Molecular marker related to pericarp thickness character in sorghum grains 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 the peel thickness character in sorghum grains and application of the molecular marker. The SNP site of the molecular marker is located at the 57954159th site of a second chromosome of a sorghum genome, and the polymorphism of the molecular marker is G / C. The molecular marker provided by the invention can quickly and accurately identify the thickness character of the pericarp in the sorghum grains, and compared with other SNP molecular markers screened in the analysis process, the SNP molecular marker site provided by the invention can more accurately identify the thickness / thinness of the pericarp in the sorghum grains, so that the identification of the thickness / thinness of the pericarp in the sorghum grains is more accurate. The breeding efficiency of the new sorghum variety can be further improved by utilizing the molecular marker-assisted breeding technology.

Description

Technical Field

[0001] This application belongs to the technical fields of molecular biology and plant molecular marker-assisted breeding research, and particularly relates to a molecular marker related to the pericarp thickness trait in sorghum grains and its application. Background Art

[0002] Sorghum (Sorghum bicolor) is the fifth largest cereal crop in the world after rice, wheat, corn, and oats. Sorghum grains are caryopses, consisting of the pericarp, testa, endosperm, and embryo. The pericarp and testa are collectively called the seed coat.

[0003] The pericarp of sorghum develops from the ovary wall and consists of three parts: the exocarp, mesocarp, and endocarp. The mesocarp is often related to the rice yield and flour yield. The thinner the mesocarp, the higher the rice yield and flour yield tend to be. Guizhou Province is the origin and main production area of Maotai-flavor liquor. It takes about 2.6 catties of wheat for making koji and 2.4 catties of waxy sorghum as raw materials to brew one catty of Maotai-flavor liquor. Generally, it is considered that sorghum for liquor should have the characteristic of being resistant to cooking to ensure that the raw materials can withstand the unique brewing process of "nine times of cooking and eight times of fermentation". Therefore, it is required that sorghum grains should have an appropriate pericarp and testa thickness to achieve good cooking resistance. Therefore, the pericarp and testa related traits of sorghum grains are the key factors affecting the brewing process and quality of Maotai-flavor liquor.

[0004] Grain traits are important factors affecting sorghum yield and uses. Although there have been relatively comprehensive studies on grain-related traits at present, as of now, there are few genetic research reports on QTL / genes related to the pericarp and testa thickness of sorghum grains. Currently, only two genes affecting the seed coat thickness have been mapped on chromosome 2 of sorghum using a genetic linkage map. One is the B2 gene that controls the appearance of the testa, and the other is the Z gene that affects the mesocarp thickness / seed luster. Regarding the Z gene, currently, using the recombinant inbred line QL399×QL41, the Z gene has been mapped in the interval of 89.9 - 104.4 cM on chromosome 2, which is tightly linked to the SSR markers txp298 and SbAGAB03 and is located in the physical segment of 57.03 - 59.10 Mb on the integrated map. Further, using the GBS technology, Hu et al. performed a GWAS analysis on 10,000 Ethiopian sorghum accessions and further mapped the Z gene to around 57,610,965 bp.

[0005] Currently, although some studies have been carried out on the B2 gene affecting the appearance of the testa and the Z gene controlling the mesocarp thickness (seed pearl luster), they are usually used as morphological markers for constructing genetic maps, and no in-depth studies on their genetic characteristics have been reported. Based on this, the purpose of this application is to provide a new molecular marker related to the mesocarp thickness trait in sorghum grains and its application. Summary of the Invention

[0006] The purpose of the present application is to provide a new molecular marker related to the pericarp thickness trait in sorghum grains, and the developed new molecular marker can quickly and effectively identify the thick / thin pericarp of sorghum grains. Using this molecular marker-assisted breeding technology can further improve the breeding efficiency of new sorghum varieties.

