GmBCM1 and GmBCM2 genes for identifying soybean hybrid yellowing and application of GmBCM1 and GmBCM2 genes

Abstract

The invention discloses GmBCM1 and GmBCM2 genes for identifying soybean hybrid yellowing and application of the GmBCM1 and GmBCM2 genes, and relates to the GmBCM1 and GmBCM2 genes and application of protein coded by the GmBCM2 genes in soybeans. The open reading frame nucleotide sequences of the GmBCM1 and GmBCM2 genes are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 in a sequence table. The GmBCM1 and GmBCM2 genes are applied to soybean molecule assisted breeding, gene selective breeding and AI breeding. The soybean GmBCM1 and GmBCM2 genes cooperatively control soybean hybrid yellowing phenotypes, and it is found that when plant individuals are Gmbcm1 / Gmbcm2 genotypes, soybean plants show phenotypes that the chlorophyll content of mature leaves is reduced, the photosynthetic efficiency of the leaves is reduced and the like. The genotype identification results of GmBCM1 and GmBCM2 related to soybean hybrid yellowing can be applied to soybean molecular assistance, gene selection breeding, AI breeding and other breeding technologies, and play a role in soybean breeding parent material optimization and advanced generation breeding material selection.

Description

Technical Field

[0001] The invention relates to application of GmBCM1 and GmBCM2 genes and encoded proteins in soybean. Background Art

[0002] Hybrid breakdown refers to the phenomenon of reduced hybrid viability or hybrid sterility in the F2 generation or higher generations produced by parental hybridization, which is often controlled by recessive gene loci. Bateson-Dobzhansky-Muller incompatibilities are currently commonly used to explain the phenomenon of hybrid breakdown. Hybrid breakdown includes hybrid weakness (weak growth, necrosis and yellowing) and hybrid sterility (male sterility, female sterility and zygotic sterility). The BDM model believes that two gene loci of species or varieties on different evolutionary branches of the same ancestor have mutated. For example, AAbb and aaBB are both derived from the ancestral genotype AABB. The fitness of the Ab and aB allele combination is the same as that of the ancestral AB allele combination. However, the fitness of aabb produced by the hybridization of species or varieties derived from the independently evolved a and b is reduced, causing hybrid sterility and hybrid weakness. This effect that causes a decrease in fitness is considered to be BDM incompatibility. Hybrid depression can be considered as a kind of reproductive isolation produced in the process of evolution. The phenomenon of hybrid depression has long been observed in animals and plants. Hybrid depression can explain the process of species domestication to a certain extent.

[0003] The phenomenon of soybean hybrid etiolation was reported as early as 1920, but the relevant regulatory genes have not been successfully cloned. Whether it is conventional hybrid breeding or molecular design breeding, the goal is to combine the excellent traits of the parents to achieve the purpose of breeding improved varieties. For soybean hybrid breeding, hybrid etiolation is undoubtedly an undesirable factor. Therefore, the identification of soybean hybrid etiolation genotypes for candidate parents and high-generation breeding materials can eliminate the possibility of hybrid etiolation in advance and guide soybean molecular-assisted breeding, gene selection breeding and AI breeding processes. Summary of the invention

[0004] The invention provides GmBCM1 and GmBCM2 genes for identifying soybean hybrid etiolation and applications thereof.

[0005] The open reading frame nucleotide sequences of the GmBCM1 and GmBCM2 genes used for identifying soybean hybrid etiolation in the present invention are shown as SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table, respectively.

[0006] Furthermore, the amino acid sequences of the proteins encoded by the GmBCM1 gene used to identify the GmBCM1 gene are shown in SEQ ID NO: 3 in the sequence listing.

[0007] Furthermore, the amino acid sequences of proteins encoded by the GmBCM2 gene used to identify the GmBCM2 gene are shown in SEQ ID NO: 4 in the sequence listing.

[0008] Further, the genotype of GmBCM1 used to identify Gmbcm1 is a mutation, that is, the open reading frame of Gmbcm1 includes the base sequence shown in SEQ ID NO:5.

[0009] Further, the Gmbcm2 genotype used to identify that Gmbcm2 is a mutant, that is, the open reading frame of Gmbcm2 includes the base sequence shown in SEQ ID NO:6.

[0010] The above is used to identify the application of GmBCM1 and GmBCM2 genes in soybean molecular assisted breeding, gene selection breeding and AI breeding.

