A molecular marker closely linked to a major qtl locus of sesame golden yellow seed and application thereof

Abstract

The present application belongs to the technical field of molecular biology and genetic breeding, and relates to a molecular marker SNP6804 closely linked to a main-effect QTL site of golden yellow sesame seeds and application thereof; the present application locates a main-effect QTL site of controlling golden yellow sesame seeds on the 6th linkage group LG06 of sesame by using phenotypes of recombinant inbred lines and high-density genetic maps, can explain 29.46% to 35.79% of the phenotypic variation rate in different environments, and is named as qSC_LG06; a SNP molecular marker closely linked to qSC_LG06 is simultaneously developed, and is named as SNP6804, the primer sequence of which is: SNP6804F1: 5'-GACAAAGTTGCGATACGCCA-3', SNP6804F2: 5'-TCGACGACAAAGTTGCGATACGACG-3', SNP6804R: 5'-TGCAGGACTCAGTCTTCATGGGTG-3'. Through the application of the molecular marker of the present application, molecular marker assisted selection breeding of golden yellow sesame seeds can be carried out, the breeding cycle is shortened, the cumbersome procedure of identifying the color of each generation in the breeding process is saved, a large amount of labor is saved, the cost is reduced, the work efficiency is improved. Meanwhile, the traditional phenotype is converted into genotype selection, and the accuracy and scientificity of selection are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker SNP6804 closely linked to a main-effect QTL site of golden yellow sesame seeds and an application thereof. BACKGROUND

[0002] Sesamum indicum L. is an important high-quality oil crop and characteristic agricultural product in China, and is known as the "Queen of Oil Crops". It has a high oil content (44%-58%) and protein content (18%-25%) (Uzun et al., 2008). In addition, the sesame seed coat is rich in antioxidant substances such as sesamin and sesamolin. Studies have shown that sesame seeds of different colors have different nutritional values. For example, white sesame seeds have a higher oil content, while dark sesame seeds are rich in anthocyanin compounds. Therefore, sesame seeds are not only an important source of high-quality edible oil, but are also widely used in food processing, healthcare, and anti-aging. In recent years, as the living standards of the Chinese people continue to improve, the demand for sesame oil has gradually increased, and the demand for edible sesame seeds with special colors and specific nutritional components has also increased. However, the sesame varieties currently on the market are mainly black and white sesame seeds. Therefore, developing sesame seed color breeding to meet the demand for sesame seeds of different colors and different nutritional components has become an important goal of sesame breeding.

[0003] Although traditional breeding methods have provided multiple excellent sesame varieties for production, the long breeding cycle and low selection efficiency of traditional breeding have been unable to fully meet the current needs of sesame production. With the rapid development of molecular biology, molecular marker-assisted selection breeding has become a new breeding method. It directly uses molecular markers closely linked to target trait genes to select individuals in advance, effectively improving selection efficiency and shortening breeding time. Currently, molecular marker-assisted selection breeding is not only applied to quality traits, but also to complex quantitative traits. Sesame seed color is a complex quantitative trait controlled by multiple major genes. Naturally mature sesame seeds have a variety of colors, including black, gray, brown, yellow, beige, and white.

[0004] With the development of molecular marker technology and the construction of linkage maps, researchers have located QTLs for sesame seed color. Zhang et al. used COI1134 (white sesame) and RXBS (black sesame) to construct an F 2:3Four QTLs (QTL1-1, QTL11-1, QTL11-2 and QTL11-3) of sesame seed color were located by population; Wang et al. (2016) located four QTLs of sesame seed color by using Zhongzhi 13 (white sesame) and ZZM2748 (black sesame) to construct a recombinant inbred line; subsequently, Wei et al. (2016) located six QTLs of sesame seed color by using Zhongzhi 13 (white sesame) and Mishuozhima (black sesame) to construct a recombinant inbred line; although the above reports located multiple QTLs related to sesame seed color, the parents used to construct the population were white sesame and black sesame, and the seed color of the hybrid offspring was complex and diverse, including a series of seed color from black to white, which caused the major QTLs located to be unable to be associated with single seed color, and was not conducive to the molecular marker assisted selection breeding of sesame seed color; in the present research, a population constructed by white sesame and golden yellow sesame was used for QTL location, aiming to identify QTL sites related to golden yellow seed of sesame, for the marker assisted selection breeding of golden yellow seed of sesame. SUMMARY

[0005] One of the purposes of the present application is to provide a major QTL site qSC_LG06 of golden yellow seed of sesame, which is located in the interval of 93.2-96.9 cM of the 6th linkage group.