[0007] According to the first aspect of the present application, the present application provides a molecular marker related to the pericarp thickness trait in sorghum grains. The SNP locus of the molecular marker is located at position 57954159 on chromosome 2 of the sorghum genome, and its polymorphism is G / C. Specifically, in some embodiments of the present application, the SNP locus of the molecular marker is located at position 57954159 on chromosome 2 of the sorghum genome, and its polymorphism is G or C, that is, its wild locus is G and the mutant locus is C.

[0008] In some embodiments of the present application, the sorghum genome is derived from LTR108.

[0009] According to the second aspect of the present application, the present application further provides a molecular marker related to the pericarp thickness trait in sorghum grains. The molecular marker includes the nucleotide sequence shown in SEQ ID NO.1, and the 101st position of the nucleotide sequence from the 5' end has polymorphism, and its polymorphism is G / C. Specifically, in some embodiments of the present application, the 101st position of the nucleotide sequence from the 5' end has polymorphism, and its polymorphism is G or C, that is, its wild locus is G and the mutant locus is C.

[0010] According to the third aspect of the present application, the present application further provides a KASP primer pair, which includes a forward competitive primer shown in SEQ ID NO.2, a forward competitive primer shown in SEQ ID NO.3, and a reverse universal primer shown in SEQ ID NO.4.

[0011] In some embodiments of the present application, the KASP primer pair is used to amplify the molecular marker according to any one of the first aspect or the second aspect of the present application.

[0012] According to the fourth aspect of the present application, the present application further provides a kit, which contains the molecular marker according to any one of the first aspect or the second aspect of the present application or / and the KASP primer pair according to any one of the third aspect of the present application.

[0013] According to the fifth aspect of the present application, the present application further provides an application of a molecular marker as described in any one of the first aspect or the second aspect of the present application and / or a KASP primer pair as described in any one of the third aspect of the present application and / or a kit as described in any one of the fourth aspect of the present application in identifying sorghum varieties and / or sorghum breeding. In some embodiments of the present application, the sorghum varieties include: sorghum varieties with thick pericarp in grains and sorghum varieties with thin pericarp in grains.

[0014] According to the fifth aspect of the present application, the present application further provides a method for identifying or assisting in identifying the thickness of the pericarp of sorghum grains. The method includes detecting the genotype of the SNP locus in the genome of the sorghum to be tested, and identifying or assisting in identifying the thickness of the pericarp of sorghum grains according to the detection result of the genotype. The SNP locus is located at position 57954159 on chromosome 2 of the sorghum genome, and its nucleotide type is G or C. In some embodiments of the present application, the SNP locus is the 101st nucleotide from the 5' end of the nucleotide sequence shown in SEQ ID NO.1.

[0015] According to the sixth aspect of the present application, the present application further provides a method for identifying or assisting in identifying the thickness of the pericarp of sorghum grains. The method includes the following steps:

[0016] (1) Extract the genomic DNA of the sorghum to be identified;

[0017] (2) Using the genomic DNA extracted in step (1) as a template, perform a PCR amplification reaction with the designed KASP primer set to obtain an amplification product;

[0018] (3) Use a fluorescence detection platform to perform fluorescence signal scanning and genotyping on the amplification product obtained in step (2);

[0019] (4) According to the genotyping result in step (3), identify the variety of the sorghum to be tested;

[0020] Wherein, the primer pair includes a forward competitive primer shown in SEQ ID NO.2, a forward competitive primer shown in SEQ ID NO.3, and a reverse universal primer shown in SEQ ID NO.4.

[0021] In some embodiments of the present application, the identifying the variety of the sorghum to be tested according to the genotyping result in step (3) includes:

[0022] When the fluorescence signal of the forward competitive primer as shown in SEQ ID NO.2 appears in the amplification product of the sorghum to be tested, while the fluorescence signal of the forward competitive primer as shown in SEQ ID NO.3 does not appear, then the sorghum variety to be tested is identified as a sorghum variety with a thin pericarp in the grain; and / or, if the genotyping result is GG, then the sorghum variety to be tested is identified as a sorghum variety with a thin pericarp in the grain;

