Molecular marker related to sorghum panicle stalk character and application of molecular marker

Abstract

The invention belongs to the technical field of molecular biology and plant molecular marker assisted breeding research, and particularly relates to a molecular marker related to sorghum panicle stalk traits and application of the molecular marker. The molecular marker comprises an SNP (Single Nucleotide Polymorphism) site located at the 59789997 site of a No.10 chromosome of a sorghum genome, and the polymorphism of the SNP site is G / C. The molecular marker provided by the invention can be used for quickly and accurately identifying the sorghum panicle stalk character, and the molecular marker site provided by the invention can be used for accurately identifying the character phenotypes of normal sorghum panicle stalk extension and abnormal sorghum panicle stalk extension. The molecular marker is widely applied to molecular marker assisted breeding and whole-genome selective breeding of sorghum, the breeding process can be further accelerated, and the breeding efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and plant molecular marker-assisted breeding research technology, specifically involving a molecular marker related to the spikelet peduncle trait of sorghum and its application. Background Technology

[0002] Sorghum [Sorghum bicolor (L.) Moench] is the world's fifth largest cereal crop. As a C4 crop, it is characterized by its strong photosynthetic capacity, high biomass yield, excellent stress resistance, and high adaptability, and is widely cultivated in semi-arid and arid regions of Africa and Asia. Sorghum belongs to the Poaceae family. The stem of Poaceae crops is mainly composed of many internodes, among which the peduncle is the last segment to elongate, directly connecting to the ear. The peduncle (the length from the flag leaf pulvinus to the base of the ear) affects the plant's height and the degree of ear exposure, thus influencing yield and quality. During mechanized sorghum harvesting, the peduncle affects the harvester's loss rate and impurity content. The longer the peduncle of the sorghum plant, the more suitable it is for mechanized harvesting; conversely, the shorter the peduncle, the less suitable it is for mechanized harvesting.

[0003] However, current understanding of the mechanisms related to the panicle stalk is mainly derived from rice. For example, hybrid rice male-sterile lines often exhibit sheathed panicle-like phenomena, caused by obstructed stalk elongation, which severely affects hybrid seed production yield. In rice, several genes related to sheathed panicle elongation have been cloned, such as esp2, M893, sui-2, sui1-4, dsp1, and esp1. These genes influence the stalk elongation trait by regulating the synthesis of plant hormones such as gibberellin, brassinolide, and abscisic acid. Furthermore, several QTLs related to the panicle stalk have been located in the genomes of barley and wheat. In wheat, a TaWUS-like gene has been cloned, which controls the wheat panicle stalk by regulating the gibberellin synthesis pathway.

[0004] Currently, there are few reports on loci and genes related to sorghum spikelets. Discovering stable and significant associated loci related to sorghum spikelet traits and using them for breeding is of great significance for achieving high and stable sorghum yields and efficient harvesting. Summary of the Invention

[0005] The purpose of this invention is to provide a molecular marker related to the spikelet peduncle trait of sorghum and its application.

[0006] According to a first aspect of the present invention, a molecular marker related to the sorghum spikelet trait is provided. This molecular marker includes an SNP locus located at position 59789997 on chromosome 10 of the sorghum genome, and the polymorphism of the SNP locus is G / C. Specifically, when the polymorphism of the SNP locus is G, the corresponding genotype is GG, and the corresponding sorghum spikelet trait is normal spikelet elongation; when the polymorphism of the SNP locus is C, the corresponding genotype is CC, and the corresponding sorghum spikelet trait is abnormal spikelet elongation. As mentioned above, the spikelet elongation state of sorghum directly affects the plant height and the degree of spikelet exposure, thereby affecting plant yield and quality. Simultaneously, the spikelet elongation state of sorghum also affects the efficiency of mechanized harvesting. Based on this, the molecular marker related to the sorghum spikelet trait provided by the present invention can be used to breed sorghum varieties with better spikelet elongation state, thereby improving sorghum yield, quality, and harvesting efficiency.

[0007] In some embodiments of the present invention, the sorghum genome is derived from the T2T genome of red tassel sorghum.

[0008] In some embodiments of the present invention, the molecular marker genotype includes two genotypes, GG and CC; wherein the nucleotide sequence corresponding to the GG genotype is shown in SEQ ID NO:3, and the nucleotide sequence corresponding to the CC genotype is shown in SEQ ID NO:4 and SEQ ID NO:5.

