Rice albino leaf and white panicle gene osal50 and related snp linkage markers and application thereof

Abstract

The present invention relates to a rice albino leaf and white panicle gene OsAL50 Linked markers with related SNPs and their applications. Rice albino leaf and panicle genes OsAL50 The CDS coding sequence is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2; and the promoter sequence 3.5 kb upstream of its start codon is shown in SEQ ID NO.3. OsAL50 Molecularly identified SNP-linked markers, including those associated with rice albino leaf and panicle genes OsAL50 Closely linked CAPS-08, CAPS-37 and CAPS-40. OsAL50 Genes can be used in hybrid rice breeding, using OsAL50 The albinism characteristics are displayed to identify and eliminate self-fertile false hybrids at the seedling stage to ensure the purity and quality of the improved varieties.

Description

Technical Field

[0001] The present invention relates to the field of rice molecular biology and genetic engineering technology, and more specifically to a rice albino leaf and white panicle gene OsAL50 Related SNP-linked markers and their applications. Background Art

[0002] 60%-80% of the nutrients required for rice grain filling come from leaf photosynthesis. Leaf albinism significantly reduces rice photosynthesis efficiency, severely impacting yield traits such as seed set and 1000-grain weight. Therefore, cloning and functional analysis of genes associated with albinism mutants is crucial for further improving photosynthesis efficiency and rice yield. Furthermore, albinism mutants have important application value in research on chloroplast structure development, photosynthetic pigment metabolism, photosynthesis mechanisms, biological development regulation, and genetic breeding. Furthermore, due to their high degree of recognition in practical applications, leaf color mutants can be used as phenotypic markers in the production of rice hybrids.

[0003] Rice leaf color mutants can be divided into eight types: albino, yellow, light green, white emerald, green and white, yellow-green, and striped. They can also be divided into total chlorophyll increase type, total chlorophyll deficiency type, chlorophyll a deficiency type, and chlorophyll b deficiency type according to the physiological mechanism of leaf color mutation. Most leaf color mutants are controlled by recessive genes in a single cell nucleus, and leaf color mutants controlled by dominant nuclear genes or cytoplasmic genes are rarely reported. Over the years, rice geneticists and breeders at home and abroad have conducted extensive research on leaf color mutants. To date, more than 50 rice leaf color-related genes have been successfully isolated and cloned from different rice mutant materials (Deng et al., 2014; Du et al., 2020). As V1 (Kusumi et al., 2011); V2 (Sugimoto et al., 2007); V3 (Yoo et al., 2009); St1 (Yoo et al., 2009); Gra(t) (Chen et al., 2009); cisc(t ) (Lan et al., 2010); YSA (Su et al., 2012); PAPST1 (Xu et al., 2013); albg (Jian et al., 2017) and VAL1 (Zhang et al., 2018) etc.

[0004] In addition, there are relatively few reports on white panicle mutants in rice. Currently, the white panicle mutant gene has been successfully isolated through map-based cloning technology. WLP1 (Song et al., 2014); WLP2 (Lv et al., 2017); St-wp (Chen et al., 2015); SLWP (Zhou et al., 2018); WP1 (Wang et al., 2016); WP2 (Wang et al., 2021); WP3 (Li et al., 2018) and WSP1 (Zhang et al., 2017). Studies have shown that these genes are mainly involved in the formation and development of chloroplast structure and the synthesis and decomposition of photosynthetic pigments. Their gene mutations and functional loss lead to the formation of albino leaves or albino panicles. The sources of albino mutants are very wide and can generally be divided into two categories: spontaneous mutations and artificial mutagenesis (including physical radiation and chemical mutagenesis). Among them, spontaneous mutations are stable heritable mutations formed under natural conditions and have a low probability of occurrence. Zhang Yi et al. discovered an albino-greening mutant from the hybrid offspring of indica rice G46B and Italian japonica rice Sirio. gra (Zhang Yi et al., 2008), Li Yuhong et al. discovered an albino mutant from the late-maturing japonica rice variety Huaidao No. 7 ir1 and Chen et al. found in japonica rice QiuFengM (Chen et al., 2007) are typical spontaneous mutants that can be stably inherited. Summary of the Invention