[0023] When the fluorescence signal of the forward competitive primer as shown in SEQ ID NO.3 appears in the sorghum to be tested, while the fluorescence signal of the forward competitive primer as shown in SEQ ID NO.2 does not appear, then the sorghum variety to be tested is identified as a sorghum variety with a thick pericarp in the grain; and / or, if the genotyping result is CC, then the sorghum variety to be tested is identified as a sorghum variety with a thick pericarp in the grain;

[0024] When the sorghum to be tested shows two fluorescence signals, then the sorghum variety to be tested is identified as a sorghum variety with a thin pericarp in the grain; and / or, if the genotyping result is GC, then the sorghum variety to be tested is identified as a sorghum variety with a thin pericarp in the grain.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] In the present application, 654 with a relatively thick pericarp in the grain is used as the male parent, and LTR108 with a relatively thin pericarp in the grain is used as the female parent. The F1 generation is obtained by hybridization, and the F2 generation is obtained by self-crossing of the F1 generation. Through BSA-seq analysis and screening, an SNP locus highly linked to the trait of sorghum grain pericarp thickness is obtained. Based on the screened SNP locus, a molecular marker related to the trait of sorghum grain pericarp thickness is provided. Through this molecular marker, the identification of the trait of sorghum grain pericarp thickness can be achieved quickly and accurately. Moreover, compared with other SNP molecular markers screened during the analysis process, the SNP molecular marker locus provided by the present application can more accurately identify the thick / thin pericarp of sorghum grains. Using this molecular marker-assisted breeding technology can further improve the breeding efficiency of new sorghum varieties. Description of the Drawings

[0027] Figure 1 It is a comparison diagram with the longitudinal section of the grains of 654 and LTR108 sorghum varieties magnified 200 times; among them, the red marking is the pericarp of the corresponding sorghum variety grains, 654 is a sorghum sample with a relatively thick pericarp in the grain, and LTR108 is a sorghum sample with a relatively thin pericarp in the grain;

[0028] Figure 2It is the positioning result of QTL-seq △SNP-index; among them, the blue line represents the 99% threshold, the green line represents the 95% threshold, the black dots represent the △SNP-index of each SNP, and the red line represents the result of sliding window (△SNP-index) analysis;

[0029] Figure 3 It is based on ED 4 Schematic diagram of QTL mapping for mesocarp thickness calculated; among them, the yellow line represents the 99% threshold line, and the black line represents the trend line;

[0030] Figure 4 It is the specific genotype typing result map of detecting the F2 segregation population using the Chr02_57954159 marker; among them, red represents the FAM fluorophore, representing the thin mesocarp type of sorghum grains, which is the LTR108 sorghum genotype type, blue represents the HEX fluorophore, representing the thick mesocarp type of sorghum grains, which is the 654 sorghum genotype type, and green represents the simultaneous presence of both FAM and HEX fluorophore signals, representing the heterozygous genotype type, representing the thin mesocarp type of sorghum grains. Detailed implementation mode

[0031] The technical solutions of the present application are further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present application. Some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the protection scope of the present application.

[0032] Example 1 Screening of SNP loci highly linked to the mesocarp thickness trait of sorghum grains

[0033] The experimental materials used in this example are as follows: 654 with a relatively thick mesocarp of grains was used as the male parent and LTR108 with a relatively thin mesocarp of grains was used as the female parent. The F1 generation was obtained by hybridization, and the F1 generation was self-crossed to obtain the F2 generation. A total of 883 F2 generation plants were obtained. Among them, the mesocarp conditions of 654 (male parent) with a relatively thick mesocarp of grains and LTR108 (female parent) with a relatively thin mesocarp of grains are as Figure 1 shown.