[0009] In some embodiments of the present invention, the molecular marker comprises at least one of the following sequences;

[0010] The nucleotide sequence shown in SEQ ID NO:3;

[0011] The nucleotide sequence shown in SEQ ID NO:4;

[0012] The nucleotide sequence shown in SEQ ID NO:5.

[0013] In some embodiments of the present invention, the genotype of the SNP site is GG or CC; wherein, the nucleotide sequence corresponding to the GG genotype is shown in SEQ ID NO:3, and the nucleotide sequence corresponding to the CC genotype is shown in SEQ ID NO:4 and SEQ ID NO:5.

[0014] In some embodiments of the present invention, the nucleotide sequence of the SNP site is at least one of the following sequences;

[0015] The nucleotide sequence shown in SEQ ID NO:3;

[0016] The nucleotide sequence shown in SEQ ID NO:4;

[0017] The nucleotide sequence shown in SEQ ID NO:5.

[0018] In some embodiments of the present invention, the molecular marker is obtained by digesting the product amplified by the upstream primer shown in SEQ ID NO:1 and the downstream primer shown in SEQ ID NO:2 with the restriction endonuclease Sac1.

[0019] According to a second aspect of the present invention, the present invention also provides a primer pair for detecting molecular markers as described in any of the first aspects of the present invention, the primer pair comprising an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2.

[0020] According to a third aspect of the present invention, the present invention also provides a detection kit comprising a molecular marker as described in any of the first aspects of the present invention and / or a primer pair as described in the second aspect of the present invention.

[0021] According to a fourth aspect of the present invention, the present invention also provides the application of molecular markers as described in any of the first aspects of the present invention, or primer pairs as described in the second aspect of the present invention, or detection kits as described in the third aspect of the present invention in the identification of sorghum varieties and / or sorghum breeding.

[0022] In some embodiments of the present invention, the sorghum varieties include: sorghum varieties with normal ear stalk extension and sorghum varieties with abnormal ear stalk extension.

[0023] In some embodiments of the present invention, the sorghum variety includes sorghum varieties with genotypes GG or CC.

[0024] In some embodiments of the present invention, the sorghum breeding includes selecting sorghum varieties with genotypes GG or CC and / or selecting sorghum varieties with normal or abnormal spikelet pedicel extension.

[0025] According to a fifth aspect of the present invention, the present invention also provides a method for identifying sorghum spikelet traits, the method comprising: detecting the genotype or nucleotide sequence of an SNP site in the genome of a sorghum genome to be tested, and identifying the sorghum spikelet trait based on the detection results of the genotype / or nucleotide sequence; wherein the SNP site is located at position 59789997 on chromosome 10 of the sorghum genome, and the corresponding genotype is GG or CC; the nucleotide sequence corresponding to the SNP site is as shown in SEQ ID NO:3, SEQ ID NO:4, and / or SEQ ID NO:5; the sorghum spikelet trait includes normal spikelet elongation and abnormal spikelet elongation.

[0026] In some embodiments of the present invention, when the genotype of the SNP site in the genome of the sorghum to be tested is GG or the nucleotide sequence corresponding to the SNP site is as shown in SEQ ID NO:3, the spikelet pedicel trait of the sorghum to be tested is determined to be normal spikelet elongation; when the genotype of the SNP site in the genome of the sorghum to be tested is CC or the nucleotide sequence corresponding to the SNP site is as shown in SEQ ID NO:4 and / or as shown in SEQ ID NO:5, the spikelet pedicel trait of the sorghum to be tested is determined to be abnormal spikelet elongation.

[0027] According to a sixth aspect of the present invention, the present invention also provides a method for identifying the characteristics of sorghum spikelets, the method comprising the following steps:

[0028] (1) Extract genomic DNA from the sorghum to be identified;

[0029] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification reaction was performed using the designed primer pair to obtain the amplification product; the amplification product was digested with the restriction endonuclease Sac1 to obtain the digested product.

