Sorghum brown midrib mutant gene bmr34 related to high digestibility and application thereof

Abstract

The invention discloses a sorghum brown midrib mutant gene bmr34 related to high digestibility and application of the sorghum brown midrib mutant gene bmr34. The haplotype of the mutant gene bmr34 can specifically reduce the lignin content by 2.99% without affecting the biological yield. The invention provides the KASP marker which can accurately locate the bmr34 gene of sorghum and is closely linked with the bmr34 gene of sorghum, so that the breeding efficiency can be greatly improved, the breeding process is accelerated, and the KASP marker has important theoretical and practical significance for improving the quality of sorghum feed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of sorghum genetic breeding, and particularly relates to a gene positioning method of a sorghum brown midrib mutant gene bmr34 and application of a KASP marker based on the positioning in sorghum breeding. BACKGROUND

[0002] Sorghum is an increasingly important biofuel feedstock and an important source of animal feed worldwide, which can be divided into two types as grain type and plant type, and the plant type is widely used as forage. With the decrease of arable land and the increase of population, the demand for animal husbandry is large, and improving the yield of sorghum is of great significance to solve the problem of animal husbandry feed. Therefore, it is necessary to vigorously carry out basic research on sorghum to help cultivate multi-functional and multi-purpose sorghum varieties to meet market demand.

[0003] Brown midrib mainly shows that the leaf veins and xylem of the plant are brownish gray or brown. The content of digestible hemicellulose and cellulose in brown midrib is high, and the content of indigestible lignin is low, which is an important genetic resource for improving the feed quality of sorghum. Developing or identifying brown midrib mutants and finding or making the lignin content of the mutants significantly lower than that of the wild type are of great significance to improve the palatability and digestibility of feed. SUMMARY

[0004] The primary object of the present application is to provide a sorghum brown midrib gene and application of the gene in sorghum breeding.

[0005] In one aspect, the present application provides a brown midrib mutant gene bmr34, the DNA sequence of which is shown in SEQ ID NO. 2, and the CDS sequence of which is shown in SEQ ID NO. 3.

[0006] It is found that the mutant gene bmr34 (Sobic.004G071000) is a key gene encoding cinnamyl alcohol dehydrogenase in the lignin synthesis pathway, which has the effect of terminating the lignin synthesis pathway.

[0007] The expression of the mutant gene in the mutant is sufficient to significantly inhibit the function of the lignin synthesis related genes, resulting in a specific decrease in the lignin content, and finally forming a stable yield and reduced lignin.

[0008] In another aspect, the present application further provides a KASP marker system developed based on the above-mentioned brown midrib mutant gene bmr34.

[0009] In a specific example of the present application, the KASP marker system has the allelic specific primers shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

[0010] The third aspect of the present application also provides the use of the KASP marker system in identifying the brown midrib variety of sorghum, specifically, using the KASP marker system, detecting the G / A mutation of the alleles shown in SEQ ID NO. 1 and SEQ ID NO. 2 by fluorescence quantitative PCR, when the detection result is G / G, it is the wild type non-brown midrib variety, when the detection result is A / A, it is the mutant brown midrib variety, and when the detection result is G / A, it is a hybrid variety.

[0011] The fluorescence quantitative PCR described in the present application can be carried out under the reaction conditions and reaction system commonly used in the art.

[0012] In one embodiment, the fluorescence quantitative PCR reaction system is 10 μL: 2 μL of 30 ng / μL DNA template, 0.14 μL of primer mixture, 5 μL of KASP Mix, and 2.86 μL of ddH2O. In the primer mixture, the mass ratio of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 is 6:6:18, and the final concentration is 0.2 μM.

[0013] In one embodiment, the fluorescence quantitative PCR amplification program is as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 65℃→55℃ annealing for 15 s, decreasing by 1℃ per cycle for a total of 10 cycles; 95℃ extension for 30 s, 57℃ extension for 60 s, 42 cycles; 30℃ incubation for 30 s, and reading the wild type and mutant fluorescence signals.