[0034] I. Identification of mesocarp thickness of F2 sorghum grains

[0035] After the harvested sorghum seeds are naturally dried, select 3 seeds of the same size for longitudinal cutting. Place the longitudinally cut seeds under a stereomicroscope, fix them with transparent tape, select the highest point on the back of the grain close to the embryo, magnify it 200 times and take a photo for preservation. Then measure the mesocarp thickness of the preserved photo and enter the data into a table. Among them, in this application, according to the phenotype of the mesocarp being thin as dominant, and based on the segregation ratio of 3:1 in the F2 generation, determine the range of the mesocarp thickness. Specifically, with the mesocarp thickness of 71.614 μm as the boundary, if the mesocarp thickness is less than 71.614 μm, it is thin; if the mesocarp thickness is greater than 71.614 μm, it is thick.

[0036] II. DNA Extraction and Quality Inspection

[0037] (1) DNA Extraction

[0038] At the three-leaf stage of the F2 generation plants, take the leaves and freeze them. Then, according to the identification results of the mesocarp thickness of sorghum grains in Step 1, respectively select the leaves of 50 individual plants with extremely thick mesocarps and 50 individual plants with extremely thin mesocarps of grains for DNA extraction. The specific steps of DNA extraction are as follows:

[0039] 1) Take the young and tender sorghum leaves and place them in a 2 ml centrifuge tube, freeze them with liquid nitrogen and grind them into powder on a tissue grinder;

[0040] 2) Add 800 ul of CTAB extraction solution to the 2 ml centrifuge tube, place it in a 65 °C water bath for 30 min, and gently shake it 5 - 8 times during this period to fully lyse the DNA;

[0041] 3) Add 800 ul of chloroform - isoamyl alcohol (volume ratio 24:1) and gently shake it for 10 min;

[0042] 4) After centrifuging at 3000 g for 10 min, take 500 ul of the supernatant and place it in a clean 96 - well deep - well plate (pay attention to the corresponding serial number);

[0043] 5) Add 500 ul of isopropanol (frozen at - 20 °C in advance), gently shake and mix well, and white DNA floccules can be seen. Place it at - 20 °C for 20 min to increase the DNA yield;

[0044] 6) After centrifuging at 3000 g for 10 min, pour out the supernatant, wash the precipitate 2 - 3 times with 70% ethanol (frozen in a - 20 °C refrigerator in advance) and air - dry it until there is no alcohol smell;

[0045] 7) Add 300 ul of ddH2O to dissolve the DNA, obtain the genomic DNA of the corresponding sorghum sample, and place it at - 20 °C.

[0046] (2) Quality Inspection

[0047] Select a part of the DNA samples obtained in step (1) and use agarose gel electrophoresis and a nucleic acid protein analyzer to measure the quality and concentration of the DNA samples.

[0048] The steps for detecting the quality of DNA by 1% agarose gel electrophoresis are as follows: 1) Weigh 1.00 g of agarose and put it into a conical flask; 2) Add 100 ml of 1×TAE to the conical flask; 3) Heat and dissolve, shake 1 - 2 times in the middle to fully dissolve the agarose; 4) When the temperature of the conical flask drops to about 60 °C, add 10 μl 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) Pull out the comb, add 5 μl of the DNA sample to the wells, set the current I = 100 mA and the voltage V = 120 V, and perform electrophoresis for 20 - 30 min; 7) After electrophoresis, use the GelDoxXR gel imaging system to take pictures and save the electrophoresis results.

[0049] Measurement of the concentration of the sample DNA: Use a nucleic acid protein analyzer to measure the concentration of the DNA sample. DNA should have a significant absorption peak at OD260. When OD260 = 1, it is equivalent to more than 50 μg / ml of double-stranded DNA. When OD260 / OD280 is 1.7 - 1.9, the purity of the obtained sample DNA is relatively high.