[0005] The present invention provides a rice albino leaf and white panicle gene OsAL50 The albinism phenotype of this mutant, linked to a related SNP marker and its application, is primarily manifested by varying degrees of albinism in leaves parallel to the veins at the three-leaf, four-leaf, and tillering stages. At the heading stage, the spikelets and peduncles become completely albinic, with the glumes exhibiting a white-green-yellow phenotype. After maturity, the spikelets and peduncles remain white, while the glumes gradually turn dark brown. This mutant's albinism trait is regulated by growth and development, unaffected by changes in external environmental conditions, and has no negative impact on rice yield. It can be used as a rice breeding marker to accurately identify and eliminate false seeds at the seedling stage, thereby ensuring the purity and quality of improved varieties.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a new rice albino leaf and white panicle gene OsAL50The CDS coding sequence is the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing, the protein coding sequence is the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, and the promoter sequence 3.5 kb upstream of the start codon of the gene is the nucleotide sequence shown in SEQ ID NO.3 in the sequence listing.

[0008] The present invention also provides a gene for albino leaves and white panicles of rice. OsAL50 Three pairs of tightly linked single nucleotide polymorphism (SNP) markers were used for molecular identification of the albinism gene. These three pairs of SNP markers were developed primarily by comparing the genome sequences of the japonica rice variety Nipponbare and the indica rice variety 9311. They are CAPS-08, CAPS-37, and CAPS-40, and consist of CAPS-08F / R, CAPS-37F / R, and CAPS-40F / R, respectively. The nucleotide sequences of the SNP markers are as follows:

[0009] The nucleotide sequence of CAPS-08F is shown in SEQ ID NO.4; the nucleotide sequence of CAPS-08R is shown in SEQ ID NO.5; the nucleotide sequence of CAPS-37F is shown in SEQ ID NO.6; the nucleotide sequence of CAPS-37R is shown in SEQ ID NO.7; the nucleotide sequence of CAPS-40F is shown in SEQ ID NO.8; and the nucleotide sequence of CAPS-40R is shown in SEQ ID NO.9.

[0010] Furthermore, the above rice albino leaf and white panicle gene OsAL50 The application is to use hybridization, backcrossing and other technical means to transform the above-mentioned albino gene OsAL50 Introduced into excellent three-line or two-line sterile lines, using OsAL50 Genetic albino traits are identified and eliminated at the seedling stage for false seeds, ensuring the purity and quality of the improved varieties. Furthermore, when hybrid rice seed is produced using superior three-line or two-line sterile lines carrying the albino trait, the resulting F1 generation seeds can also be phenotypically identified at the seedling stage, thereby eliminating false hybrids resulting from self-pollination. This effectively reduces the risks and losses associated with the seed production process for three-line or two-line hybrid rice.

[0011] The present invention obtains a spontaneous and stable hereditary rice albino leaf and panicle mutant from the high-generation breeding material of japonica rice variety GP50, and names it osal50 Compared with the wild type, the mutant osal50The leaves show varying degrees of whitening in stripes parallel to the veins at the three-leaf, four-leaf, and tillering stages of the seedling stage; the spikelets and stalks turn completely white at the heading stage, and the glume exhibits a white-yellow-green phenotype; at maturity, the spikelets and stalks remain white, and the glume gradually turns dark brown. The albinism trait of this mutant is only regulated by growth and development and is not affected by changes in external environmental conditions. In addition, this albinism trait has no negative effect on rice yield. In addition, compared with the wild-type GP50, the mutant osal50 The chlorophyll content in different albino tissues (including leaves and ears) at different growth and development stages decreased significantly. Transmission electron microscopy analysis showed that compared with wild-type green tissues, osal50 The morphological and structural development of chloroplasts in the albino parts of leaves and ears is severely hindered, with a significant reduction in the number and volume of chloroplasts. The mutant also lacks tightly packed thylakoid lamellae and orderly arranged grana. Through genetic analysis and gene mapping, it was determined that the albino trait of the mutant is controlled by a recessive nuclear gene, which was temporarily named OsAL50 The gene was precisely located in the 160kb physical interval of rice chromosome 1. Expression analysis showed that the candidate gene LOC_Os01g20110 was expressed in the mutant relative to the wild-type GP50. osal50 Expression levels were significantly downregulated in leaves and ears during the albinism period. Sequence analysis revealed no base mutations in the CDS coding region of LOC_Os01g20110, but 20 mutations were identified in its promoter, primarily single-base substitutions and deletions of varying lengths. Protein sequence and phylogenetic tree analysis revealed that LOC_Os01g20110 encodes an endoribonuclease E-like protein localized to the thylakoids. Its primary function is to participate in RNA processing in plastids and play a crucial role in chloroplast structural development.

