Application of endo-beta-mannase coding gene GhMAN7 and SNP associated with endo-beta-mannase coding gene GhMAN7 in regulation and control of plant seed vigor

Abstract

The invention provides an application of an endo-beta-mannase coding gene GhMAN7 and an SNP (Single Nucleotide Polymorphism) associated with the endo-beta-mannase coding gene GhMAN7 in regulation and control of plant seed vigor, and belongs to the technical field of molecular breeding. The endo-beta-mannase coding gene GhMAN7 and SNP associated with the activity of the cotton seeds are analyzed and excavated through co-localization of GWAS and eQTL, the cDNA sequence of the gene is as shown in SEQ ID NO.1, and the genomic sequence of the gene is as shown in SEQ ID NO.2. The invention also discloses an application of the endo-beta-mannase coding gene GhMAN7 and SNP associated with the activity of the cotton seeds. The SNP is located at the D05: 16685321 position of a genome sequence, and the polymorphism is A or G. The invention further discloses a method for detecting the SNP. The research finds that the root length and germination rate traits between two different SNP types of materials in cotton have significant difference, the correlation between the SNP locus and the gene GhMAN7 associated with the SNP locus and the cotton seed vigor is proved, and the SNP locus and the gene GhMAN7 can be used for identifying, screening, culturing or improving cotton varieties with high seed vigor.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding, and specifically relates to the application of an endo-β-mannanase encoding gene GhMAN7 and its associated SNP in regulating plant seed vitality. Background Art

[0002] Cotton (Gossypium hirsutum L.) is an economic crop with rich use value. It not only provides natural fiber raw materials for the textile industry, but its seeds are also an important source of oil. Gu Zenghui et al. (1982) proposed that seed vigor is the sum of seed germination rate, germination rate, seedling growth potential, plant stress resistance and production potential, and is an important indicator for measuring seed quality. Seed imbibition germination is the first step in plant growth and development, affecting the subsequent growth process of the plant. Therefore, in-depth exploration of the external phenotypic changes and internal molecular mechanisms of seeds during germination will help to clarify the internal causes and influencing factors of differences in seed vigor, which is of great significance for promoting the genetic improvement of cotton varieties.

[0003] Genome-wide association studies (GWAS) can use single nucleotide polymorphisms (SNPs) of the whole genome as molecular markers to perform statistical analysis on phenotypic differences at the population level, thereby screening out sites associated with trait variation. Expression quantitative trait loci (eQTLs) refer to regions on chromosomes that can specifically regulate mRNA and protein expression levels, and can be used to analyze the relationship between gene expression differences and genetic variation. eQTLs are generally SNPs, and are divided into cis-eQTLs and trans-eQTLs based on the correlation between SNPs and gene expression levels and the distance between SNPs and genes. GWAS and eQTLs can be co-localized by comparing the overlap of the signals to determine sites with co-localized signals, thereby more accurately identifying regulatory genes associated with the phenotype.

[0004] Mannan is a natural hemicellulose polysaccharide widely distributed in yeast and plant tissues. Mannan present in the primary cell wall of plants is the source of the strong mechanical resistance of the seed radicle protruding from the ground during germination. In plants, endo-β-mannanase (MAN) is a key enzyme that modifies mannan by randomly hydrolyzing the internal β-1,4D-rhamnose linkages in the mannan skeleton. The first MAN gene (SlMAN1) was isolated from tomato seeds and is mainly expressed in the endosperm after germination. It may provide nutrients for seedling growth by decomposing galactomannan in the endosperm cell wall (Bewley et al., 1997). At present, 8 MAN genes have been identified in Arabidopsis, of which AtMAN3 and AtMAN4 are mainly expressed in siliques, while AtMAN2, AtMAN5, AtMAN6 and AtMAN7 are expressed in roots, stems, leaves, siliques and seeds (Iglesias-Fernandez et al., 2011). Raquel et al. (2011) detected that the transcripts of AtMAN6 and AtMAN7 in Arabidopsis seeds accumulated only in the micropylar endosperm and radicle, and disappeared soon after the radicle appeared, proving that these two genes can promote the hydrolysis of mannan-rich endosperm cell walls, which is very important for the germination of Arabidopsis seeds. Wu Qi et al. (2023) found that AtMAN7 encodes an endo-β-mannanase homologous to AtMAN3, which can regulate Arabidopsis tolerance to cadmium through a mannose-dependent pathway. It can be seen that different MAN genes differ in function. There are no reports on the function of GhMAN7 in cotton. Summary of the invention

[0005] The purpose of the present invention is to explore the function of cotton MAN gene and provide a scheme for regulating cotton seed vitality.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of an endo-β-mannanase encoding gene GhMAN7 in regulating plant seed vigor. The cDNA sequence of the gene GhMAN7 is shown as SEQ ID NO.1, and the genome sequence is shown as SEQ ID NO.2.