[0011] The soybean varieties of Gmbcm1 / GmBCM2 and Gmbcm1 / GmBCM2 genotypes showed normal leaf color. The soybean varieties of these two genotypes were used as parents for hybridization. The F2 generation was obtained after self-pollination of the F1 generation. The Gmbcm1 / Gmbcm2 genotype individuals in the F2 generation or higher generations showed a phenotype of leaf yellowing. The characteristic indicators of soybean yellowing are the reduction of chlorophyll content in mature soybean leaves and the significant decrease in the energy conversion efficiency in the photochemical activity of the leaves.

[0012] The soybean GmBCM1 and GmBCM2 genes of the present invention synergistically control the chlorotic phenotype of soybean hybrids. It is found that when the plant individuals are of the Gmbcm1 / Gmbcm2 genotype, the soybean plants exhibit phenotypes such as reduced chlorophyll content in mature leaves and reduced photosynthetic efficiency of leaves. The results of the identification of the GmBCM1 and GmBCM2 genotypes related to soybean hybrid chlorosis can be applied to soybean molecular-assisted breeding, gene selection breeding, AI breeding and other breeding technologies, and play a role in the optimization of soybean breeding parent materials and the final selection of high-generation breeding materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 35 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Phenotype diagram of 168-Y;

[0014] Figure 2 45 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Phenotype diagram of 168-Y;

[0015] Figure 3 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F2:5 Phenotype diagram of 168-Y;

[0016] Figure 4 35 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis chart of the first and sixth compound leaves of 168-Y;

[0017] Figure 5 45 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis chart of the first and sixth compound leaves of 168-Y;

[0018] Figure 6 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis chart of the first and sixth compound leaves of 168-Y;

[0019] Figure 7 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M )Analysis diagram;

[0020] Figure 8 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Analysis of the effective quantum yield [Y(II)] of photosystem II of the first and sixth compound leaves of 168-Y;

[0021] Fig. 9 To use F 2:5 168 Population and BSA analysis located one of the candidate genes on chromosome 11;

[0022] Fig.10 35 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5 Phenotype diagram of 175-Y;

[0023] Fig.11 45 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5 Phenotype diagram of 175-Y;

[0024] Fig.12 55 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5Phenotype diagram of 175-Y;

[0025] Fig.13 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 Chlorophyll content analysis chart of the first and sixth compound leaves of 175-Y;

[0026] Fig.14 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 The maximum chlorophyll fluorescence efficiency (F) of the first and sixth compound leaves of 175-Y V / F M )Analysis diagram;

[0027] Fig.15 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 Analysis of the effective quantum yield [Y(II)] of photosystem II of the first and sixth compound leaves of 175-Y;

[0028] Fig.16 To use F 2:5 175 Population and BSA analysis located one of the candidate genes on chromosome 1;

[0029] Fig.17 For the fine mapping of candidate genes on chromosome 11;

[0030] Fig.18 For the fine mapping of candidate genes on chromosome 1;

[0031] Fig.19 For the sequence analysis of GmBCM1 and GmBCM2 genes in Dongnong 50 and Yanji;

[0032] Fig. 20 is the position of the sgRNA target sequence in the GmBCM1 gene;

[0033] Fig.21 Schematic diagram of sequence variation of Gmbcm1-12 and Gmbcm1-26 mutants;

[0034] Fig. 22 To analyze the variation of GmBCM1 protein structure in Gmbcm1-12 mutant;

[0035] Fig.23 For the variation analysis of GmBCM1 protein structure in Gmbcm1-26 mutant;

[0036] Fig.24To analyze the phenotypes of Gmbcm1-12 and Gmbcm1-26 mutants obtained by gene editing;

[0037] Fig.25 Chlorophyll content analysis of Gmbcm1-12 and Gmbcm1-26 mutants;

[0038] Fig.26 Analysis of the effective quantum yield [Y(II)] of photosystem II in Gmbcm1-12 and Gmbcm1-26 mutants;

[0039] Fig. 27 Phenotypic analysis of GmBCM2 gene overexpression materials;

[0040] Fig.28 Analysis of chlorophyll content in GmBCM2 gene overexpression materials;

[0041] Fig.29 Analysis of the effective quantum yield [Y(II)] of photosystem II in GmBCM2 gene overexpression materials;

[0042] Fig.30 Overexpression of the GmBCM2 gene can complement the phenotype of the gmbcm1 mutant;

[0043] Fig.31 Analysis of chlorophyll content in materials complementing the gmbcm1 mutant by overexpression of the GmBCM2 gene;

[0044] Fig.32 Analysis of the effective quantum yield [Y(II)] of photosystem II in materials complementing the gmbcm1 mutant with overexpression of the GmBCM2 gene. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0047] Specific implementation method 1: The open reading frame nucleotide sequences of the GmBCM1 and GmBCM2 genes used for identifying soybean hybrid etiolation in this implementation method are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing, respectively.