[0030] (3) The enzyme digestion products were detected by gel electrophoresis;

[0031] (4) Based on the gel electrophoresis detection results, the electrophoretic bands present in the enzyme digestion product are confirmed, and the spikelet characteristics of the sorghum to be identified are identified based on the confirmation results; wherein, the identification steps include: if the enzyme digestion product contains a homozygous band of 294bp or contains a heterozygous band of 294bp and 239bp, the spikelet characteristics of the sorghum to be identified are determined to be normal spikelet extension; if the enzyme digestion product contains only a homozygous band of 239bp, the spikelet characteristics of the sorghum to be identified are determined to be normal or abnormal spikelet.

[0032] The primer pair includes an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention uses the Shp-I sorghum mutant with abnormal pedicel extension as the female parent and the wild-type Red Tassel sorghum with normal pedicel extension as the male parent. The F1 generation is obtained through hybridization, and the F2 generation is obtained through self-crossing of the F1 generation. BSA-seq analysis reveals a new SNP locus highly linked to the sorghum pedicel trait. Based on the screened SNP locus, a molecular marker associated with the sorghum pedicel trait is developed. This molecular marker enables rapid and accurate identification of the sorghum pedicel trait. Furthermore, the SNP molecular marker locus provided by this invention can accurately identify the phenotypic trait of normal and abnormal sorghum pedicel extension. The state of sorghum pedicel extension directly affects plant height and the degree of ear exposure, thus impacting yield and quality. Simultaneously, the state of sorghum pedicel extension also affects the efficiency of mechanized harvesting. The molecular markers related to sorghum pedicel traits provided by this invention allow for the selection and breeding of sorghum varieties with better pedicel extension, thereby improving yield, quality, and harvesting efficiency. These molecular markers can be widely used in molecular marker-assisted breeding and genome-wide selection breeding of sorghum, accelerating the breeding process and improving breeding efficiency. Attached Figure Description

[0035] Figure 1 The pedicel phenotypes of the male parent (wild-type Red Tassel) (HYZ) and the female parent (mutant Shp-I) (Shp-I) in this embodiment of the invention are shown. Among them, "HYZ" represents the pedicel phenotype of the male parent (wild-type Red Tassel), that is, the pedicel extends normally, and "Shp-I" represents the pedicel phenotype of the female parent (mutant Shp-I), that is, the pedicel extends abnormally.

[0036] Figure 2 The results of BSA-seq analysis obtained in this embodiment of the invention are regarding the correlation region of sorghum spikelets.

[0037] Figure 3 The results of electrophoretic analysis of enzyme digestion products from 30 sorghum plants in the F2 generation populations of wild-type red sorghum (HYZ), mutant Shp-I sorghum (Shp-I), and paternal-wild-type red sorghum and maternal-mutant Shp-I in this embodiment of the invention; wherein, Figure 3 The "HYZ-294" indicates that the agarose gel electrophoresis pattern of the enzyme digestion product corresponding to wild-type red tassel sorghum contains a 294bp band. Figure 3The phrase "Shp-I-239 and 55bp" indicates that the agarose gel electrophoresis pattern of the enzyme digestion product of the mutant Shp-I sorghum contains two electrophoretic bands of 239bp and 55bp; the numbers 1-30 represent the electrophoretic band patterns of the enzyme digestion products of 30 sorghum plants in the F2 generation population. Detailed Implementation

[0038] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0039] Example 1: Mining of SNPs Related to Sorghum Spike Traits

[0040] A mutant with abnormally elongated peduncle, resulting in the base of the ear being enclosed by the flag leaf sheath, forming a sheathed panicle, was identified from the EMS mutant library of *Symplocos rubra*. This mutant underwent multiple generations of self-pollination and field testing in Guiyang, Guizhou and Ledong, Hainan, demonstrating that the mutant trait is stably inherited. This mutant was named Shp-I. Wild-type *Symplocos rubra* was used as the male parent (corresponding peduncle phenotype as shown in the image). Figure 1 As shown in “HYZ”, the pedicel extends normally. The mutant Shp-I is the maternal parent (the corresponding pedicel phenotype is shown in the figure). Figure 1 As shown in “Shp-I”, the spikelet stalk extends abnormally. F1 generation seeds are obtained by hybridization, and F1 generation plants are bagged and self-pollinated to harvest F2 generation.