[0050] III. Pool construction

[0051] The steps for pool construction are as follows:

[0052] After the DNA extracted in Step 2 passed the quality inspection and its corresponding concentration was measured using a nucleic acid protein analyzer, the DNA of the leaves of 50 individual plants with extremely thick mesocarps of the grains was mixed to obtain a thick mesocarp pool of the grains, and the DNA of the leaves of 50 individual plants with extremely thin mesocarps of the grains was mixed to obtain a thin mesocarp pool of the grains. Then it was sent to a sequencing company for deep sequencing, and its sequencing process was as follows: The DNA of the two parents and the pooled samples was randomly fragmented by an ultrasonic high-performance sample processing system, followed by end repair of the DNA fragments, addition of an "A" base at the 3' end, and addition of library adapters at both ends; the ligated library was linearly amplified; an appropriate amount of the amplified product was taken for single-strand separation and circularization treatment, and the circularized library was replicated by rolling circle to generate DNA nanoballs. After passing the quality control, an optimized combined probe-anchored polymerization technology and an improved DNA nanoball core sequencing technology were used to perform high-throughput sequencing on each qualified library; for the original image data obtained by sequencing, it was converted into original sequence data by the BGISEQ-T7 base recognition software, and the original sequencing data was quality-controlled and filtered using Soapnuke (v1.65) to obtain high-quality CleanData data. Among them, the original data filtering conditions were as follows: (1) Remove reads containing adapters; (2) Remove low-quality reads (reads with a base quality value less than or equal to 20 accounting for 40%); (3) Remove reads with a proportion of N bases greater than 5%. The obtained high-quality clean data was used for subsequent analysis, and a quality control program was applied to the original data, including removing adapters and low-quality sequences.

[0053] After filtering, the cleandata was aligned to the reference genome LTR108 using the "mem" algorithm of the BWA alignment software. The SAMtools software (v1.9) was used to sort the alignment results. The HaplotypeCaller algorithm (local haplotype assembly) based on the GATK software (Version 4.1.2) was used for SNP and InDel variant detection. The SnpEff software (Version 5.1) was used to annotate and predict the variant sites.

[0054] IV. BSA-seq analysis

[0055] (1) SNP-index

[0056] The degree of sequence difference between the offspring population and the parents is expressed by SNP-index, which is equal to the ratio of the number of reads of SNPs different from the reference parent to the total number of all reads to the same position. The SNP-index and Δ(SNP-index) of all chromosome positions are calculated using a 1Mb sliding window and a 500kb step size. A SNP index value of 0 indicates no variation, a SNP index value of 1 indicates that all SNPs belong to one parent, and a SNP index value of 0.5 indicates that both parents contribute equally to the variation. The SNP-inedx 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 follows:

[0057] △SNPindex=SNP index1-SNP index2;

[0058] In the formula, SNP index1 and SNP index2 represent the degree of difference between the offspring populations in the two extreme pools and their parents.

[0059] (2)ED

[0060] ED (Euclidean distance) is the abbreviation of Euclidean distance. It calculates the frequency distance of each mutant type between different mixed 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:

[0061]

[0062] In the formula, mut and wt represent the mutant pool and wild-type pool, respectively, which are the two extreme pools of this screening. A, C, G, and T represent the proportion of sequencing reads occupied by each mutant type of the marker site. Based on the SNP site set and genotype depth information between the pools, the difference in mutation frequency between the pools, i.e., the ED value, is calculated.

[0063] To amplify the difference and reduce background noise, increase the ED value to ED 4 , as shown below:

[0064] ED 4 =[(Amut-Awt) 2 +(Cmut-Cwt) 2 +(Gmut-Gwt) 2 +(Tmut-Twt) 2 ] 2 ;

[0065] The results obtained by the above calculation and analysis method are as follows Figure 2 , Figure 3 As shown, according to Figure 2The QTL-seq △SNP-index mapping results shown indicate that there is a continuous and relatively high peak within the region of 57002093 - 58449776 on chromosome 2. Within this region, the maximum value of △SNP-index is -0.97; further, according to Figure 3 the schematic diagram of QTL mapping for mesocarp thickness calculated based on ED 4 as shown, there are significantly continuous peaks at 57000709 - 58466642 on chromosome 2, and at 57437832 bp on chromosome 2, the ED 4 value shows the maximum peak, and ED 4 = 3.493. Its trend line is greater than the 99% threshold line. The two algorithms have an intersection at 57002093 - 58449776 on chromosome 2. Therefore, this interval is used as the genetic region for BSA-seq analysis.