[0008] Preferably, the amino acid sequence of the protein encoded by the gene GhMAN7 is shown in SEQ ID NO.3.

[0009] The invention provides a SNP site associated with plant seed vigor traits. The SNP site is associated with the endo-β-mannanase encoding gene GhMAN7 and is located at D05:16685321 of the genome sequence. The polymorphism is A or G.

[0010] Preferably, the upstream primer of the primer pair for detecting the SNP site is shown as SEQ ID NO.6, and the downstream primer is shown as SEQ ID NO.7.

[0011] The present invention provides the use of the endo-β-mannanase encoding gene GhMAN7 or the above-mentioned SNP site in identifying, screening, cultivating or improving plant varieties with high seed vigor.

[0012] Preferably, the method for identifying or screening plant varieties with high seed vigor is: detecting the SNP site, and the plant with base A at position D05:16685321 of the genome sequence is a plant variety with high seed vigor.

[0013] Preferably, the method for cultivating or improving a plant variety with high seed vigor is: transferring the gene GhMAN7 into the plant by overexpression or gene knock-in.

[0014] The present invention provides a set of primer pairs that can be used to detect the SNP sites.

[0015] The invention also provides the use of the primer pair in identifying or screening plant varieties with high seed vigor.

[0016] Preferably, the plant is cotton.

[0017] The present invention has discovered an endo-β-mannanase encoding gene GhMAN7 associated with cotton seed vigor through GWAS and eQTL co-localization analysis, and the associated SNP (D05:16685321) is related to the root length trait and germination rate during seed germination. According to the GWAS results and eQTL co-localization analysis, the SNP base mutated from A to G, and the corresponding amino acid changed from aspartic acid to glycine, and the root length and germination rate of the mutated genotype decreased significantly. It can be seen that the SNP can regulate the root length and germination rate phenotypes of cotton seeds during germination, and is closely related to the seed vigor trait.

[0018] In the present invention, the expression level analysis of GhMAN7 in different tissues and developmental stages of cotton was obtained by transcriptome sequencing. The gene was dominantly expressed during ovule development and seed imbibition germination, indicating that the gene was related to the constituent factors of seed vigor traits.

[0019] The present invention verifies the SNP genotype of GhMAN7 in populations with relatively high and low seed vigor by PCR technology, which is easy to operate, has high sensitivity and good accuracy, and is easy to screen or identify varieties with high seed vigor.

[0020] The present invention divides the variety population into two major categories, haplotype GhMAN7 (A) and haplotype GhMAN7 (G), according to the different SNP genotypes of GhMAN7, and finds through statistical analysis methods that there are significant differences in the root length and germination rate traits between the two groups, further proving the correlation between the gene and cotton seed vigor. Furthermore, molecular markers are designed based on the two haplotypes of SNPs associated with the gene GhMAN7, which can effectively identify the cotton seed vigor traits and have great application value in the research of high seed vigor cotton variety breeding. On the other hand, the gene GhMAN7 can be transferred into the cotton variety by means of genetic engineering in the form of overexpression or gene knock-in to improve the seed vigor of cotton, thereby cultivating new cotton varieties with high seed vigor. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 GWAS and eQTL co-localization analysis of cotton root length and germination rate traits.

[0022] The figure shows the Manhattan plot of root length and germination rate in the GWAS results, and the Manhattan plot of the gene expression in the eQTL results. The arrows point to the sites where the SNP and eQTL are located, and the boxes indicate the co-localization of the GWAS and eQTL results. The horizontal axis indicates the position on the chromosome (Mb), and the vertical axis indicates the significance of the association of the SNP site, expressed as -log 10 (Pvalue) indicates.

[0023] Figure 2 Interval relationship diagram of GhMAN7 and its associated SNPs.

[0024] Figure 3 Statistical graph of the gene expression level of GhMAN7 in different tissues and developmental stages of cotton.

[0025] Figure 4 Results of the analysis of the differences in root length and germination rate between different haplotypes of SNPs associated with GhMAN7 in the population.