[0048] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the amino acid sequence of the protein encoded by the GmBCM1 gene is shown in SEQ ID NO: 3 in the sequence table. Other aspects are the same as those of specific embodiment 1.

[0049] Specific embodiment 3: This embodiment is different from specific embodiment 1 in that the amino acid sequence of the protein encoded by the GmBCM2 gene is shown in SEQ ID NO: 4 in the sequence table. Other aspects are the same as those of specific embodiment 1.

[0050] Specific embodiment 4: This embodiment is different from specific embodiment 1 in that Gmbcm1 is a mutant GmBCM1 genotype, that is, the open reading frame of Gmbcm1 includes the base sequence shown in SEQ ID NO: 5. Other aspects are the same as those of specific embodiment 1.

[0051] Specific embodiment 5: This embodiment is different from specific embodiment 1 in that Gmbcm2 is a mutant GmBCM2 genotype, that is, the open reading frame of Gmbcm2 includes the base sequence shown in SEQ ID NO: 6. Other aspects are the same as specific embodiment 1.

[0052] Specific embodiment six: Application of the GmBCM1 and GmBCM2 genes identified as described in specific embodiment one in soybean molecular assisted breeding, gene selection breeding and AI breeding.

[0053] Specific embodiment seven: Application of the mutant genotype as described in specific embodiment three or four in soybean molecular assisted breeding, gene selection breeding and AI breeding.

[0054] Example 1 Discovery of Soybean Hybrid Yellowing Mutants

[0055] First, we used the Chinese soybean variety Dongnong 50 with normal leaf color and the Japanese soybean variety Yanji to crossbreed and obtain the F1 generation. After self-pollination of the F1, we obtained the F2 generation population. A mutant with yellowing leaves was found in the F2 generation population, and its occurrence rate was about 1 / 16. According to Mendel's law of inheritance, the hybrid yellowing mutant was controlled by two recessive genes, and the mutant was named dry (double recessive yellowing).

[0056] Second, in this group of F 2:3 、F 2:4 and F 2:5 We found the dry mutant in all generations, indicating that the phenotype of this mutant is controlled by genes rather than environmental factors.

[0057] 3. Using F 2:5 The 168th strain in the RIL population (named F2:5 168) observed the phenotype of the dry mutant. 2:5 The ratio of the number of individuals with normal leaf color to the number of individuals with yellow leaves in 168 was 3:1. 2:5 168 The plant with normal leaf color (named F 2:5 168-G) and plants with yellow leaves (designated F 2:5 168-Y) phenotype was observed and it was found that F 2:5 The first compound leaf of 168-Y showed yellowing phenotype, and F 2:5 The sixth compound leaf of 168-Y did not show the phenotype of leaf yellowing, such as Figure 1 ( Figure 1 35 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 168-Y phenotype). On the 45th day after seed germination, F 2:5 The first compound leaf of 168-Y showed obvious yellowing phenotype, while the sixth compound leaf did not show yellowing phenotype. Figure 2 ( Figure 2 45 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 168-Y phenotype). On the 55th day after seed germination, F 2:5 The 168-Y plants showed a whole-plant yellowing phenotype, such as Figure 3 ( Figure 3 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 168-Y phenotype).

[0058] Chlorophyll content is often closely related to leaf color. 2:5 168-G and F 2:5 The chlorophyll content of the first and sixth compound leaves of 168-Y was measured. 2:5 Compared with 168-G plants, F 2:5 The chlorophyll content of the first compound leaf of 168-Y was significantly reduced; 2:5 The chlorophyll content of the sixth compound leaf of 168-Y is higher than that of Dongnong 50, Yanji and F 2:5 168-G The median value of chlorophyll content in the sixth compound leaf, such as Figure 4 ( Figure 4 35 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis of the first and sixth compound leaves of 168-Y). On the 45th day after germination, F2:5 The chlorophyll content of the first compound leaf of 168-Y was significantly lower than that of Dongnong 50, Yanji and F 2:5 The chlorophyll content of the first compound leaf of 168-G plant; 2:5 The chlorophyll content of the sixth compound leaf of 168-Y is similar to that of Iwahime and F 2:5 The chlorophyll content of the sixth compound leaf of the 168-G plant is comparable, e.g. Figure 5 ( Figure 5 45 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis of the first and sixth compound leaves of 168-Y). On the 55th day after germination, F 2:5 The chlorophyll contents of the first and sixth compound leaves of 168-Y were significantly lower than those of Dongnong 50, Yanji and F 2:5 168-G plants, such as Figure 6 ( Figure 6 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Chlorophyll content analysis of the first and sixth compound leaves of 168-Y).