[0006] The second purpose of the present application is to provide a molecular marker SNP6804 closely linked to the major QTL site related to golden yellow seed of sesame and primer sequences thereof.

[0007] The third purpose of the present application is to provide an application method of the above-mentioned molecular marker SNP6804 in the marker assisted selection breeding of golden yellow seed of sesame.

[0008] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0009] A method for obtaining a major QTL site qSC_LG06 of golden yellow seed of sesame, comprising the following steps:

[0010] (1) F1 seeds are obtained by hybridizing a white seed variety and a golden yellow seed variety, and F2 is obtained by selfing, and a recombinant inbred line population RIL is obtained by 8 years and 8 generations of selfing by single seed transmission method;

[0011] (2) the RIL population is planted in three different geographical locations respectively, and the sesame seed color is investigated after mature harvesting to obtain the phenotype data;

[0012] (3) total DNA of leaves of the white seed variety, the golden yellow seed variety and the RIL population is extracted;

[0013] (4) Re-sequencing the parents and RIL population, developing SNP markers by BWA software and GATK software, screening homozygous variation sites different between the parents, then according to the genetic principle, encoding the polymorphic molecular markers according to the parents, and performing quality filtering to screen high-quality molecular markers, obtaining the genotype data of the RIL population;

[0014] (5) Combining the genotype data of the RIL population, using Mstmap software to construct a genetic linkage map;

[0015] (6) Combining the genotype data of the RIL population, the genetic linkage map and the sesame seed color phenotype data, using R / qtl software to perform QTL analysis, locating a major QTL site controlling sesame golden seed on the LG06 linkage group, and repeatedly detecting in three different geographical environments, respectively explaining 35.68%, 35.79% and 29.46% of the phenotypic variation, and named as qSC_LG06.

[0016] A major QTL site qSC_LG06 associated with sesame golden seed is identified by the above method, and a molecular marker SNP6804 closely linked to qSC_LG06 is developed, the primer sequence of which is: SNP6804F1: 5'-GACAAAGTTGCGATACGCCA-3', SNP6804F2: 5'-TCGACGACAAAGTTGCGATACGACG-3', SNP6804R: 5'-TGCAGGACTCAGTCTTCATGGGTG-3'; using the primer for PCR amplification, only a 105bp fragment can be amplified in white seed 'Yuzhi No.8'; only a 110bp fragment can be amplified in golden yellow seed material 'Yanzhou No.2 red skin'; in the heterozygous genotype of golden yellow seed material, two fragments can be amplified, one of which is 105bp in length and the other is 110bp in length.

[0017] The above molecular marker SNP6804 is applied in marker-assisted selection of sesame golden seed, and the specific mode is to use the primer sequence of molecular marker SNP6804 for PCR amplification, and the amplified sample is total DNA of sesame breeding offspring single leaf or other tissues.

[0018] Further, the system of the PCR amplification is 10μL, containing 25-50ng of template DNA, 0.1μL of 5U / μL Taq enzyme, 1μL of 10×PCR buffer, 0.2μL of 10mM / μL dNTPs, 0.2μL of 10μM / μL forward primer, 0.2μL of 10μM / μL reverse primer, and the rest is supplemented with ddH2O to 10μL.

[0019] Further, the PCR amplification procedure is: pre-denaturation 94 DEG C for 1 min; denaturation 94 DEG C for 30 s, annealing 57 DEG C for 30 s, extension 72 DEG C for 30 s, 35 cycles, extension 72 DEG C for 10 min.

[0020] Further, the PCR amplification product is separated by electrophoresis, i.e. 9% non-denaturing polyacrylamide gel electrophoresis separation; the electrophoresis buffer during the electrophoresis separation is 0.5xTBE, and the electrophoresis separation is 150V constant power electrophoresis separation; the result judging method is: if only 105bp fragments are amplified, the sample is homozygous material of white seeds; if only 110bp fragments are amplified, the sample is homozygous material of golden yellow seeds; if 105bp and 110bp fragments are amplified, the sample is heterozygous material of golden yellow seeds.

[0021] The present application has the beneficial effects that:

[0022] 1. The present application firstly locates a main QTL site qSC_LG06 controlling golden yellow seed color of sesame in sesame, and develops a SNP molecular marker SNP6804 closely linked to the qSC_LG06.

[0023] 2. The present application locates a main QTL site controlling golden yellow seed color of sesame in the 6th linkage group LG06 of sesame by using phenotypes and high-density genetic maps of recombinant inbred lines, which can explain 29.46% to 35.79% of the phenotypic variation rate in different environments, and the site can be used as the basis of molecular markers of golden yellow seed color of sesame and the application basis of auxiliary selection breeding of sesame seed color.