[0014] The present application also provides the use of the brown midrib mutant gene bmr34 and the KASP marker system in sorghum breeding.

[0015] (1) The bmr34 gene discovered in the present application is a brown midrib mutant, which has significantly lower lignin content than the wild type, thereby improving the palatability and digestibility of feed, and not affecting plant yield, which has more application value in production practice.

[0016] (2) The KASP marker provided by the present application can accurately locate the sorghum bmr34 gene and is closely linked thereto, greatly improving the breeding efficiency and thereby accelerating the breeding process, which has important theoretical and practical significance for improving the quality of sorghum feed.

[0017] (3) The brown midrib bmr34 sorghum identified by the present application, the lignin of the brown midrib plant is found to be significantly reduced, the plant height, stem diameter, tillering, fresh weight and dry weight of the brown midrib bmr34 sorghum plant have no significant difference with the wild type, that is, the bmr34 haplotype first reveals the phenomenon of lignin reduction and stable biological yield in the brown midrib mutant of sorghum, and provides a new target for high-quality forage sorghum breeding. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is the section and phenotype of wild type and bmr34 mutant; wherein A is the comparison of WT and bmr34 plants, B is the midrib of WT and bmr34 leaves, C is the comparison of stems, D is the comparison of root systems, E is the comparison of leaf midribs, and F is the comparison of stem sections.

[0019] Figure 2 Figure 2 is the agronomic traits and lignin content determination of wild type and bmr34 mutant; wherein ABCDE are the plant height, stem diameter, fresh weight, dry weight and lignin content of BTx623 and bmr34 mutant plants determined at the heading stage, respectively.

[0020] Figure 3 Figure 3 is the gene location of the brown midrib bmr34 mutant; wherein A is the DNA sequencing result, and B is the CDS sequencing result.

[0021] Figure 4 Figure 4 is the comparison of WT and bmr34 location and gray value ratio results; wherein A is the comparison of WT and bmr34 peak charts, B is the gene structure chart, C is the WT and bmr34 in 2% agarose gel electrophoresis chart, and D is the ratio of wild type bands in WT and bmr34.

[0022] Figure 5 Figure 5 is the genotyping result of bmr34 mutant population, wherein the red dots are the paternal genotype, the blue dots are the maternal genotype, and the green dots are the heterozygous genotype. DETAILED DESCRIPTION

[0023] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified. In the following examples, the quantitative tests are set up with three repeated experiments, and the results are averaged.

[0024] In the following examples, the sorghum used is BTx623, which is a known variety and can be obtained commercially.

[0025] Example 1 Mutant Creation

[0026] 1. Obtaining of Brown Midrib bmr34 Mutant Sorghum

[0027] Select plump and uniformly sized BTx623 seeds; place the seeds in 0.1% EMS and shake on a shaker for 12 hours (50 rpm); add 100 mL of distilled water and shake on a shaker for 30 minutes, then discard the water solution; wash the seeds repeatedly using the above method, 8-10 times, until the EMS residue on the seed surface is completely removed; after drying the washed seeds, plant them in the field.

[0028] A phenotypic survey was conducted during the heading stage of sorghum, and a mutant plant, bmr34, was found with brown midribs in its leaves or stems.

[0029] 2. Determination of agronomic traits and lignin content in wild-type and BMR34 mutants

[0030] There was no significant difference in overall plant characteristics between wild-type sorghum and mutant sorghum. Figure 1 A). Wild-type sorghum has white roots, green stems, and white midribs on its leaves; mutant sorghum has brown midribs on its leaves, stems, and roots. Figure 1 B, C, D) indicate that brown expression is prominent in the mutant.

[0031] To investigate the effects of the BMR34 mutant on leaf midribs and stems, leaf midribs and stem sections of both BMR34 mutant and wild-type plants were observed using a stereomicroscope. Phloroglucinol staining results showed that the midribs of the leaves from the BMR34 mutant showed deeper staining. Figure 1 E,F).

