCCCGCTCCTCTCCCTCCATCGCGCCGCGGGGAACCCCAACCCGGCCGCACTCTCCTTCCCGCCTCCCCTCCGCGCGCCCCCGCTCCGCTCCCGCGCCGCCGCTGCCGCGGCCTCCGCCGCAGCACCGCCCGCCGAGACCATCCCCGACATGCCCGAGGAGACGGAGGGGACGGGGATTCCGATGCCGTCGTCGATTGGGGATGATGGGGAGCAGTTGGCACCCAAGCAGAAGATCAGAATTAAGCTGAGGTCTTAC TGGGTACCATTGATCGAGGACTCCTGTAAGAAGATCATTGAAGCTGCAAAAACAACAAATGCAAAGACGATGGGTCCAGTTCCTCTGCCAACCAAGCGAAGAGTATATTGTGTGCTCAATTCTCCTCACGTGCACAAAGATTCAAGGTTCCATTTTGAGATCAGAACGCACCAGCGATTGATTGATATCATGTACCCGACTGCACAAACAATAGATTCCTTGATGCAGCTCCAGCTCCCTGCCGGGGTAGATGTTGAGGTTAAGCTATGA。

[0029] SEQ ID NO.2:

[0030] ATGGCCGTCTCCACCTCCGCCTCCTCCACCCTCCCGCTCCTCTCCCTCCATCGCGCCGCGGGGAACCCCAACCCGGCCGCACTCTCCTTCCCGCCTCCCCTCCGCGCGCCCCCGCTCCGCTCCCGCGCCGCCGCTGCCGCGGCCTCCGCCGCAGCACCGCCCGCCGAGACCATCCCCGACATGCCCGAGGAGACGGAGGGGACGGGGATTCCGATGCCGTCGTCGATTGGGGATGATGGGGAGCAGTTGGCACCCAAGCAGAAGATCAGAATTAAGCTGAGGTTTTACTGGGTACCATTGATCGAGGACTCCTGTAAGAAGATCATTGAAGCTGCAAAAACAACAAATGCAAAGACGATGGGTCCAGTTCCTCTGCCAACCAAGCGAAGAGTATATTGTGTGCTCAATTCTCCTCACGTGCACAAAGATTCAAGGTTCCATTTTGAGATCAGAACGCACCAGCGATTGATTGATATCATGTACCCGACTGCACAAACAATAGATTCCTTGATGCAGCTCCAGCTCCCTGCCGGGGTAGATGTTGAGGTTAAGCTATGA。

[0031] SEQ ID NO.3:

[0032] MAVSTSASSTLPLLSLHRAAGNPNPAALSFPPPLRAPPLRSRAAAAAASAAAPPAETIPDMPEETEGTGIPMPSSIGDDGEQLAPKQKIRIKLRSYWVPLIEDSCKKIIEAAKTTNAKTMGPVPLPTKRRVYCVLNSPHVHKDSRFHFEIRTHQRLIDIMYPTAQTIDSLMQLQLPAGVDVEVKL。

[0033] SEQ ID NO.4:

[0034] MAVSTSASSTLPLLSLHRAAGNPNPAALSFPPPLRAPPLRSRAAAAAASAAAPPAETIPDMPEETEGTGIPMPSSIGDDGEQLAPKQKIRIKLRFYWVPLIEDSCKKIIEAAKTTNAKTMGPVPLPTKRRVYCVLNSPHVHKDSRFHFEIRTHQRLIDIMYPTAQTIDSLMQLQLPAGVDVEVKL.

[0035] Example 1 Isolation and genetic analysis of mutant oslcd2

[0036] The conventional japonica rice Xiushui 134 was induced with EMS and the leaf albino mutant oslcd2 was screened out from the progeny of the mutant library. After multiple generations of backcrossing and selfing in Hangzhou and Hainan, the genetically stable material of the mutant trait was obtained.