[0041] I. DNA Extraction and Quality Inspection

[0042] From the F2 generation population, 30 individual plants exhibiting extreme phenotypes from both parents were selected. DNA was extracted from the leaves of these 30 selected individual plants exhibiting extreme phenotypes from both parents, as well as from the leaves of the parental sample plants. The specific DNA extraction steps are as follows:

[0043] (1) Take tender sorghum leaves and place them in a 2ml centrifuge tube. Freeze them in liquid nitrogen and grind them into powder using a tissue homogenizer. (2) Add 800ul of CTAB extraction solution to a 2ml centrifuge tube and place it in a 65℃ water bath for 30min. Gently shake it 5-8 times during the process to ensure complete DNA lysis. (3) Add 800ul of chloroform-isoamyl alcohol (volume ratio 24:1) and gently shake for 10min. (4) After centrifuging at 3000g for 10min, take 500ul of the supernatant and place it in a clean 96-well plate (note the corresponding serial number). (5) Add 500ul of isopropanol (pre-frozen at -20℃) and gently shake to mix. White DNA flocculents will be observed. Place it at -20℃ for 20min to increase DNA yield. (6) After centrifuging at 3000g for 10min, discard the supernatant. Wash the precipitate 2-3 times with 70% ethanol (pre-frozen at -20℃) and air dry until there is no alcohol smell. (7) Add 300ul of chloroform-isoamyl alcohol to a 2ml centrifuge tube and grind it into powder using a tissue homogenizer. DNA was dissolved in ddH2O to obtain the genomic DNA of the corresponding sorghum sample, and then stored at -20℃.

[0044] Genomic DNA extracted using the above method was selected, and a portion of the samples were analyzed for quality and concentration using agarose gel electrophoresis and a nucleic acid protein analyzer.

[0045] The steps for detecting DNA quality by 1% agarose gel electrophoresis are as follows: (1) Weigh 1.00g of agarose and put it into an Erlenmeyer flask; (2) Add 100ml of 1×TAE to the Erlenmeyer flask; (3) Heat to dissolve, shaking 1-2 times in the middle to fully dissolve the agarose; (4) When the temperature of the Erlenmeyer flask drops to about 60℃, add 10μl of nucleic acid dye; (5) Pour the agarose solution into the mold with the comb inserted, and wait for the agarose to solidify into a gel; (6) Remove the comb, add 5μl of DNA sample to the well, current I = 100mA, voltage V = 120V, and perform electrophoresis for 20-30min; (7) After the electrophoresis is completed, use the GelDoxXR gel imaging system to take pictures and save the electrophoresis results.

[0046] The steps for determining the concentration of DNA in a sample are as follows: The concentration of the DNA sample is determined using a nucleic acid protein analyzer. The DNA should be within the OD range. 260 It exhibits a significant absorption peak, OD 260 When OD = 1, it is equivalent to more than 50 μg / ml of double-stranded DNA, when OD 260 / OD 280 When the pH is 1.7–1.9, the DNA purity of the sample is relatively high.

[0047] II. Mixed Pool Construction

[0048] After the DNA extracted in step one passed quality inspection and its corresponding concentration was measured using a nucleic acid protein analyzer, the leaf DNA of 30 plants with the paternal phenotype of wild-type *Rhododendron simsii* was mixed to construct a wild-type mixed pool; the leaf DNA of 30 plants with the paternal phenotype of mutant *Shp-I* was mixed to construct a mutant mixed pool; the leaf DNA of the paternal phenotype of wild-type *Rhododendron simsii* was mixed to obtain a paternal parent mixed pool; and the leaf DNA of the paternal phenotype of mutant *Shp-I* was mixed to obtain a maternal parent mixed pool.

[0049] The mixed pool constructed above was sent to a sequencing company for deep sequencing. The sequencing process is as follows: the DNA in the mixed pool was randomly fragmented using a high-performance ultrasonic sample processing system, followed by DNA fragment end repair, with an "A" base added to the 3' end and library adapters added to both ends; the library after adapter ligation was linearly amplified; an appropriate amount of amplification product was taken for single-strand separation and circularization, and the circularized library was replicated by rolling circle to generate DNA nanospheres. After quality control, optimized combined probe anchoring polymerization technology and improved DNA nanosphere core sequencing technology were used to perform high-throughput sequencing on each qualified library; the raw image data obtained from sequencing was converted into raw sequence data using BGISEQ-T7 base recognition software, and the raw sequencing data was quality controlled and filtered using Soapnuke (v1.65) to obtain high-quality CleanData data. The raw data filtering conditions are as follows: (1) reads containing adapters were removed; (2) low-quality reads (40% of the bases had a base quality value of less than or equal to 20) were removed; (3) reads containing more than 5% of N bases were removed. The resulting high-quality, clean data is used for subsequent analysis. Quality control procedures are applied to the raw data, including the removal of adapters and low-quality sequences.