[0066] V. Development of Molecular Markers for Grain Mesocarp Thickness

[0067] (1) SNP Locus Screening

[0068] According to the BSA-seq analysis results in step four, further screen the SNP data within the associated region (the interval of 57002093 - 58449776 on chromosome 2) for SNP loci that can be developed into molecular markers. The screening criterion is that there are no other SNPs within 100 bp upstream and downstream of the SNP locus.

[0069] The results are shown in Table 1:

[0070] Table 1 SNP Locus Information

[0071]

[0072] Through the above method, a total of 28 SNP loci highly linked to the grain mesocarp thickness performance of sorghum were screened in this example. And in the above analysis of this application, LTR108, one of the parents, was used as the reference genome to further improve the accuracy of the alignment data.

[0073] Example 2 Development of SNP Molecular Markers for Identifying Grain Mesocarp Thickness of Sorghum

[0074] (1) Primer Design

[0075] Based on the SNP loci shown in Table 1 obtained by screening, use Tbtools to retrieve the sequence information of 100 bp upstream and downstream of the above 28 SNP loci. Then use PrimerPicker Lite for KASPar v 0.26 (https: / / www.biosearchtech.com / ) to design multiple sets of amplification primers. Further use the genomic database to detect the primer specificity. The primer sequences that can specifically recognize the target loci and meet the requirements of PCR amplification principles are converted into KASP primers. Each set of primers in the KASP primer set sequence consists of 3 sequences, including: Forward competitive primer 1: FAM fluorescent tag sequence + amplification primer sequence; Forward competitive primer 2: HEX fluorescent tag sequence + amplification primer sequence; Reverse universal primer: amplification primer sequence. After primer design and screening, there are 11 SNP loci for which primers can be designed.

[0076] The primer sequences are shown in Table 2:

[0077] Table 2 KASP primer information corresponding to molecular markers

[0078]

[0079] Note: The underlines are fluorescent sequence tags

[0080] (2) Marker development and validation

[0081] Develop markers that are polymorphic between parents and in extreme materials using 654 and LTR108. Then randomly select 89 materials from the F2 population of 654×LTR108 for marker validation. Extract genomic DNA from each sorghum sample using the CTAB method and dissolve it in sterilized ddH2O; perform quality detection of DNA by 0.8% agarose gel electrophoresis. For the extracted DNA, clear bands, no impurities, and no degradation are required; after measuring the DNA concentration, uniformly dilute it to a concentration of about 50 ng / μl; perform PCR amplification using the KASP primer set designed in the above step (1). The reaction system, amplification program, and genotype typing method are as follows:

[0082] 1) The PCR reaction system is as follows: The total volume is 10 μL, including 2.5 μL of sample DNA (120 ng / μl), 5 μL of 2×KASPMaster Mix (LGC Genomics, Hoddeston, UK), 0.14 μL of KASPAssay Mix, and 2.36 μL of ddH2O; among them, the preparation method of KASPAssay Mix is as follows: Each 100 μL of KASPAssay Mix contains 12 μL of each of the two forward competitive primers with a concentration of 100 μM, 30 μL of the reverse universal primer with a concentration of 100 μM, and 46 μL of ddH2O;

[0083] 2) The PCR reaction program is as follows: heat activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 61 - 55°C for 60 s (10 Touch-down cycles, with a decrease of 0.6°C per cycle); denaturation at 95°C for 20 s, renaturation at 55°C for 60 s, 27 cycles, and the amplified product is incubated at 25°C.

[0084] 3) Genotype typing: Use the fluorescence quantitative PCR instrument Applied Biosystems ABI Viia7 Real TimePCR System (Thermo Scientific, USA) to scan the fluorescence signals and perform genotype typing. The specific steps are as follows: The fluorescence type of the samples with fluorescence signal intensity values aggregated near the X-axis and showing red represents the FAM fluorophore, which is the genotype type of LTR108 sorghum; the fluorescence type of the samples with fluorescence signal intensity values aggregated near the Y-axis and showing blue represents the HEX fluorophore, which is the genotype type of 654 sorghum; the color in the middle of the X-axis and Y-axis that is green is heterozygous.