[0024] 3. The present application develops a molecular marker SNP6804 closely linked to the main QTL site of golden yellow sesame seed color and primer sequences thereof, uses PCR amplification technology, the template DNA is denatured into single strand after heating, the primer is combined with the corresponding complementary sequence of the single strand, then the extension is carried out under the action of DNA polymerase, the purpose product is obtained after the repeated cycle and extension, and the golden yellow sesame seed color varieties are screened according to the size of the product fragments.

[0025] 4. The application method of the present application overcomes the problem that the phenotypic identification in conventional breeding needs to be observed after seed harvesting, and the sesame seed color can be identified at the seedling stage by using molecular markers, so that the unnecessary materials can be removed early, the manpower and material resources are saved, the early generation selection of sesame seed color is realized, the breeding period is shortened, the sesame material with golden yellow seed color is screened faster, and the breeding efficiency is improved significantly. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure shows the positioning result of the main QTL site qSC_LG06 controlling golden yellow seed color of sesame.

[0027] Figure 2 Schematic diagram of detection of amplification product of molecular marker SNP6804 in RIL population. DETAILED DESCRIPTION

[0028] The method for obtaining the main-effect QTL site qSC_LG06 of golden yellow sesame seeds in the application comprises the following steps:

[0029] (1) F1 seeds are obtained by crossing the white seed variety 'Yuzhi No. 8' and the golden yellow seed variety 'Yanzhou No. 2 Red Skin', and F2 is obtained by selfing. A recombinant inbred line (RIL) population is obtained by using single-seed transmission method and selfing for 8 years and 8 generations;

[0030] (2) The RIL population is planted in Zhumadian, Henan, Nanyang, Henan and Sanya, Hainan, respectively, and the sesame seed color is investigated after mature harvest to obtain the phenotype data;

[0031] (3) The total DNA of the leaves of 'Yuzhi No. 8', 'Yanzhou No. 2 Red Skin' and the RIL population is extracted;

[0032] (4) The parents and the RIL population are resequenced, SNP markers are developed by using BWA software and GATK software, and the homozygous variation sites different between the parents are screened. Then, according to the principle of genetics, the molecular markers of polymorphism are coded according to the parents, and high-quality molecular markers are screened by quality filtering to obtain the genotype data of the RIL population;

[0033] (5) The Mstmap software is used to construct a genetic linkage map in combination with the genotype data of the RIL population;

[0034] (6) The R / qtl software is used to analyze QTL in combination with the genotype data of the RIL population, the genetic linkage map and the sesame seed color phenotype data, a main-effect QTL site controlling golden yellow sesame seeds is located on the LG06 linkage group, and the site is repeatedly detected in Zhumadian, Nanyang, Henan and Sanya, Hainan, which respectively explain 35.68%, 35.79% and 29.46% of the phenotypic variation, and is named as qSC_LG06.

[0035] Using the above method, the applicant finally identified a major QTL associated with golden yellow sesame seeds q SC_LG06, and independently developed a molecular marker SNP6804 closely linked to q SC_LG06, the primer sequence of which is: SNP6804F1: 5'-GACAAAGTTGCGATACGCCA-3', SNP6804F2: 5'-TCGACGACAAAGTTGCGATACGACG-3', SNP6804R: 5'-TGCAGGACTCAGTCTTCATGGGTG-3'; using the primer for PCR amplification, only a 105bp fragment can be amplified in white seed 'Yuzhi No. 8'; only a 110bp fragment can be amplified in golden yellow seed material 'Yanzhou No. 2 red skin'; in the hybrid genotype of golden yellow seed material, two fragments can be amplified, one of which is 105bp in length and the other is 110bp in length.

[0036] The application method of the above-mentioned molecular marker SNP6804 in marker-assisted selection of golden yellow sesame seeds is to amplify the total DNA of a single plant leaf or other tissues of the sesame breeding offspring with the marker primer.

[0037] The PCR amplification system is 10μL, containing 25-50ng of template DNA, 0.1μL of 5U / μL Taq enzyme, 1μL of 10×PCR buffer, 0.2μL of 10mM / μL dNTPs, 0.2μL of 10μM / μL forward primer, 0.2μL of 10μM / μL reverse primer, and the rest is supplemented with ddH2O to 10μL.

[0038] The PCR amplification program is: pre-denaturation 94℃ for 1min; denaturation 94℃ for 30s, annealing 57℃ for 30s, extension 72℃ for 30s, 35 cycles, extension 72℃ for 10min.