[0037] Mutants and wild-type plants were grown under different artificial temperature control conditions (22°C and 28°C), such as Figure 1 As shown in A, the mutant oslcd2 plants were found to be sensitive to low temperature, that is, they showed obvious white leaf symptoms at 22°C, while they were light green at 28°C. The chlorophyll content was further measured, and the results were as follows Figure 1 As shown in B, the chlorophyll and carotenoid contents of the mutant oslcd2 plants were basically the same as those of the wild-type plants at 28°C, while the chlorophyll a, chlorophyll b, carotenoid and total chlorophyll contents of the mutant oslcd2 plants were only 20.67%, 19.85%, 33.58% and 20.11% of those of the wild-type control at 22°C, respectively. These results show that the mutant albino leaf trait is sensitive to low temperature.

[0038] The oslcd2 plant was used as the female parent and was hybridized with Xiushui 134 and the indica rice restorer line 93-11 to obtain F1. The F1 plants performed normally. The F2 offspring obtained by self-pollination of the F1 showed wild-type and mutant phenotypes, with the segregation ratio of wild type: mutant = 3:1, indicating that the mutant albino leaf trait is controlled by a single recessive nuclear gene.

[0039] Example 2 OsLCD2 gene map-based cloning

[0040] 1. Fine positioning of OsLCD2 gene

[0041] 500 pairs of SSR markers and 50 pairs of InDel markers distributed on 12 rice chromosomes were selected, and the polymorphism of each marker between the mutant oslcd2 plant and 93-11 was analyzed to obtain polymorphic molecular markers evenly distributed on 12 chromosomes. The molecular markers linked to the target gene OsLCD2 were determined by the BSA method. The results showed that the SSR markers RM14487, RM14545 and RM14572 on the short arm of rice chromosome 3 were closely linked to the gene OsLCD2. Then, these three molecular markers were used to analyze the genotypes of 169 recessive extreme plants in the oslcd2 / 93-11F2 population. At the same time, combined with the position of each molecular marker in the chromosome, the physical arrangement of these three markers and the OsLCD2 gene on chromosome 3 was determined, namely RM14487-OsLCD2-RM14545-RM14572 (such as Figure 2 A).

[0042] In order to further precisely locate the OsLCD2 gene, 10 pairs of SSR markers were designed between RM14487 and RM14545, of which 2 pairs of markers were polymorphic between the parents. These 2 pairs of markers were used to perform genotyping on recessive plants in the F2 population that had single exchange with RM14487 and RM14545. Finally, the OsLCD2 gene was located between SSR markers RM14525 and RM14531, with a physical distance of about 198 kb, containing 36 genes (such as Figure 2 B).

[0043] 2. Leaf albinism mutation gene prediction and sequencing analysis

[0044] The 36 genes in the mutant and wild-type control regions were cloned by PCR and sequenced for comparative analysis. The results showed that the 284th base C of the ORF (open reading frame) of the candidate gene LOC_Os03g10060 was mutated to T, resulting in the mutation of the threonine it encodes to phenylalanine (such as Figure 2 C and Figure 2 D).

[0045] 3. Genetic Complementation Verification of Mutant oslcd2

[0046] Construction of gene complementation vector: A pair of primers were designed based on the sequence of the full-length cDNA of the OsLCD2 gene in GenBank (TIGR Locus: LOC_Os03g10060):

[0047] Upstream primer: OsLCD2F1: 5′-GGAGGTGGAGACGGCCAT-3′;

[0048] Downstream primer: OsLCD2R1: 5′-TCGAGCTCGGTACCCAGCTCCACGTGAATCATG-3′;

[0049] The total RNA from the leaves of Xiushui 134 was used as a template, and the cDNA fragment of the OsLCD2 gene was obtained by RT-PCR.

[0050] 1 st The cDNA reaction system is shown in the following table:

[0051] Table 1 Reaction system

[0052]

[0053] After the sample is mixed, it is incubated at 37°C for 5 minutes, and then 4 μL of 5×Easy RT MasterMix is ​​added, mixed and incubated at 42°C for 20 minutes, and then the cDNA fragment of the OsLCD2 gene is obtained by conventional PCR using the reaction product as a template. The PCR reaction system is as follows:

[0054]

[0055] Reaction condition parameters: 98°C denaturation for 10 seconds, 56°C annealing for 15 seconds, 72°C extension for 45 seconds, a total of 40 cycles. 72°C extension for 5 minutes.