[0059] The results of the above two parts show that the chlorophyll content of the young leaves of the dry mutant (the sixth compound leaves on the 35th and 45th days after germination) is similar to that of Dongnong 50, Yanji or F 2:5 The chlorophyll content of the sixth compound leaf of 168-G plants at the same time was similar. The chlorophyll content of the mature leaves of the dry mutant (the first compound leaves on the 35th, 45th and 55th days after germination and the sixth compound leaf on the 55th day) was significantly lower than that of Dongnong 50, Yanji or F 2:5 Chlorophyll content of leaves of 168-G plants at the same time. This result proves that the two regulatory genes of dry mutant mainly control the chlorophyll degradation process of mature leaves.

[0060] 4. Chloroplasts are the main photosynthetic organs. Leaf color and chlorophyll content will affect the photosynthetic rate of plants. 2:5 168-G and F 2:5 The photosynthetic rate of 168-Y was evaluated and the results showed that on the 55th day after germination, F 2: The maximum chlorophyll fluorescence efficiency (F V / F M ) and the effective quantum yield of photosystem II [Y(II)] were significantly lower than those of Dongnong 50, Yanji and F 2:5 168-G, such as Figure 7 ( Figure 7 55 days after germination, Dongnong 50, Iwahime, F 2:5168-G and F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M )Analysis diagram) and Figure 8 ( Figure 8 55 days after germination, Dongnong 50, Iwahime, F 2:5 168-G and F 2:5 Analysis of the effective quantum yield of photosystem II [Y(II)] of the first and sixth compound leaves of 168-Y. The results show that due to F 2:5 The yellowing of leaves of 168-Y resulted in a decrease in its photosynthetic rate.

[0061] Example 2: Preliminary localization of the gene controlling soybean hybrid yellowing mutant.

[0062] 1. The dry mutant appeared in about 1 / 16 of the F2 population of the cross between Dongnong 50 and Yanji. The dry mutant was controlled by two recessive genes. 2:5 The ratio of individuals with normal leaf color to individuals with yellow leaves in the RIL population of 168 was 3:1, indicating that a recessive gene had been fixed in this population. 2:5 168-G leaves were mixed in the pool, and 20 F 2: The 5168-Y leaf pool was used to extract the genome of the leaf pool using the CTAB method, and the genome resequencing was performed on an Illumina HiSeq instrument by Annoroad Gene Technology Co., Ltd. The obtained Illuminasequencing short sequences were aligned to the soybean Glycine max Wm82.a2.v1 reference genome [G.max Wm82.a2.v1:Phytozome(doe.gov)], and then the data obtained from sequencing were used for BSA sequencing analysis. The results of the BSA analysis showed that one of the genes controlling soybean hybrid yellowing was located at the front end of chromosome 11, such as Fig. 9 ( Fig. 9 To use F 2:5 168 population and BSA analysis located one of the candidate genes on chromosome 11).