[0085] Among them, the mutation sites of each marker are shown in Table 3:

[0086] Table 3 Mutation sites of each marker

[0087]

[0088]

[0089] The above 11 pairs of primers were respectively used for marker development between parents and in extreme materials. The results showed that 7 markers were not clearly typed or could not be typed in the parents, and 4 markers were clearly typed between the parents.

[0090] Based on the above genotype typing results, for the 4 molecular markers with clear typing (Chr02_57549233, Chr02_57757493, Chr02_57908646, Chr02_57954159), typing and accuracy calculation of the typing results were performed in the validation population. The results are shown in Table 4:

[0091] Table 4 Accuracy of mesocarp thickness identification in the validation population of 4 molecular markers

[0092]

[0093] Combined with Table 3 and Table 4, for the marker Chr02_57549233, if the detected locus is AA and AG, the mesocarp is thin; if it is GG, the mesocarp is thick, and the accuracy rate in the validation population is 79.77%; for Chr02_57757493, if the detected locus is AA and AC, the mesocarp is thin; if it is CC, the mesocarp is thick, and the accuracy rate in the validation population is 76.40%; for Chr02_57908646, if the detected locus is AA and AG, the mesocarp is thin; if it is GG, the mesocarp is thick, and the accuracy rate in the validation population is 74.5%; for the marker Chr02_57954159, if the detected locus is GG and GC, the mesocarp is thin; if it is CC, the mesocarp is thick, and the accuracy rate in the validation population is 100%. Among them, in this application, based on the phenotype of the mesocarp being thin as dominant, and according to the segregation ratio of 3:1 in the F2 generation, the range of the mesocarp thickness is determined. Specifically, taking the mesocarp thickness of 71.614 μm as the boundary, if the mesocarp thickness is less than 71.614 μm, it is thin; if the mesocarp thickness is greater than 71.614 μm, it is thick.

[0094] The results of phenotype identification of each material based on the marker Chr02_57954159 are shown in Table 5:

[0095] Table 5 Identification results of 89 materials

[0096]

[0097]

[0098]

[0099] To sum up, based on the above method, molecular marker verification was carried out on 89 sorghum samples with known grain mesocarp thickness. The specific genotype typing results of the marker Chr02_57954159 are as Figure 4 shown. Red represents the FAM fluorophore, representing the type of thin grain mesocarp, which is the genotype type of LTR108 sorghum; blue represents the HEX fluorophore, representing the type of thick grain mesocarp, which is the genotype type of 654 sorghum; green represents the simultaneous presence of two fluorescence signals of FAM fluorophore and HEX fluorophore, which is the heterozygous genotype type. According to Figure 3The KASP genotyping results shown, as well as the phenotype identification results (Table 4), among the 89 F2 generation plants, 14 plants have the genotype CC, and the pericarp thickness of their grains is thick; 28 plants have the genotype GG, and the pericarp thickness of their grains is thin; and 47 plants have the genotype GC at this molecular locus, and the pericarp thickness of their grains is thin. This result indicates that the above-mentioned molecular marker provided in this application can obtain stable PCR products in 89 individual plants of the F2 segregation population, and the three genotypes can be clearly distinguished and clustered. The 57954159-G / C marker is consistent with the phenotype identification results of the pericarp thickness of the grains, and the accuracy rate can reach 100%. This marker can be applied to molecular marker-assisted breeding of sorghum pericarp thickness with high efficiency and low cost.

[0100] It can be understood that this application is described through some embodiments. As is known to those skilled in the art, without departing from the scope of this application, various changes or equivalent replacements can be made to these features and embodiments. In addition, under the teaching of this application, these features and embodiments can be modified to adapt to specific situations and materials without departing from the scope of this application. Therefore, this application is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application belong to the scope protected by this application.