[0050] After filtering, the cleandata was aligned to the T2T reference genome of *Sorghum sorghum* using the "mem" algorithm of the BWA alignment software. The alignment results were sorted using SAMtools software (v1.9). Variant sites were detected using the HaplotypeCaller algorithm (local haplotype assembly) based on GATK software (Version 4.1.2). Variant sites were annotated and predicted using SnpEff software (Version 5.1).

[0051] III. BSA-seq Analysis

[0052] (1) SNP-index

[0053] The degree of sequence difference between the offspring population and the parents is represented by the SNP-index. The SNP-index is equal to the ratio of the number of reads with SNPs different from the reference parent to the total number of reads at the same position. A 1Mb sliding window and a 500kb step size are used to calculate the SNP-index and Δ(SNP-index) for all chromosome positions. An SNP index value of 0 indicates no variation, an SNP index value of 1 indicates all SNPs belong to one parent, and an SNP index value of 0.5 indicates that both parents contribute equally to the variation. The SNP-index between the two mixed pools is calculated separately, and the difference between the SNP-index of the two extreme pools is used to obtain the ΔSNP-index, as detailed below:

[0054] △SNPindex=SNP index1-SNP index2;

[0055] In the formula, SNP index1 and SNP index2 represent the degree of difference between the offspring populations and the parents in the two extreme pools.

[0056] (2)ED

[0057] ED (Euclidean distance) is an abbreviation for Euclidean distance. It calculates the frequency distance between mutants in different pools and uses the distance difference to reflect the linkage strength between the marker and the target region. The specific calculation method is as follows:

[0058]

[0059] In the formula, mut and wt represent the mutant pool and wild-type pool, respectively, which are the two extreme pools in this screening. A, C, G, and T represent the proportion of sequencing reads of each mutant at the marker site. Based on the obtained SNP locus set and genotype depth information between the pools, the mutation frequency difference between the pools, i.e., the ED value, is calculated.

[0060] To amplify the differences and reduce background noise, the ED value was increased to ED. 4 As shown below:

[0061] ED 4 =[(Amut-Awt) 2 +(Cmut-Cwt) 2 +(Gmut-Gwt) 2 +(Tmut-Twt) 2 ] 2 ;

[0062] The results obtained through the above calculation and analysis methods are as follows: Figure 2 As shown, according to Figure 2The results showed a continuous and high peak across the entire genome on chromosome 10, identifying a sorghum spikelet-associated region. Finally, SNP data within this region were screened to develop SNP sites for dCAPS markers. The specific screening steps were as follows: A total of 836,507 SNPs and 207,726 InDel variant sites were obtained from four sample pools. Using the delta-SNP-index algorithm, a significant candidate region (0.29 Mb, 59.65 Mb–59.94 Mb) containing 32 genes was detected on chromosome 10. ED was used... 4 The algorithm detected two significant candidate regions on chromosomes 9 and 10, with lengths of 1.02 Mb (60.93 Mb–61.68 Mb) and 0.27 Mb (59.65 Mb–59.92 Mb), respectively, containing a total of 128 genes. The intersection of the two algorithms identified a candidate region (0.27 Mb, 59.65 Mb–59.92 Mb) on chromosome 10 associated with the spikelet, containing 28 genes. Referring to the sequence information, annotation information, and resequencing results of the *Sorghum sorghum* genome, SNP sites were screened within the candidate region, and the presence of related restriction enzyme sites was checked based on the sequence of variants. Finally, a SNP site significantly associated with the spikelet trait of sorghum was found. This SNP site is located at position 59789997 on chromosome 10 of the *Sorghum sorghum* genome. This SNP site has a GC mutation and can be digested by the Sac1 enzyme.