[0012] It can be seen from the above description of the present invention that, compared with the prior art, the present invention has the following advantages:

[0013] Rice albino leaf and white panicle gene of the present invention OsAL50 Its CDS coding sequence is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, the promoter sequence 3.5 kb upstream of the start codon is shown in SEQ ID NO.3, and the three pairs of single nucleotide polymorphism (SNP) marker sequences closely linked to the gene are shown in SEQ ID NOs.4-9. OsAL50 The gene encodes an endoribonuclease E-like protein located on the thylakoid, which is mainly involved in the processing of RNA in the plastid and plays a vital role in the structural development of chloroplasts. osal50The mutant's phenotype primarily manifests as varying degrees of albinism in stripes parallel to the veins on leaves at the three-leaf, four-leaf, and tillering stages of the seedling stage. At the heading stage, the spikelets and peduncles become completely albinic, with the glumes exhibiting a white-yellow-green phenotype. After maturity, the spikelets and peduncles remain white, while the glumes gradually turn dark brown. The albino phenotype of this mutant differs significantly from that of existing albino mutants. This albinism is regulated solely by growth and development and is unaffected by environmental changes. Furthermore, this albino phenotype has no negative effects on key rice yield traits. Therefore, this gene can be used in the breeding of new hybrid rice varieties and seed purity testing. The albino phenotype it exhibits can serve as an effective marker for accurately identifying and eliminating false seeds at the seedling stage, providing a new gene and method for ensuring the purity and quality of improved rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Wild-type GP50 and albino mutant osal50 Comparison of phenotypic traits; in the figure, a: wild-type GP50 and albino mutant osal50 Phenotype at the seedling and one-leaf stage; b: wild-type GP50 and albino mutant osal50 Phenotype at the 2-leaf stage of seedlings; c: wild-type GP50 and albino mutant osal50 Phenotype at the 3-leaf stage of seedlings; d: wild-type GP50 and albino mutant osal50 Phenotype at the 4-leaf stage; e: albino mutant osal50 Three types of striped albino leaves with different degrees at the 4-leaf stage; f: wild-type GP50 and albino mutant osal50 Phenotype at the 7-leaf stage; g: wild-type GP50 and albino mutant osal50 Plant phenotype at the tillering stage; h: Leaf types of wild type GP50 at the tillering stage and albino mutant osal5 Three types of striped albino leaves at different degrees at the 0-tillering stage; i: wild-type GP50 and albino mutant osal50 Plant phenotypes at heading stage; j: wild-type GP50 and albino mutant osal50 Ear phenotype at heading stage; k: wild-type GP50 and albino mutant osal50 Grain phenotype at heading stage; l: wild-type GP50 and albino mutant osal50 Grain phenotype at maturity.

[0015] Figure 2 Wild-type GP50 and albino mutant osal50 Comparison of main agronomic traits; in the figure, a: wild-type GP50 and albino mutant osal50 Plant height; b: Wild-type GP50 and albino mutant osal50 Effective panicle number; c: wild-type GP50 and albino mutant osal50Spike length; d: wild type GP50 and albino mutant osal50 Grain length; e: wild-type GP50 and albino mutant osal50 Grain width; f: wild-type GP50 and albino mutant osal50 Fruiting rate; g: wild type GP50 and albino mutant osal50 Number of grains per ear; h: wild-type GP50 and albino mutant osal50 1000-grain weight; i: wild-type GP50 and albino mutant osal50 Flag leaf length; j: wild type GP50 and albino mutant osal50 The sword leaves are wide.

[0016] Figure 3 Wild-type GP50 and albino mutant osal50 Photosynthetic pigment content and chloroplast ultrastructure; Figure a: wild-type GP50 and albino mutant osal50 Light and pigment content in leaves at the 4-leaf stage; b: wild-type GP50 and albino mutant osal50 Light and pigment content in leaves during the tillering stage; c: wild-type GP50 and albino mutant osal50 Photosynthetic pigment content in the hull of ears at the heading stage; d: wild-type GP50 and albino mutant osal50 Photosynthetic pigment content in spikelets at the heading stage; ej: chloroplast ultrastructure of wild-type GP50 in leaves (e, f), spikelets (g, h), and glumes (i, j); kp: mutant osal50 Chloroplast ultrastructure in leaves (k, l), peduncles (m, n), and glumes (o, p). osal50 -1, -2, and -3 represent three different degrees of striped white leaf types. osal50 -1: light stripe type, osal50 -2: Heavy stripe type, osal50 -3: almost completely white type; CW: cell wall; A: starch granules; OG: osmiophilic granules; Ch: chloroplasts; G: grana; N: nucleus.