[0063] 2. InDel molecular markers DY11_1943000, DY11_3136020 and DY11_4156783 were designed at both ends and in the middle of the candidate segment of chromosome 11. The sequences of the molecular markers are shown in Table 1. These three primer pairs were used to identify F 2:5 The genotypes of the previous generation individuals of other lines that could isolate the dry mutant in the first generation RIL population were used to obtain the individual F with the leaf yellowing genotype in the candidate segment 11. 2:4 175. Then to F 2:5 175-G and F2:5 Phenotypic analysis and physiological index determination of 175-Y plants were performed. 2:5 The first compound leaves of 175-Y showed yellowing phenotype, and F 2:5 The sixth compound leaf of 175-Y did not show the phenotype of leaf yellowing, such as Fig.10 ( Fig.10 35 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5 175-Y phenotype). On the 45th day after seed germination, F 2:5 The first and sixth compound leaves of 175-Y showed obvious leaf yellowing phenotypes, such as Fig.11 ( Fig.11 45 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5 175-Y phenotype). On the 55th day after seed germination, F 2:5 The 175-Y plants showed a whole-plant yellowing phenotype, such as Fig.12 ( Fig.12 55 days after germination, Dongnong 50, Iwahime, F 2:5 175-G and F 2:5 The results of chlorophyll content measurement were the same as those of phenotypic observation, namely: 2:5 The chlorophyll content of the young leaves (the sixth compound leaf 35 days after germination) of 175-Y and F 2:5 175-G is similar, while F 2:5 The chlorophyll content of mature leaves (the first compound leaf after 35 days after germination and the sixth compound leaf after 45 days after germination) in 175-Y was significantly lower than that in F 2:5 175-G, such as Fig.13 ( Fig.13 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 Chlorophyll content analysis of the first and sixth compound leaves of 175-Y). 2:5 175-G and F 2:5 The photosynthetic rate of 175-Y was evaluated and the results showed that on the 35th day after germination, F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M ) and the effective quantum yield of photosystem II [Y(II)] and F 2:5 175-G; on the 45th day after germination, F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M) and the effective quantum yield of photosystem II [Y(II)] were significantly lower than those of F 2:5 175-G, F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M ) and the effective quantum yield of photosystem II [Y(II)] and F 2:5 175-G; on the 55th day after germination, F 2:5 The maximum chlorophyll fluorescence efficiency (F V / F M ) and the effective quantum yield of photosystem II [Y(II)] were significantly lower than those of F 2:5 175-G, such as Fig.14 ( Fig.14 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 The maximum chlorophyll fluorescence efficiency (F) of the first and sixth compound leaves of 175-Y V / F M )Analysis diagram) and Fig.15 ( Fig.15 F at 35, 45 and 55 days after germination 2:5 175-G and F 2:5 Analysis of the effective quantum yield of photosystem II [Y(II)] of the first and sixth compound leaves of 175-Y. The results show that due to F 2:5 The yellowing of leaves of 175-Y led to a decrease in its photosynthetic rate. This result indicates that 2:5 The phenotype of the dry mutant in the RIL population of 175 was similar to that of the F 2:5 The phenotypes of dry mutants in the RIL population of 168 were similar and were regulated by the same target gene.

[0064] Table 1 Primer sequences for identification of chromosome 11 segment.

[0065]

[0066] 3. The above results prove that F 2:4 The genotype of plant 175 was a leaf yellowing genotype in the chromosome 11 segment, F 2:5 The dry mutant was isolated from strain 175, and the isolation ratio was about 1:4. 2:5 175-G mixed pool, 20 strains of F 2:5175-Y pool, the genome of the leaf pool was extracted using the CTAB method, and the genome resequencing was performed on an Illumina HiSeq instrument by Annoroad Gene Technology Co., Ltd. The obtained Illuminasequencing short sequences were aligned to the soybean Glycinemax Wm82.a2.v1 reference genome [G.max Wm82.a2.v1:Phytozome(doe.gov)], and then the data obtained by sequencing were used for BSA sequencing analysis. The results of BSA analysis showed that one of the genes controlling soybean hybrid yellowing was located at the end of chromosome 1, such as Fig.16 ( Fig.16 To use F 2:5 175 population and BSA analysis located one of the candidate genes on chromosome 1).

[0067] Example 3: Fine mapping of genes controlling soybean hybrid yellowing mutants.

[0068] 1. Design 15 new molecular markers in the candidate segment of chromosome 11, the sequences are shown in Table 2. 1386 F2 population individuals and 2147 F 2:6 DNA of individuals from the RIL population of 168. The 15 molecular markers in Table 2 and the 3 molecular markers in Table 1 were used to identify the genotypes of the above 3553 individuals. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 35s, a total of 35 cycles, and then 72℃ extension for 10min. The size of the PCR product was identified by agarose gel electrophoresis. According to the recombination rate of phenotype and genotype, the candidate gene was located in the segment of about 55kb between the two molecular markers DY11_3159120 and DY11_3214107, as shown in Figure 2. Fig.17 ( Fig.17 (Refer to the fine positioning of candidate genes on chromosome 11). According to the annotation information of the genome, this segment contains 4 genes: Glyma.11G043200, Glyma.11G043300, Glyma.11G043400 and Glyma.11G043500. The annotation information of these 4 genes is shown in Table 3.

[0069] Table 2 Molecular marker sequences for fine mapping of candidate gene No. 11.

[0070]

[0071]

[0072] Table 3 Annotation information of candidate genes on chromosome 11.