[0063] Example 2: Development of Molecular Markers

[0064] The development and design of corresponding molecular markers based on the SNP sites screened in Example 1 are as follows:

[0065] I. Genomic DNA of wild-type red-tasseled sorghum and mutant Shp-I sorghum was analyzed using the CTAB method. The specific steps are as follows:

[0066] (1) Take tender leaves of the corresponding sorghum variety samples and place them in 2ml centrifuge tubes. Freeze them in liquid nitrogen and grind them into powder using a tissue homogenizer. (2) Add 800ul CTAB extraction solution to 2ml centrifuge tubes and place them in a 65℃ water bath for 30min. Gently shake 5-8 times during the process to ensure complete DNA lysis. (3) Add 800ul chloroform-isoamyl alcohol (volume ratio 24:1) and gently shake for 10min. (4) After centrifuging at 3000g for 10min, take 500ul of the supernatant and place it in a clean 96-well plate (note the corresponding serial number). (5) Add 500ul isopropanol (pre-frozen at -20℃) and gently shake to mix. White DNA flocculents will be observed. Place the plate at -20℃ for 20min to increase DNA yield. (6) After centrifuging at 3000g for 10min, discard the supernatant. Wash the precipitate 2-3 times with 70% ethanol (pre-frozen at -20℃) and air dry until there is no alcohol smell. (7) Add 300ul DNA was dissolved in ddH2O to obtain the genomic DNA of the corresponding sorghum sample, which was then stored at -20°C for later use.

[0067] II. Primer Design

[0068] Based on the SNP sites screened in Example 1, the sequence information of the upstream and downstream 250 bp of each SNP site was retrieved. Multiple sets of amplification primers were then designed using Primer Premier5 (http: / / www.premierbiosoft.com / primerdesign / ). The quality of the designed primers was then evaluated using DNAMAN, and primer specificity was further detected using a genomic database. The primer sequence information is as follows:

[0069] The upstream primer sequence is: 5'-CATCTCGGCTCTTCTTCTTCTC-3' (SEQ ID NO:1);

[0070] The downstream primer sequence is: 5'-CTTCTGGTTCCTCCACAGC-3' (SEQ ID NO:2).

[0071] III. PCR Amplification

[0072] Based on the primer pairs designed above, the genomic DNA of wild-type red sorghum and mutant Shp-I sorghum extracted in step one was amplified to obtain the corresponding PCR amplification products. The PCR reaction system was as follows: 10 μl of 2×PCRMix, 0.2 μl of 10 mol / μl upstream primer, 0.2 μl of 10 mol / μl downstream primer, 1 μl of template DNA, and 8.6 μl of sterile double-distilled water. The PCR reaction program was as follows: 94℃ for 1 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s. This process was repeated for 30 cycles, with a final extension at 72℃ for 5 min.

[0073] IV. Enzyme digestion

[0074] The corresponding amplification products obtained by PCR amplification in step 3 were subjected to enzyme digestion to obtain the corresponding enzyme digestion products. The enzyme digestion reaction system consisted of 1.5 μl of 10× Buffer, 0.3 μl of Sacll enzyme, 10 μl of PCR product, and 3.2 μl of sterile double-distilled water. The enzyme digestion reaction program was 37℃ for 12 h.

[0075] V. Electrophoresis Detection

[0076] The enzyme digestion products obtained in step four were analyzed by 2% agarose gel electrophoresis to observe the differences in band positions. The enzyme digestion products of wild-type red-tasseled sorghum showed one band on the agarose gel electrophoresis pattern, corresponding to 294 bp (the corresponding nucleotide sequence is shown in SEQ ID NO:3), which corresponds to the genotype GG, denoted as G, and the corresponding sorghum variety has normal spikelet extension. The enzyme digestion products of mutant Shp-I sorghum showed two bands on the agarose gel electrophoresis pattern, namely 239 bp (the corresponding nucleotide sequence is shown in SEQ ID NO:4) and 55 bp (the corresponding nucleotide sequence is shown in SEQ ID NO:5), which correspond to the genotype CC, denoted as C, and the corresponding sorghum variety has abnormal spikelet extension.