[0017] Figure 4 Albino mutant gene OsAL50 The chromosome fine positioning map; In the figure, a: molecular marker genetic linkage map of rice chromosome 1; b: OsAL50 Chromosome fine positioning of genes; c: OsAL50 Physical map of the gene; d: OsAL50 The gene was defined in a 160 kb physical region between the single nucleotide polymorphism (SNP) markers CAPS-08 and CAPS-37; e: A total of 26 candidate genes were predicted in this fine-mapping interval; the darker and lighter rectangles in a represent the centromere and OsAL50 The approximate location of the gene on the chromosome; cM represents the genetic distance; n represents the size of the F2 recessive population used for positioning.

[0018] Figure 5 for OsAL50 Candidate gene analysis diagram; Figure a: RT-PCR analysis of candidate genes in wild-type GP50 and albino mutant osal50 b: RT-PCR analysis of the expression level of candidate gene LOC_Os01g20110 in wild-type GP50 and albino mutant osal50 c: qRT-PCR analysis of the expression of the candidate gene LOC_Os01g20110 in the wild-type GP50 and the albino mutant osal50 Expression levels in leaves at the 4-leaf stage (left), glumes at the heading stage (middle), and peduncles at the heading stage (right); d, e: wild type GP50, Ningjing 44, 9311, Nipponbare, and albino mutants osal50 Promoter sequence analysis of the candidate gene LOC_Os01g20110.

[0019] Figure 6 for OsAL50 Schematic diagram of gene function verification. In the figure, a: Vector construction of the CRISPR-Cas9 gene editing system. LB is the left border of the vector, UBI is the UBI promoter, Cas9 is the Cas9 gene, gRNA is the guide RNA, rU6 is the rice U6 promoter, 35S is the 35S promoter, Hygro is the hygromycin gene, and RB is the right border of the vector. b: PCR identification of T0-generation positive transgenic plants using the specific functional markers FMHPT-F / R (left) and FMCas9-F / R (right); c: Phenotypic identification of two positive transgenic lines; d: Analysis of mutation types at the target site of the candidate gene LOC_Os01g20110 in the two positive transgenic lines.

[0020] Figure 7 for OsAL50 Analysis diagram of the protein sequence encoded by the gene; in the figure, a: amino acid sequence comparison analysis of OsAL50 protein with homologous proteins in grass plants such as maize, sorghum, barley, wheat, Brachypodium and millet; b: phylogenetic tree analysis of OsAL50 protein with homologous proteins in monocotyledons (including maize, sorghum, barley, wheat, Brachypodium and millet, etc.) and dicotyledons (Arabidopsis, tomato, potato and alfalfa, etc.). DETAILED DESCRIPTION

[0021] The following describes specific embodiments of the present invention with reference to the accompanying drawings. Numerous details are provided below to provide a comprehensive understanding of the present invention, but those skilled in the art will appreciate that the present invention can be practiced without these details. Well-known components, methods, and processes are not described in detail below.