[0056] Using Xiushui 134 genomic DNA as a template, the OsLCD2 fragment (including the gene ORF and its promoter sequence) was amplified by PCR using the following specific primers.

[0057] Upstream primer: OsLCD2F2: 5′-CTCTAGAGGATCCCTCTACTGCTAGCAATCTTCC-3′;

[0058] Downstream primer: OsLCD2R2: 5′-CATGATTCACGTGGAGCT-3′;

[0059] PCR reaction system:

[0060]

[0061] The Agrobacterium plasmid pCAMBIA1300-Nos was digested with SmaI, and then the cDNA fragment of OsLCD2 and its promoter were introduced into the linearized pCAMBIA1300-Nos using In-Fusion technology to form pOsLCD2. The correct plasmid was obtained by digestion and sequencing, and Agrobacterium-mediated transformation of Agrobacterium EHA105 was used to transform the mutant oslcd2 and obtain transgenic offspring (oslcd2-T1, oslcd2-T2). The results showed that the leaf color and chlorophyll content of the transgenic offspring transformed with pOsLCD2 returned to normal (such as Figure 2 E and Figure 2 F).

[0062] Example 3 Transformation of the albino leaf three-line sterile line

[0063] Xiushui 134 is a maintainer line, and its mutant oslcd2 is also a maintainer line. It can be hybridized with the three-line sterile line Xiushui 134A to obtain F1, which is a sterile line with normal leaf color carrying the oslcd2 gene. Then F1 is backcrossed with the mutant oslcd2 to obtain a BC1F1 population, and Xiushui 134A carrying albino leaf markers (i.e., plants carrying the homozygous oslcd2 genotype) is obtained from the offspring population (such as Figure 3 As shown in A). Figure 3 As shown in B, under low temperature conditions, the total chlorophyll content of Xiushui 134A carrying the albino leaf marker is only 23.13% of that of normal Xiushui 134A.

Claims

1. Application of rice leaf albino gene OsLCD2 in regulating rice leaf color traits, characterized in that: The nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.

1.

2. Application of the rice leaf albino gene OsLCD2 in molecular breeding of rice leaf color traits, characterized in that: The nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.

1.

3. Application of the rice leaf albino gene OsLCD2 in the purity identification of hybrid rice F1 generation and / or the purity identification of sterile line self-pollinated seeds, characterized in that: The nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.

1.

4. A low temperature sensitive rice leaf albinism gene oslcd2, characterized in that: The nucleotide sequence of the oslcd2 gene is shown in SEQ ID NO.

2.

5. Use of the oslcd2 gene as claimed in claim 4 in molecular breeding of rice leaf color traits.

6. Use of the oslcd2 gene as claimed in claim 4 in the purity identification of hybrid rice F1 generation hybrids and / or the purity identification of sterile line self-pollinated seeds.

7. Application of OsLCD2 protein in regulating rice leaf color traits, characterized in that: The amino acid sequence of OsLCD2 protein is shown in SEQ ID NO.

3.

8. A method for removing false hybrids in cross breeding, characterized in that: The following steps are involved: (1) constructing rice leaf albino gene OsLCD2 mutant plants in which the gene was disrupted, knocked out, or silenced; the nucleotide sequence of the OsLCD2 gene is shown in SEQ ID NO.1; (2) hybridizing and backcrossing the mutant plants constructed in step (1) with rice maintainer lines and sterile line materials to cultivate three-line and / or two-line sterile lines with low-temperature-sensitive leaf albino traits, and producing seeds to obtain F1 hybrids; The hybrid F1 generation obtained from seed production was identified at the seedling stage. Plants with albino leaf phenotypes were pseudo-hybrids of the sterile line self-pollination and were removed.

9. The method according to claim 8, characterized in that In step (1), a mutant plant containing the low-temperature-sensitive rice leaf albino gene oslcd2 as described in claim 4 is constructed.

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