[0073] Gene ID Description Glyma.11G043200 C2H2-type zinc finger Glyma.11G043300 Acetylornithine deacetylase / N-acetylornithinase Glyma.11G043400 CAAX protease self-immunity(Abi) Glyma.11G043500 DNA-binding bromodomain-protein

[0074] 2. Design 18 molecular markers in the candidate segment of chromosome 1, the sequences are shown in Table 4. 3542 F 2:6 The 18 molecular markers in Table 4 were used to identify the 3542 F 2:6 175 individuals and 1386 F2 individuals were genotyped. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 35s, a total of 35 cycles, and then 72℃ extension for 10min. The size of the PCR product was identified by agarose gel electrophoresis. Based on the recombination rate of phenotype and genotype, the candidate gene was located in the approximately 70kb segment between the two molecular markers DY01_53219326 and DY01_53289170, as shown in Figure 2. Fig.18 ( Fig.18 (Refer to the fine positioning of candidate genes on chromosome 1). According to the annotation information of the genome, this segment contains 5 genes: Glyma.01G198500, Glyma.01G198600, Glyma.01G198700, Glyma.01G198800 and Glyma.01G198900. The annotation information of these 5 genes is shown in Table 5.

[0075] Table 4 Molecular marker sequences for fine mapping of candidate gene No. 1.

[0076]

[0077]

[0078]

[0079] Table 5 Annotation information of candidate genes on chromosome 1.

[0080] Gene ID Description Glyma.01G198500 CAAX protease self-immunity Glyma.01G198600 Acetylornithine deacetylase / N-acetylornithinase Glyma.01G198700 C2H2-type zinc finger Glyma.01G198800 AT-hook motif nuclear localized protein 21-related Glyma.01G198900 AT hook motif DNA-binding family protein

[0081] 3. Based on the annotation information of the candidate genes of chromosomes 11 and 1, the Glyma.11G043400 and Glyma.01G198500 genes were identified as the optimal candidate genes. Then, using the information from the genome sequencing of Donnong 50 and Iwaji, the genotypes of the Glyma.11G043400 and Glyma.01G198500 genes in the parents were examined. The results showed that in Donnong 50, the Glyma.11G043400 gene lacked a G at base 383, causing a frameshift and premature termination of translation. In Iwaji, the Glyma.01G198500 gene mutated from G to A at base 1055, causing a variant splicing site variation, which ultimately caused premature termination of translation after a frameshift. Fig.19 ( Fig.19 (Figure 2) Sequence analysis of GmBCM1 and GmBCM2 genes in Dongnong 50 and Yanji.

[0082] 4. According to the gene annotation information, Glyma.11G043400 and Glyma.01G198500 genes are members of the CAAX protease gene family. The homologous gene of the CAAX protease gene in Arabidopsis is named BCM gene, so the Glyma.11G043400 gene is named GmBCM1, and the Glyma.01G198500 gene is named GmBCM2. The nucleotide sequences of the GmBCM1 and GmBCM2 genes are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence list. The amino acid sequences of the proteins encoded by the GmBCM1 and GmBCM2 genes are shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence list. In the dry mutant, the nucleotide sequence of the GmBCM1 gene lacks the G base at position 383. The open reading frame of this mutant GmBCM1 gene is shown in the base sequence of SEQ ID NO: 5 in the nucleic acid sequence list, and it is named Gmbcm1. The 1055th base of the nucleotide sequence of the GmBCM2 gene changes from G to A. The open reading frame of this mutant GmBCM2 gene is shown in the base sequence of SEQ ID NO: 6 in the nucleic acid sequence table, and it is named Gmbcm2.

[0083] Example 4: Verification of the functions of GmBCM1 and GmBCM2 genes.

[0084] 1. The functions of GmBCM1 and GmBCM2 genes were verified using the parental genetic population. The F1 generation obtained by hybridizing Yanji and Dongnong 50 did not show the phenotype of rapid decrease in chlorophyll content. After self-pollination, a total of 2547 F2 populations were obtained, of which 151 showed a hybrid yellowing phenotype similar to dry, with a segregation ratio of 1:15. The genotypes of GmBCM1 and GmBCM2 of F2 plants were detected using molecular markers DY11_3159120 and DY01_53224597. Soybean leaf DNA was extracted using the CTAB method; the PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 35 seconds, a total of 35 cycles, and then extension at 72°C for 10 minutes. The size of the PCR product was identified by agarose gel electrophoresis. The results showed that only GmBCM1 - / - / GmBCM2 - / - Individuals of the genotype showed the phenotype of hybrid yellowing, as shown in Table 6. Three individual plants were selected as representatives in each genotype, and their offspring were observed after planting to see whether hybrid yellowing plants could be separated. The results showed that only GmBCM1 + / - / GmBCM2 + / - 、GmBCM1 + / - / GmBCM2 - / - and GmBCM1 - / - / GmBCM2 + / - Individuals with hybrid etiolation can be isolated. In addition, GmBCM1, which exhibits hybrid etiolation in F2 - / - / GmBCM2 - / - All offspring of individuals with the same genotype showed hybrid yellowing.