[0026] The unit of root length is pixel (px); the unit of germination rate is percentage (%); the box plot represents the difference in root length (left) and germination rate (right) between different haplotypes in the population. There are two haplotypes, AA and GG, and the numbers represent the number of individuals in different haplotype samples. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] Mining of the endo-β-mannanase encoding gene GhMAN7 associated with cotton seed vigor:

[0030] Seeds from 356 different varieties of cotton materials were used for swelling and germination experiments. Phenotypic data related to seed size within 24 hours after seed swelling began, as well as phenotypic data related to germination and root length development within 120 hours were collected. The phenotypic data obtained were analyzed with the genotypic data by GWAS, and a total of 541 candidate loci were obtained. Among them, the SNP (D05:16685321) located at the loci368 site had a high correlation with the root length and germination rate during seed germination. The SNP was associated with 14 genes, namely GH_D05G1948, GH_D05G1949, GH_D05G1950 to GH_D05G1961. In order to further accurately lock in genes closely related to phenotypic traits, transcriptome sequencing was performed on the radicle of seeds germinated for 36 hours, and the data obtained were used for GWAS and eQTL co-localization analysis (such as Figure 1 As shown in the figure), we finally found that GH_D05G1958 (i.e., GhMAN7) was closely related to cotton seed vigor.

[0031] like Figure 2 As shown: the gene GhMAN7 is in the same linkage disequilibrium interval with SNP (D05:16685321) in both root length and germination rate traits, and this SNP is the most significant in this region. This indicates that this SNP can regulate the root length and germination rate phenotypes of cotton seeds during germination, and is closely related to seed vigor traits.

[0032] Example 2

[0033] Obtaining the endo-β-mannanase encoding gene GhMAN7:

[0034] The cDNA sequence (SEQ ID NO.1) and genomic sequence (SEQ ID NO.2) of GhMAN7, as well as the amino acid sequence (SEQ ID NO.3) of the protein encoded by GhMAN7 were obtained from the genomic sequence of TM-1, a genetic standard line of upland cotton.

[0035] The cDNA sequence of GhMAN7 (1296 bp) is shown in SEQ ID NO.1:

[0036] SEQ ID NO.1:

[0037]

[0038] The GhMAN7 genomic sequence (2134 bp) is shown in SEQ ID NO.2:

[0039] SEQ ID NO.2:

[0040]

[0041] The amino acid sequence (431aa) of the protein encoded by GhMAN7 (SEQ ID NO.1) is shown in SEQ ID NO.3:

[0042] SEQ ID NO.3:

[0043] MKHWGLILLLFLFQQGNFLLRVKADDGFIKTKGLQLMLHGSPFYANGFNAYWLMYMAADSSQRSKVSSAFQQAKEHGLTIARTWAFSDGGDRPLQ YSPGSYNEQMFQGLDFVVSEAKRYGIKLVLSFANNYDQFGGKKQYVSWARNEGQSIGSDDDFFTNSVVKEYYKNHIKAVLTRRNTLTGVAYKDEPTI MAWELMNEPRCLSDDPSGKTMQAWITEMASHVKSIDGNHLLEAGLEGFYGPSSSQKQQYNPNFQVGTDFIANNQIPGIDFATVHSYPDQWLQSSSDESQIAFLNNWLYNHIQDAQNILQK PLLFAEFGKSLKIAGPPNQRDELYNTVYTAIYSSARGGGAAIGGLFWQLLAEGMDSYGDGYEVIMSQGTSTVDLITQESQKLNRIRKMYVRLRDIEKWNKAREIRRAQWWSGNGVSNTGN.

[0044] Primers F1: (5'-3') ATGAAGCATTGGGGTCTGATTTTG (SEQ ID NO. 4) and R1: (5'-3') TCAATTTCCCGTGTTGCTGAC (SEQ ID NO. 5) were designed based on the gene cDNA to clone the full length of the gene.

[0045] The PCR reaction procedure is as follows: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30sec, 58℃ annealing for 1min, 72℃ extension for 45sec, for a total of 35 cycles; and finally 72℃ thorough extension for 10min. The PCR amplification product is then sequenced and further compared with the reference cDNA sequence to determine the accuracy of the cloned sequence. If the sequence alignment is successful, the endobacterial β-mannanase encoding gene GhMAN7 is obtained.