[0022] Example 1: Rice albino mutant osal50 Phenotypes and agronomic traits

[0023] Albino mutant osal50 The natural field mutation of the wild-type japonica rice variety GP50 breeding material mainly manifests as: different degrees of striped whitening parallel to the veins on the leaves at the 3-leaf and 4-leaf stages of the seedling (refer to Figure 1 At the 5-leaf stage, the leaves begin to turn green gradually, and at the 7-leaf stage, the leaves turn green gradually. osal50 The seedlings showed a normal green phenotype that was completely consistent with the wild type (refer to Figure 1 f); During the tillering stage, the mutant showed a mixed phenotype, with green leaves and a large number of striped albino leaves appearing simultaneously, with some leaves showing an almost completely albino phenotype (refer to Figure 1 gh); the spikelets are completely white at the heading stage, and the husks show a white-yellow-green phenotype (refer to Figure 1 During the mature stage, the spikelets remain white, and the husks gradually turn dark brown ( Figure 1 In addition, the study showed that the albino characteristics of this mutant are only regulated by growth and development, and are not affected by changes in external environmental conditions (including temperature and light). In addition, the albino characteristics are significantly different from the albino phenotypes of existing albino mutants. In order to study the impact of this mutant phenotype on rice yield, the wild-type GP50 and the mutant were investigated. osal50 The main agronomic traits of the wild type and mutants were plant height, effective number of ears, ear length, grain length, grain width, seed setting rate, number of grains per ear, 1000-grain weight, sword leaf length and sword leaf width. The results showed that there were no significant differences in all yield traits between the wild type and the mutant (refer to Figure 2 ), indicating that the mutant osal50 The albino phenotype exhibited had no negative effect on rice yield.

[0024] Example 2: Rice albino mutant osal50 Determination of photosynthetic pigment content and observation of chloroplast ultrastructure

[0025] osal50 The albino leaf phenotype of the mutant was most obvious at the 4-leaf stage and the tillering stage, and the degree of albino change in different leaves of the same plant at these two growth stages was significantly different. osal50The photosynthetic pigment content in the striped albino leaves of plants at different stages and degrees. We selected three different types of striped albino leaves at the 4-leaf stage and the tillering stage: osal50 -1 (light streak type); osal50 -2 (heavy stripe type) and osal50 -3 (almost completely albino type) (refer to Figure 1 The results showed that compared with the green leaves of wild type GP50, osal50 The contents of chlorophyll a, chlorophyll b and carotenoids in leaves of three mutants with different albinism degrees decreased by 26.01-94.91%, 33.56-96.87% and 40.67-98.19% respectively at the seedling stage and the fourth leaf stage (refer to Figure 3 in a); in the tillering stage, the decreases were 29.96-89.03%, 35.43-91.89% and 42.93-96.33% respectively (refer to Figure 3 In addition, we also measured the contents of photosynthetic pigments in the glumes and stalks of ears at the heading stage.

[0026] The results showed that compared with wild-type GP50, osal50 The contents of chlorophyll a, chlorophyll b and carotenoids in the husks of the ears and branches decreased by 54.06%, 49.09% and 61.79% respectively (refer to Figure 3 c) and 95.81%, 97.4% and 92.07% (ref. Figure 3 In order to explore osal50 The physiological basis of the decreased photosynthetic pigment content was observed by transmission electron microscopy in wild-type GP50 and albino mutants. osal50 The ultrastructure and morphological development of chloroplasts in leaves at the 4-leaf stage and ears (including husks and stalks) at the heading stage. The results showed that the chloroplasts in green leaves and ears of wild type GP50 were morphologically and structurally complete (refer to Figure 3 (ej in Chinese) osal50 The morphological and structural development of chloroplasts in albino leaves and albino ears (including husks and stalks) is severely hindered, mainly manifested by a significant reduction in the number and volume of chloroplasts, and a lack of tightly packed thylakoid lamellae and orderly arranged grana (refer to Figure 3 kp), indicating osal50 The abnormal chloroplast structure development in the mutant is the main reason for the formation of its albino leaf and white ear phenotypes and the decrease in photosynthetic pigment content.

[0027] Example 3: Rice Albino Mutant Gene OsAL50 Genetic analysis and fine mapping of

[0028] Using mutants osal50 Four reciprocal cross combinations were formulated with japonica rice varieties GP50 and Ningjing 44 and indica rice variety 9311: GP50 / osal50 、 osal50 / GP50, Ningjing 44 / osal50 and 9311 / osal50 , and obtain the F1 generation. After the F1 generation self-pollinates, four independent F2 generation segregating population pairs are constructed. osal50 The results showed that the F1 plants obtained from the four hybrid combinations all showed a normal green phenotype, indicating that the albinism trait is recessive. In addition, in the four independent F2 hybrid populations, green plants (wild type) and albino plants (mutant) showed clear segregation, and all conformed to the 3:1 segregation ratio (χ2) controlled by Mendelian single genes. 2 =<χ 2 0.05 =3.84; P >0.05) (see Table 1). The above results show that osal50 The albinism trait is controlled by a single recessive gene in the nucleus. osal50 The mutant albino gene is tentatively named OsAL50 .