[0085] Table 6 The genotypes of GmBCM1 and GmBCM2 were analyzed using the F2 generation of self-pollination after hybridization between Yanji and Dongnong 50.

[0086]

[0087] Note: #Yes means the plant exhibits the same phenotype as dry, *Yes means the phenotypes of all offspring are the same as dry.

[0088] Second, the functions of GmBCM1 and GmBCM2 genes were verified using high-generation RIL populations. 2:9The population with a segregation ratio of 15:1 (a total of 140 plants) was selected in the next generation to verify the accuracy of the positioning of the GmBCM1 and GmBCM2 genes. The molecular markers DY11_3159120 and DY01_53224597 were used to detect the genotypes of GmBCM1 and GmBCM2 in the F2 plants. Soybean leaf DNA was extracted by the CTAB method; the PCR reaction conditions were as follows: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 35s, a total of 35 cycles, and then 72℃ extension for 10min. The size of the PCR product was identified by agarose gel electrophoresis. The results showed that only the individuals with the dry phenotype were genotyped as GmBCM1. - / - / GmBCM2 - / - , as shown in Table 7.

[0089] Table 7 Analysis of the genotypes of GmBCM1 and GmBCM2 using the RIL population.

[0090]

[0091] 3. Use mutants to verify the gene function of GmBCM1. The genotype of Dongnong 50 is GmBCM1 + / + / GmBCM2 - / - , and Dongnong 50 is a soybean variety that can be genetically transformed. Therefore, the GmBCM1 gene mutant in the Dongnong 50 background can be obtained, that is, the Gmbcm1 / Gmbcm2 double mutant, and the function of the GmBCM1 gene can be verified using the double mutant. Targeting the GmBCM1 gene sequence, Cas9 target sgRNA was designed and synthesized. The target sequence of sgRNA is 5'-TTCAGGGATTTATGGTGCCA-3', which is located at the junction of the first intron and the second exon in the GmBCM1 gene sequence, such as Fig. 20 ( Fig. 20is the position of the sgRNA target sequence in the GmBCM1 gene). A CRISPR-Cas9 knockout vector specifically targeting the GmBCM1 gene was constructed. The constructed vector was transformed into EHA105 Agrobacterium, and Dongnong 50 was used as the recipient material. The soybean transformation experiment was carried out by the soybean cotyledon node genetic transformation method. After obtaining the resistant T0 generation plants, primers were designed to amplify the DNA fragment containing the target position. The forward primer sequence was 5'-AACTTTGCTTTTTCTAGCTT-3', and the reverse primer sequence was 5'-TGTCAGCTCATTAATTAGAA-3'. The PCR reaction conditions were 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension for 35s, a total of 35 cycles, and then 72℃ extension for 10min. After the PCR products were separated by agarose gel electrophoresis, the gel strips of the target size were cut and sent to the sequencing company for sequencing using amplification primers. Based on the GmBCM1 gene sequence, the target site DNA in the T0 generation resistant plants was compared to see if it was edited. Two successfully edited Gmbcm1 mutants were obtained from the candidate transgenic plants and named Gmbcm1-12 and Gmbcm1-26, respectively. The GmBCM1 gene in Gmbcm1-12 was missing 12 bp, and the GmBCM1 gene in Gmbcm1-26 was missing 26 bp. Fig.21 ( Fig.21 Schematic diagram of sequence variation of Gmbcm1-12 and Gmbcm1-26 mutants). In the Gmbcm1-12 mutant, the GmBCM1 protein lacks 4 amino acids, the first loop located in the membrane lacks 2 amino acids, and the second transmembrane domain lacks 2 amino acids, such as Fig. 22 ( Fig. 22 For the structural variation analysis of GmBCM1 protein in Gmbcm1-12 mutant). In Gmbcm1-26 mutant, the translation of GmBCM1 protein terminated prematurely, resulting in significant changes in the protein sequence, such as Fig.23 ( Fig.23 (Figure 2A) Variation analysis of the GmBCM1 protein structure in the Gmbcm1-26 mutant.

[0092] The phenotypes of Gmbcm1-12 and Gmbcm1-26 mutants were observed, and the results showed that both mutant lines showed a yellow leaf phenotype, such as Fig.24 ( Fig.24 Phenotypic analysis of the Gmbcm1-12 and Gmbcm1-26 mutants obtained by gene editing). The chlorophyll content and effective quantum yield of photosystem II [Y(II)] of Dongnong 50 and Gmbcm1-12 and Gmbcm1-26 mutants were measured. The results showed that these two indicators in the mutants were significantly lower than those in Dongnong 50. Fig.25 ( Fig.25 Chlorophyll content analysis of Gmbcm1-12 and Gmbcm1-26 mutants) and Fig.26 ( Fig.26 The results above indicate that GmBCM1 is one of the control genes of yellowing phenotype in soybean hybrids.