[0077] Example 3: Validation of the application of molecular markers

[0078] The application verification of the molecular markers developed in Example 2 was carried out, and the specific steps are as follows:

[0079] I. Genomic DNA was extracted from 30 sorghum plants with known spikelet characteristics in the F2 population using the CTAB method (15 plants with normal spikelet elongation, corresponding to genotype GG, denoted as G; 15 plants with abnormal spikelet elongation, corresponding to genotype CC, denoted as C). The specific steps are as follows:

[0080] (1) Take tender leaves from the corresponding sorghum plant samples and place them in 2ml centrifuge tubes. Freeze them in liquid nitrogen and grind them into powder using a tissue homogenizer; (2) Add 800ul (2) Place the CTAB extract in a 2ml centrifuge tube and in a 65℃ water bath for 30min, gently shaking 5-8 times during the process to ensure complete DNA lysis; (3) Add 800ul chloroform-isoamyl alcohol (volume ratio 24:1) and gently shake for 10min; (4) After centrifuging at 3000g for 10min, take 500ul of the supernatant and place it in a clean 96-well deep plate (note the corresponding serial number); (5) Add 500ul isopropanol (pre-frozen at -20℃) and gently shake to mix. White DNA flocculents will be observed. Place it at -20℃ for 20min to increase DNA yield; (6) After centrifuging at 3000g for 10min, discard the supernatant. Wash the precipitate 2-3 times with 70% ethanol (pre-frozen at -20℃) and air dry until there is no alcohol smell; (7) Add 300ul ddH2O to dissolve the DNA to obtain the genomic DNA of the corresponding sorghum plant. Store it at -20℃ for later use.

[0081] II. PCR Amplification

[0082] The genomic DNA of each sorghum plant was amplified using primer pairs designed in Example 2 (as shown in SEQ ID NO:1 and SEQ ID NO:2) to obtain the corresponding PCR amplification products. The PCR reaction system was as follows: 10 μl of 2×PCRMix, 0.2 μl of 10 mol / μl upstream primer, 0.2 μl of 10 mol / μl downstream primer, 1 μl of template DNA, and 8.6 μl of sterile double-distilled water. The PCR reaction program was as follows: 94℃ for 1 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s. This process was repeated 30 times, with a final extension at 72℃ for 5 min.

[0083] III. Enzyme Digestion

[0084] The corresponding amplification products obtained from PCR amplification were subjected to enzyme digestion reactions to obtain the corresponding enzyme digestion products. The enzyme digestion reaction system consisted of 1.5 μl of 10× Buffer, 0.3 μl of Sacllase, 10 μl of PCR product, and 3.2 μl of sterile double-distilled water. The enzyme digestion reaction program was 37℃ for 12 h.

[0085] IV. Electrophoresis Detection

[0086] The enzyme digestion products obtained in step three were analyzed by 2% agarose gel electrophoresis. The genotype of the corresponding sorghum plant was determined based on the electrophoresis results: if the enzyme digestion product contained a homozygous band of 294 bp, the genotype of the corresponding sorghum plant was determined to be GG or GC, and the pedicel of the tested sorghum plant would extend normally; if the enzyme digestion product contained only a homozygous band of 239 bp, the genotype of the corresponding sorghum plant was determined to be CC, and the pedicel of the tested sorghum plant would extend abnormally. The electrophoresis results are shown below. Figure 3 As shown, according to Figure 3 The electrophoresis results shown indicate that, among the 30 sorghum plants from 30 F2 populations, 15 sorghum plants had enzyme digestion products containing a band of 239 bp (i.e., corresponding to...). Figure 3 The sorghum plants with the reference number 16-30 were identified as having abnormal spikelet elongation, with the genotype CC, corresponding to the maternal parent's phenotype; the enzyme digestion products of the 15 sorghum plants contained 294 bp (i.e., the corresponding...). Figure 3 The sorghum plants with the number 1-15 were identified as having normal spikelet stalk extension, genotype GG, and corresponding phenotype of the male parent.

[0087] The phenotypic information and corresponding genotype identification results of the 30 sorghum plants identified in Example 3 are shown in Table 1:

[0088] Table 1

[0089]

[0090] Combination Figure 3 As shown in Table 1, the results of genotyping of 30 sorghum plants from 30 F2 populations based on the molecular markers developed above are completely consistent with the actual spikelet phenotypes of the 30 sorghum plants, with a detection accuracy of 100%. This demonstrates that molecular markers further developed based on the SNP sites discovered in this invention can achieve accurate detection and identification of spikelet traits in sorghum.