[0046] Example 3

[0047] Analysis of the expression level of gene GhMAN7 in different tissues and developmental stages of cotton:

[0048] In this experiment, RNA samples from different tissues and developmental stages of cotton were used for transcriptome sequencing. Sample materials include roots, stems, leaves, seeds, cotyledons and ovules. Furthermore, samples of roots, stems and leaves were taken from the four-leaf stage, seeds included samples of 0 hours, 5 hours and 10 hours of imbibition germination, cotyledon growth and root elongation 24 hours, 48 ​​hours, 72 hours to 120 hours, and ovule tissues included 3 and 1 days before flowering, the day of flowering, and 1 to 35 days after flowering.

[0049] The results show (such as Figure 3 ): The gene GhMAN7 was dominantly expressed during ovule development and seed germination, indicating that this gene is related to seed vigor traits.

[0050] Example 4

[0051] Application of gene GhMAN7 in identifying high seed vigor cotton varieties and improving seed vigor traits:

[0052] Genotyping was performed according to the SNP (D05: 16685321) of the regulatory gene GhMAN7. Based on the location of the SNP on the chromosome, amplification primers were designed at both ends of the SNP, and the primer sequences were F2 (SEQ ID NO. 6): (5'-3') ATGATGTGGCAAGTTTTTATTGGTACCTAAAAGCC and R2 (SEQ ID NO. 7): (5'-3') AAAACTTGCCACATCATTAAATTTTAGAGATATGAAATTATT. This pair of primers was used to perform PCR amplification and sequencing in the population varieties.

[0053] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 45 sec, for a total of 35 cycles; and final complete extension at 72°C for 10 min.

[0054] The PCR products were sequenced, and the genotypes of each variety population at the SNP site were analyzed based on the sequencing results. The base at the SNP (D05: 16685321) site changed from A to G, and the corresponding amino acid changed from aspartic acid to glycine. Based on the base information of the SNP site on the genome sequences of 356 varieties, 183 materials with haplotype GhMAN7 (A), 87 materials with haplotype GhMAN7 (G), and 59 heterozygous materials were identified (the identification results are shown in Figure 4and Table 1, only materials with both phenotypes and haplotypes were retained). The correlation of seed vigor traits between the two haplotypes was calculated using the t-test method, and it was found that compared with the non-mutated haplotype GhMAN7(A), the root length and germination rate of the mutated haplotype GhMAN7(G) were significantly decreased, by 41.9% (P=1.328187e-03) and 39.5% (P=6.810692e-03), respectively ( Figure 4 ).

[0055] Table 1 Identification of high-vigor and low-vigor haplotypes in population variety materials

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] The above results show that the endo-β-mannanase encoding gene GhMAN7 has important research value in improving cotton seed vigor traits and cultivating new cotton varieties with high seed vigor. On the one hand, molecular markers can be designed based on the two haplotypes of SNPs associated with the gene GhMAN7 to effectively identify cotton seed vigor traits, which has great application value in the identification and breeding research of high seed vigor cotton varieties. On the other hand, the GhMAN7 gene can be transferred into cotton varieties by overexpression or gene knock-in through genetic engineering to improve cotton seed vigor and cultivate new cotton varieties with high seed vigor.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the endo-β-mannanase encoding gene GhMAN7 in regulating plant seed vigor, characterized in that: The cDNA sequence of the gene GhMAN7 is shown in SEQ ID NO.1, and the genome sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the gene GhMAN7 is shown in SEQ ID NO.

3.

3. A SNP locus associated with a plant seed vigor trait, characterized in that: The SNP site is associated with the endo-β-mannanase encoding gene GhMAN7 described in claim 1, is located at D05:16685321 of the genome sequence, and its polymorphism is A or G.

4. The SNP site according to claim 3, characterized in that: The upstream primer of the primer pair for detecting the SNP site is shown as SEQ ID NO.6, and the downstream primer is shown as SEQ ID NO.

7.

5. Use of the endo-β-mannanase encoding gene GhMAN7 according to claim 1 or the SNP site according to claim 3 in identifying, screening, cultivating or improving plant varieties with high seed vigor.

6. The use according to claim 5, characterized in that The method for identifying or screening plant varieties with high seed vigor is: detecting the SNP site, and the plant with base A at position D05:16685321 of the genome sequence is a plant variety with high seed vigor.

7. The use according to claim 5, characterized in that The method for cultivating or improving a plant variety with high seed vigor is: transferring the gene GhMAN7 into the plant by overexpression or gene knock-in.

8. The primer pair for detecting the SNP site according to claim 4.

9. Use of the primer pair according to claim 8 in identifying or screening plant varieties with high seed vigor.

10. The use according to claim 9, characterized in that: The plant is cotton.

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