[0093] 4. Verify the function of the GmBCM2 gene by genetic complementation. The Iwahime soybean variety cannot be genetically transformed, so the function of the GmBCM2 gene was verified by genetic complementation. Using Iwahime as the material, the total RNA of the leaves was extracted using the operating manual of the Omini Plant RNA Kit (CW25985) purchased from Comverse Century. 1 μg of total RNA was taken for cDNA synthesis. The cDNA synthesis operation was carried out according to the manual of the TransScript One-Step gDNARemoval and cDNA Sythesis SuperMix Kit (AT311-03) purchased from Quanshijin Company to obtain cDNA. The obtained cDNA was used as a template to amplify the GmBCM2 gene by the forward primer 5'-ATGGAACTGTTGTCTCTGAGACTC-3' and the reverse primer 5'-GTTGGAGTTTTTTTCTTCTG-3'. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 35 s, for a total of 38 cycles, and then extension at 72°C for 10 min. The PCR products were sequenced on an ABI3130 sequencer (ABI).

[0094] Through the genetic transformation method of soybean cotyledon node, the GmBCM2 gene overexpression material was obtained, which showed significant difference in leaf color phenotype ( Fig. 27 Phenotypic analysis of GmBCM2 gene overexpression materials), chlorophyll content ( Fig.28 Chlorophyll content analysis of GmBCM2 gene overexpression materials) and effective quantum yield of photosystem II [Y(II)] ( Fig.29 The effective quantum yield of photosystem II [Y(II)] analysis of GmBCM2 gene overexpression materials was consistent with that of Dongnong 50. By hybridization of soybeans, a material with GmBCM2 gene overexpression in the background of Gmbcm1 mutant was obtained, named GmBCM2-OE3 / Gmbcm1-26. The results of phenotypic observation experiments showed that GmBCM2 can complement the yellowing phenotype of leaves of Gmbcm1 mutant, such as Fig.30 ( Fig.30The results of physiological index determination showed that GmBCM2 could complement the phenotype of gmbcm1 mutant with reduced chlorophyll content and effective quantum yield of photosystem II [Y(II)], such as Fig.31 ( Fig.31 Analysis of chlorophyll content in materials complementing the gmbcm1 mutant by overexpression of GmBCM2) and 32 ( Fig.32 Analysis of the effective quantum yield of photosystem II (Y(II)) in materials complementing the gmbcm1 mutant by overexpression of GmBCM2. These results indicate that GmBCM2 is another gene controlling the yellowing phenotype of soybean hybrids.

Claims

1. The GmBCM1 and GmBCM2 genes for identifying soybean hybrid etiolation, characterized in that The nucleotide sequences of the open reading frames of the GmBCM1 and GmBCM2 genes are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, in the sequence listing.

2. The protein encoded by the GmBCM1 and GmBCM2 genes for identifying soybean hybrid etiolation according to claim 1, characterized in that The amino acid sequences of the proteins encoded by the GmBCM1 gene are shown in SEQ ID NO: 3 in the sequence listing.

3. The protein encoded by the GmBCM1 and GmBCM2 genes for identifying yellowing of soybean hybrids according to claim 1, characterized in that The amino acid sequences of the proteins encoded by the GmBCM2 gene are shown in SEQ ID NO: 4 in the sequence listing.

4. The GmBCM1 and GmBCM2 genes for identifying soybean hybrid etiolation according to claim 1, characterized in that Gmbcm1 is a mutant GmBCM1 genotype, and Gmbcm1 is a gene whose open reading frame includes the base sequence shown in SEQ ID NO:

5.

5. The GmBCM1 and GmBCM2 genes for identifying soybean hybrid etiolation according to claim 1, characterized in that Gmbcm2 is a mutant GmBCM2 genotype, and Gmbcm2 is a gene whose open reading frame includes the base sequence shown in SEQ ID NO:

6.

6. Use of the GmBCM1 and GmBCM2 genes identified as claimed in claim 1 in soybean molecular assisted breeding, gene selection breeding and AI breeding.

7. Application of the mutant genotype as claimed in claim 3 or 4 in soybean molecular assisted breeding, gene selection breeding and AI breeding.

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