[0091] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present invention are protected by the present invention.

[0092]

Claims

1. A molecular marker associated with a Sorghum peduncle trait, comprising, The molecular marker comprises a SNP site located at 59789997 of chromosome 10 of a sorghum genome, and the polymorphism of the SNP site is G / C.

2. The molecular marker of claim 1, wherein The sorghum genome is derived from a T2T genome of red plume sorghum.

3. The molecular marker of claim 1, wherein The genotype of the molecular marker comprises GG and CC; the nucleotide sequence corresponding to the GG genotype is shown as SEQ ID NO: 3, the nucleotide sequences corresponding to the CC genotype are shown as SEQ ID NO: 4 and SEQ ID NO: 5; and / or the molecular marker comprises at least one of the following sequences: the nucleotide sequence shown as SEQ ID NO: 3; the nucleotide sequence shown as SEQ ID NO: 4; the nucleotide sequence shown as SEQ ID NO:

5.

4. The molecular marker of claim 1, wherein The molecular marker is obtained by amplifying the product with the upstream primer shown as SEQ ID NO: 1 and the downstream primer shown as SEQ ID NO: 2, and then cutting with a restriction enzyme Sacll.

5. A pair of primers, characterized in that, The primer pair for detecting the molecular marker of any one of claims 1-4 comprises the upstream primer shown as SEQ ID NO: 1 and the downstream primer shown as SEQ ID NO:

2.

6. A test kit characterized in that, The detection kit comprises the molecular marker of any one of claims 1-4 or the primer pair of claim 5.

7. Use of the molecular marker of any one of claims 1-4 or the primer pair of claim 5 or the detection kit of claim 6 in identifying sorghum varieties and / or breeding sorghum. Preferably, the sorghum variety comprises: sorghum varieties with normal panicle stalk extension and sorghum varieties with abnormal panicle stalk extension; Preferably, the sorghum varieties comprise sorghum varieties with genotypes of GG or CC. Preferably, the breeding of sorghum comprises breeding sorghum varieties with genotypes of GG or CC and / or breeding sorghum varieties with normal or abnormal panicle stalk extension.

8. A method for identifying the characteristics of sorghum spike stalks, characterized in that, The identification method comprises detecting the genotype or nucleotide sequence of the SNP site in the sorghum genome to be detected, and identifying the panicle stalk trait according to the detection result of the genotype or nucleotide sequence; the SNP site is located at 59789997 of chromosome 10 of the sorghum genome, and the genotype thereof is GG or CC; the nucleotide sequence corresponding to the SNP site is shown as SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5; and the panicle stalk trait comprises normal panicle stalk extension and abnormal panicle stalk extension.

9. The method of claim 8, wherein, When the genotype of the SNP site in the sorghum genome to be detected is GG or the nucleotide sequence corresponding to the SNP site is shown as SEQ ID NO: 3, it is determined that the panicle stalk trait of the sorghum to be detected is normal panicle stalk extension; when the genotype of the SNP site in the sorghum genome to be detected is CC or the nucleotide sequence corresponding to the SNP site is shown as SEQ ID NO: 4 and / or SEQ ID NO: 5, it is determined that the panicle stalk trait of the sorghum to be detected is abnormal panicle stalk extension.

10. A method of identifying a stalk trait in sorghum, comprising, The identification method comprises the following steps: (1) extracting genomic DNA of the to-be-identified sorghum; (2) using the genomic DNA obtained in step (1) as a template, performing a PCR amplification reaction using a designed primer pair to obtain an amplification product; performing an enzyme cutting reaction on the amplification product using a restriction enzyme Sacll to obtain an enzyme cutting product; (3) performing gel electrophoresis detection on the enzyme cutting product; (4) confirming the existence of an electrophoresis band in the enzyme cutting product according to the gel electrophoresis detection result, and identifying the ear stalk trait of the to-be-identified sorghum according to the confirmation result; wherein the identification step comprises: if the enzyme cutting product contains a homozygous band of 294 bp, it is determined that the ear stalk trait of the to-be-identified sorghum is that the ear stalk normally extends, and if the enzyme cutting product contains only a homozygous band of 239 bp, it is determined that the ear stalk trait of the to-be-identified sorghum is that the ear stalk is normally abnormal; wherein the primer pair comprises an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2.

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