[0032] Five agronomic traits (plant height, stem diameter, tillering, fresh weight per plant, and dry weight per plant) and lignin content were measured in the BMR34 mutant and wild type at the heading stage. The results showed that there were no significant differences in plant height, stem diameter, tillering, fresh weight per plant, and dry weight per plant between the BMR34 mutant and wild type at the heading stage. However, the lignin content was significantly different between the two types, decreasing by 2.99% compared to the wild type. Figure 2 This indicates that the brown midrib significantly reduces lignin content without affecting sorghum biomass.

[0033] 3. Localization of the sorghum bmr34 gene

[0034] (1) Group construction and phenotypic analysis

[0035] F1 generation was obtained by crossing the bmr34 mutant (maternal parent) with the wild-type Tx623 (paternal parent), and the F2 segregating population was constructed through self-pollination. The color of the midrib of the leaves (brown / white) was observed under a stereomicroscope. Combined with the brown phenotype of the stems and roots, 30 plants with extreme brown midribs (mutant) and 30 plants with white midribs (wild-type) were selected for gene pool construction.

[0036] (2) Combined Group Analysis (BSA) and Initial Positioning

[0037] Genomic DNA was extracted from the parents and individual plants using a DP320-03 kit at a concentration of 30 ng / μL.

[0038] Thirty mutant DNA samples were mixed in equal proportions to form a mutant pool, and 30 wild-type DNA samples were mixed in equal proportions to form a wild pool. SNP sites were detected using whole-genome resequencing.

[0039] ΔSNP-Index calculations revealed that the ΔSNP-Index value in the 0-10Mb range of chromosome 4 was significantly lower than the threshold (P<0.01), preliminarily locating the bmr34 gene on chromosome 4.

[0040] (3) Fine mapping and candidate gene validation

[0041] InDel markers were designed within the initial localization interval. Using an F2 population of 143 individual plants, the target interval was narrowed down to chromosome 4, 5,731,148-5,731,548 bp (physical distance 400 bp).

[0042] Functional annotation of candidate genes in this region revealed that the Sobic.004G071000 gene encodes cinnamyl alcohol dehydrogenase (CAD), the product of which is involved in the lignin biosynthesis pathway. Sequencing analysis showed that in the mutant, a single-base mutation G→A occurred at position 15 downstream of exon 3 of this gene (the genomic sequence changes from SEQ ID NO.1 to SEQ ID NO.2, and the mutated CDS region sequence is shown in SEQ ID NO.3). Figure 3 , Figure 4 (A, B). This mutation causes the coding sequence to prematurely generate a stop codon, resulting in premature termination of protein translation and loss of function. Combined with gene localization and sequencing results, a unique correspondence between this gene mutation and the trait in Example 1 can be established. Since CAD is a key enzyme in the lignin synthesis pathway, its loss of function significantly affects lignin accumulation and cell wall structure.

[0043] cDNA from wild-type and mutant strains was amplified by PCR and detected by 2% agarose gel electrophoresis. Figure 4 C) The wild-type mutant had only one band, but the mutant bmr34 had two bands (the upper band was the mutant band with a 33bp insertion, and the lower band was the same size as the wild-type. bmr34-1 and bmr34-2 are the same mutant. The two experiments were repeated to ensure accuracy).

[0044] In mutant BMR34-1, the grayscale ratio of wild-type to mutant is 1:1.785, with wild-type bands accounting for 36.20% and mutant bands for 63.80%. In mutant BMR34-2, the grayscale ratio of wild-type to mutant bands is 1:1.71, with wild-type bands accounting for 36.95% and mutant bands for 63.05%. Figure 4 D). The residual expression of the wild-type gene in this mutant partially compensates for the impact of the mutant gene on the metabolic pathways related to biomass production, thereby maintaining a normal biomass production level. Therefore, the biomass production of haplotype bmr34G3015A sorghum is no different from that of the wild type.

[0045] Below is an application example of gene mapping of bmr34 in the brown midrib of sorghum and the development of KASP markers.