[0101]

Claims

1. A molecular marker related to the thickness of the mesocarp in sorghum grains, characterized in that: The SNP site of the molecular marker is located at position 57954159 of chromosome 2 of the sorghum genome, and its polymorphism is G / C.

2. The molecular marker according to claim 1, characterized in that The sorghum genome is derived from LTR108.

3. A molecular marker related to the thickness of the mesocarp in sorghum grains, characterized in that: The molecular marker includes a nucleotide sequence as shown in SEQ ID NO.

1. The nucleotide sequence as shown in SEQ ID NO.1 has a polymorphism at the 101st position from the 5' end, and the polymorphism is G / C.

4. A KASP primer pair, characterized in that: The primer pair includes a forward competitive primer as shown in SEQ ID NO.2, a forward competitive primer as shown in SEQ ID NO.3 and a reverse universal primer as shown in SEQ ID NO.

4.

5. The KASP primer pair according to claim 4, characterized in that: The KASP primer pair is used to amplify the molecular marker according to any one of claims 1-3.

6. A kit, characterized in that: It comprises the molecular marker as described in any one of claims 1 to 3 or / the KASP primer pair as described in any one of claims 4 to 5.

7. Use of the molecular marker according to any one of claims 1 to 3 or / the KASP primer pair according to any one of claims 4 to 5 or / the kit according to claim 6 in identifying sorghum varieties and / or sorghum breeding; preferably, the sorghum varieties include: Sorghum varieties with thick pericarp in the grain and sorghum varieties with thin pericarp in the grain.

8. A method for identifying or assisting in identifying the thickness of the mesocarp of sorghum grains, characterized in that: The method comprises detecting the genotype of a SNP site in the sorghum genome to be tested, and identifying or assisting in identifying the pericarp thickness of sorghum grains according to the detection result of the genotype, wherein the SNP site is at position 57954159 of chromosome 2 of the sorghum genome, and the nucleotide type thereof is G or C; preferably, the SNP site is the 101st nucleotide of the nucleotide sequence shown in SEQ ID NO.

1.

9. A method for identifying or assisting in identifying the thickness of the mesocarp of sorghum grains, characterized in that: The method comprises the following steps: (1) extracting genomic DNA of sorghum to be identified; (2) using the genomic DNA extracted in step (1) as a template and using the designed KASP primer set to perform a PCR amplification reaction to obtain an amplified product; (3) using a fluorescence detection platform to perform fluorescence signal scanning and genotyping on the amplified product obtained in step (2); (4) identifying the variety of the sorghum to be tested according to the typing results in step (3); The primer pair includes a forward competitive primer as shown in SEQ ID NO.2, a forward competitive primer as shown in SEQ ID NO.3, and a reverse universal primer as shown in SEQ ID NO.

4.

10. The method according to claim 9, characterized in that The step of identifying the variety of the sorghum to be tested according to the typing result in step (3) comprises: When the fluorescent signal of the forward competitive primer shown in SEQ ID NO.2 appears in the amplified product of the sorghum to be tested, but the fluorescent signal of the forward competitive primer shown in SEQ ID NO.3 does not appear, the sorghum variety to be tested is identified as a sorghum variety with a thin mesocarp in the grain; and / or, when the genotyping result is GG, the sorghum variety to be tested is identified as a sorghum variety with a thin mesocarp in the grain; When the sorghum to be tested shows a fluorescent signal of the forward competitive primer as shown in SEQ ID NO.3, but does not show a fluorescent signal of the forward competitive primer as shown in SEQ ID NO.2, the sorghum variety to be tested is identified as a sorghum variety with thick mesocarp in the grain; and / or, when the genotyping result is CC, the sorghum variety to be tested is identified as a sorghum variety with thick mesocarp in the grain; When the sorghum to be tested shows two fluorescent signals, the sorghum variety to be tested is identified as a sorghum variety with a thin mesocarp in the grain; and / or, when the genotyping result is GC, the sorghum variety to be tested is identified as a sorghum variety with a thin mesocarp in the grain.

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