[0029]

[0030] Table 1 osal50 Segregation analysis of hybrid combinations

[0031] Application of Ningjing 44 / osal50 The obtained F2 segregating population is positive for the albino gene OsAL50 Preliminary positioning was carried out. First, 356 pairs of SSR primers evenly distributed on 12 rice chromosomes were used to detect polymorphisms of the parental and extreme gene pools. Then, 526 F2 recessive individual plants were genotyped and linkage analysis was performed using Mapmaker / Exp 3.0. The results showed that the Ningjing 44 / osal50 The obtained F2 segregating population identified a total of OsAL50 Five pairs of linked polymorphic SSR markers were identified: RM562, RM6642, RM10701, RM10710, and Rm10747. OsAL50 The gene was initially located on rice chromosome 1 and between Rm10710 and Rm10747, with relative genetic distances of 0.4 cM and 1.8 cM, respectively (refer to Figure 4 Further application of 9311 / osal50 Obtain F2 population pairs OsAL50The gene was precisely located and the genome sequences of japonica rice variety Nipponbare and indica rice variety 9311 were compared. A total of 95 pairs of insertion / deletion (InDel) markers and single nucleotide polymorphism (SNP) markers were developed between markers Rm10710 and Rm10747. osal50 , gene pool and 685 F2 recessive single plants were subjected to polymorphism detection and genotype analysis to obtain OsAL50 There are 14 pairs of tightly linked specific InDel and SNP markers: InDel-01, CAPS-08, CAPS-40, CAPS-37, InDel-07, InDel-08, ZZY-01, ZZY-04, InDel-11, InDel-13, InDel-15, InDel-18, InDel-20 and InDel-29. OsAL50 Fine mapping was performed between SNP markers CAPS-08 and CAPS-37, and the relative genetic distance was 0.1 cM (refer to Figure 4 (a, b) where the SNP marker CAPS-40 is OsAL50 Refer to the BAC clone sequences of Nipponbare and separate them into OsAL50 The positions of linked molecular markers on chromosomes were anchored on the corresponding BAC clones, thus constructing a coverage OsAL50 Physical map of gene loci (refer to Figure 4 Middle c).

[0032] Example 4: Rice Albino Leaf and White Ear Genes OsAL50 Candidate gene analysis

[0033] Combined with the RGAP rice genome annotation database, the physical distance of this fine-mapping interval corresponding to the japonica rice variety Nipponbare is 160 kb (refer to Figure 4 d), and a total of 26 candidate genes were annotated (refer to Figure 4 Among them, three genes (LOC_Os01g19970, LOC_Os0120140 and LOC_Os01g20160) encode MYB transcription factor, nuclear antigen protein and OsHKT1;5-Na +transporter, 15 genes (LOC_Os01g19910, LOC_Os01g19940, LOC_Os01g19950, LOC_Os01g19980, LOC_Os01g19990, LOC_Os01g20000, 10 genes (LOC_Os01g19920, LOC_Os01g19930, LOC_Os01g19960, LOC_Os01g20040, LOC_Os01g20050, LOC_Os01g20070, LOC_Os01g20080, LOC_Os01g20090, LOC_Os01g20010, LOC_Os01g20110, and LOC_Os01g20120) encode hypothetical proteins of unknown function. 16 candidate genes) encoding transposons or retrotransposon proteins. Since no transposons or retrotransposon proteins have been found to be associated with the formation of albino leaf or panicle phenotypes in rice, we focused on analyzing the other 16 candidate genes. Semi-quantitative PCR (RT-PCR) and real-time fluorescence quantitative PCR (qRT-PCR) analyses revealed that the candidate gene LOC_Os01g20110 was significantly expressed in wild-type GP50 compared to wild-type GP50. osal50 The expression levels of the mutant in leaves at the 4-leaf stage and ears (including husks and stalks) at the tillering stage were extremely significantly down-regulated. The other 15 candidate genes were significantly down-regulated in wild-type GP50 and mutant. osal50 There was no significant difference in the expression level or no expression (reference Figure 5 Therefore, we speculate that LOC_Os01g20110 is OsAL50 Further PCR amplification and sequencing technology were used to identify candidate genes from rice varieties GP50, Ningjing 44, Nipponbare, 9311 and osal50 The CDS coding sequence of the candidate gene LOC_Os01g20110 and the 3.5 kb DNA sequence upstream of the start codon were cloned. Sequencing analysis revealed that no base mutations were found in the CDS coding sequence of all rice varieties, but osal50 A total of 20 mutation sites were detected in the promoter region of the mutant LOC_Os01g20110 gene (refer to Figure 5 Therefore, combined with the above analysis results, we further speculate that LOC_Os01g20110 is OsAL50 Gene.