[0046] Example 2: Development of KASP markers based on bmr34 gene mutations

[0047] 1. Primer design: Based on the G / A mutation site after exon 3 of the Sobic.004G071000 gene, primers were designed...

[0048] KASP allel-specific primers:

[0049] Forward primer BTx623-FAM (SEQ ID NO.4):

[0050] 5'-GAAGGTGACCAAGTTCATGCTTAGTTTTCAGTTTCAAGAAACTCAC-3'

[0051] Forward primer BTx623-VIC (SEQ ID NO.5):

[0052] 5'-GAAGGTCGGAGTCAACGGATTTAGTTTTCAGTTTCCAAGAAACTCAT-3'

[0053] Reverse primer BTx623-R (SEQ ID NO.6):

[0054] 5'-GCGGCTTCGCCTCCACCATGGTCGTCGAC-3'.

[0055] 2. KASP-PCR reaction system (10μL system): 2μL DNA template (30ng / μL), 0.14μL primer mixture (FAM:VIC:R=6:6:18, final concentration 0.2μM), 5μL KASP Mix (containing Taq enzyme, dNTPs, and fluorescent probe), 2.86μL ddH2O.

[0056] 3. Amplification program: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 65℃→55℃ annealing for 15 s (1℃ decrease per cycle, 10 cycles in total); 95℃ extension for 30 s, 57℃ extension for 60 s, 42 cycles; 30℃ incubation for 30 s, read FAM (wild type) and VIC (mutant) fluorescence signals.

[0057] 4. Experimental Results: To verify the specificity of the developed KASP marker, 40 brown midrib plants and 2 plants from each parent were randomly selected from the F2 population for validation. Good genotyping results were obtained, with 10 heterozygous genotypes, 11 paternal genotypes, and 19 maternal genotypes. Figure 5 The results demonstrate that the genotype and phenotype of the KASP marker described in this invention are consistent, and that this marker can be used for systematic breeding of highly digestible brown midrib sorghum.

[0058] 5. Technical advantages: Single-tube dual fluorescence detection can simultaneously distinguish between wild type (FAM+), mutant type (VIC+), and heterozygous type (FAM+ / VIC+), with high accuracy and sensitivity, making it suitable for rapid screening of traits during the seedling stage.

Claims

1. A brown midrib mutant gene bmr34, the DNA sequence of which is shown in SEQ ID NO.2 and the CDS sequence of which is shown in SEQ ID NO.

3.

2. The application of the mutant gene bmr34 as described in claim 1 in sorghum breeding.

3. A KASP marker system for the mutant gene bmr34 as described in claim 1.

4. The KASP tagging system according to claim 3, characterized in that, Primers with allele specificity as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

5. The application of the KASP marking system as described in claim 3 or 4 in the identification of brown midrib varieties of sorghum.

6. The application according to claim 5, characterized in that, The KASP marker system was used to detect G / A mutations in the alleles shown in SEQ ID NO.1 and SEQ ID NO.2 by real-time PCR. When the detection result was G / G, it was a wild-type non-brown midrib variety; when the detection result was A / A, it was a mutant brown midrib variety; and when the detection result was G / A, it was a heterozygous variety.

7. The application according to claim 6, characterized in that, The 10 μL system for real-time PCR reaction consisted of 2 μL of 30 ng / μL DNA template, 0.14 μL of primer mixture, 5 μL of KASP Mix, and 2.86 μL of ddH2O.

8. The application according to claim 7, characterized in that, Primer mixture containing SEQ ID NO.4 and SEQ ID The mass ratio of NO.5 to SEQ ID NO.6 is 6:6:18, and the final concentration is 0.2 μM.

9. The application according to claim 6, characterized in that, Real-time PCR amplification program: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s; 65℃→55℃ annealing for 15 s, decreasing by 1℃ per cycle, for a total of 10 cycles; 95℃ for 30s extension, 57℃ for 60s extension, 42 cycles; 30℃ for 30s incubation, and read the fluorescence signals of wild type and mutant.

10. The application of the KASP marker system as described in claim 3 or 4 in sorghum breeding.

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