[0034] Example 5: Rice Albino Mutant Gene OsAL50 Functional analysis

[0035] To further verify that LOC_Os01g20110 is OsAL50 Gene, the CRISPR-Cas9 gene editing system was used to knock out the LOC_Os01g20110 gene in wild-type GP50 (refer to Figure 6 (a) Two positive transgenic lines with homozygous mutations, OsAL50-Cas9-1, were obtained. # and OsAL50-Cas9-2 # (refer to Figure 6 b), and both independent transgenic lines showed the same osal50 Consistent albino phenotype (refer to Figure 6 c), further confirming that the candidate gene LOC_Os01g20110 is OsAL50 , and regulate mutants osal50 Sequencing analysis showed that OsAL50-Cas9-1 # and OsAL50-Cas9-2 # The two transgenic lines had 3 and 11 base deletion mutations in the target sequence of the LOC_Os01g20110 gene, respectively (refer to Figure 6 d), which leads to a frameshift mutation, thus affecting the normal translation and function of the protein. To preliminarily analyze the function of the OsAL50 protein, combined with rice bioinformatics, a comparison search was conducted to obtain multiple homologous proteins in different plant species. Multiple amino acid sequence alignment analysis showed that OsAL50 has a high degree of sequence similarity with homologous proteins in maize, sorghum, wheat, barley, Brachypodium and millet (refer to Figure 7 According to the annotation results of NCBI and Phytomozome databases, the homologous genes in these grasses are predicted to encode an endoribonuclease E-like protein located in the thylakoid. So far, no albino mutants related to the loss of function of this protein have been reported in rice. In the studies of Arabidopsis and other grasses, the function of this protein is mainly involved in the processing of RNA in the plastid, and plays a vital role in the structural development of chloroplasts. Phylogenetic tree analysis showed that OsAL50 and homologous proteins in monocots were clustered into one category, while homologous proteins in dicots (including Arabidopsis, potato, tomato and alfalfa, etc.) were clustered into another category (refer to Figure 7(b) The OsAL50 protein sequence is highly conserved among higher plants, particularly in the grass family. These results suggest that OsAL50 likely encodes an endoribonuclease E-like protein localized in chloroplasts, which plays a crucial role in chloroplast structural development.

[0036] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited to this. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. Rice albino leaf and panicle genes OsAL50 , characterized in that: The CDS coding sequence is shown in SEQ ID NO.1, the encoded protein sequence is shown in SEQ ID NO.2; and the promoter sequence 3.5 kb upstream of the start codon is shown in SEQ ID NO.

3.

2. A rice albino leaf and panicle gene for use in claim 1 OsAL50 Molecularly identified SNP-linked markers characterized by: Including the rice albino leaf and white panicle gene OsAL50 The three pairs of tightly linked single nucleotide polymorphism (SNP) markers are CAPS-08, CAPS-37, and CAPS-40; The CAPS-08 is composed of CAPS-08F and CAPS-08R, and the nucleotide sequences thereof are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; The CAPS-37 is composed of CAPS-37F and CAPS-37R, and their nucleotide sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively; The CAPS-40 is composed of CAPS-40F and CAPS-40R, and the nucleotide sequences thereof are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

3. The rice albino leaf and white panicle gene according to claim 1 OsAL50 The application in hybrid rice breeding is characterized by: Through hybridization and backcrossing techniques OsAL50 Introduce excellent three-line or two-line sterile lines, and use OsAL50 The albinism characteristics are displayed to identify and eliminate self-fertile false hybrids at the seedling stage to ensure the purity and quality of the improved varieties.

4. The use according to claim 3, characterized in that: The rice albino leaf and white panicle gene OsAL50 The albinism characteristics include: different degrees of whitening of leaves in stripes parallel to the veins at the 3-leaf stage, 4-leaf stage and tillering stage; complete whitening of the spike stalks at the heading stage, and the glumes showing a white-green-yellow phenotype; after maturity, the spike stalks remain white, and the glumes gradually turn to black-brown.

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