[0039] The electrophoretic separation is 9% non-denaturing polyacrylamide gel electrophoresis separation.

[0040] The electrophoresis buffer during the electrophoretic separation is 0.5×TBE, and the electrophoretic separation is 150V constant power electrophoretic separation.

[0041] The electrophoresis separation is adopted, and the determination method of the result is as follows: if only the 105 bp fragment is amplified, it indicates that only the same allele as 'Yuzhi No. 8' exists at the qFT_LG06 locus, and the sample is a homozygous material with white seeds; if only the 110 bp fragment is amplified, it indicates that only the same allele as 'Yanzhou Erhongpi' exists at the qFT_LG06 locus, and the sample is a homozygous material with golden yellow seeds; if the 105 bp and 110 bp fragments are amplified, it indicates that a heterozygous allele exists at the qSC_LG06 locus, and the sample is a heterozygous material with golden yellow seeds.

[0042] The following examples are used to specifically illustrate the present application, and all reagent components involved in the implementation process can be obtained from commercial channels, and are used according to the conditions in the experimental manual or the conditions suggested by the manufacturers of the reagents used.

[0043] Example 1

[0044] A method for obtaining a major QTL locus of golden yellow sesame seeds, and a method for developing and applying a molecular marker SNP6804 closely linked to the major QTL locus of golden yellow sesame seeds, specifically includes the following contents:

[0045] (1) RIL population construction: taking 'Yuzhi No. 8' as the female parent and 'Yanzhou Erhongpi' as the male parent, F1 seeds are obtained by hybridization, and F2 seeds are obtained by selfing. By using single seed transmission method, a recombinant inbred line population RIL is obtained after 8 years and 8 generations of selfing.

[0046] (2) Grain color phenotype identification: the RIL population is planted in Zhumadian, Nanyang and Sanya, Henan, respectively, using randomized block design. Each RIL individual material is planted in one row, and two replicates are set. Ten mature single plants are randomly mixed and harvested in each row, and the grain color is observed after the seeds are dried to obtain the phenotype data.

[0047] (3) DNA extraction: The CTAB method was used to extract the DNA of single plants of 'Yuzhi 8', 'Yanzhou Erhongpi' and the RIL population. The specific steps were as follows: 0.5 g of leaf was ground in liquid nitrogen, and the powder was added to a 2 ml centrifuge tube. 1 ml of extraction buffer was added, and the tube was placed on ice for 10 min. It was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. 600 μl of lysis buffer was added, mixed well, and incubated at 65°C for 30-60 min. 1 ml of phenol: chloroform: isoamyl alcohol (25:24:1, V / V / V) was added and mixed well for 30 times. After standing for 5 min, it was centrifuged at 12000 rpm for 5 min, and the supernatant was aspirated. An equal volume of isopropanol was added, mixed well, and placed for 10 min. It was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. It was washed twice with 75% ethanol, blown dry in the fume hood, dissolved in 1xTE (500 μl), and added with two volumes of phenol: chloroform: isoamyl alcohol (25:24:1, V / V / V). It was mixed well for 50 times, stood for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was aspirated, mixed with an equal volume of chloroform for 50 times, stood for 5 min, and centrifuged at 12000 rpm for 5 min. The supernatant was aspirated, added with one-tenth volume of 3 mol / L NaAc (pH 5.2), and mixed with an equal volume of isopropanol. It was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. It was washed twice with 75% ethanol, blown dry in the fume hood, dissolved in an appropriate amount of TE containing RNase (50 μl), and incubated at 37°C for 30 min to digest RNA. The DNA concentration was detected by Nanodrop analyzer, and the DNA integrity was detected by agarose gel electrophoresis. It was stored at -20°C for standby.

[0048] (4) Genomic resequencing and SNP marker development: The DNA of single plants of 'Yuzhi 8', 'Yanzhou Erhongpi' and the RIL population was fragmented into random fragments by ultrasonic wave. The fragmented DNA was sequentially subjected to end repair, 3' A addition, and ligation of sequencing adapters. Then, the fragments with a genome length of about 400 bp were enriched by magnetic bead adsorption, amplified by PCR to form a sequencing library, and sequenced on the Illumina HiSeq 2000 platform (Shanghai Meiji Biomedical Technology Co., Ltd.). Raw data was obtained, and Clean Reads were obtained by filtering with fastp. Then, the Clean Reads were aligned to the Zhongzhi 13 reference genome by using BWA software (Wang et al. 2014). The SNP markers were detected by using the Best Practices process of GATK4.0 software. Then, the homozygous SNP markers that existed differences in the parents were screened. According to the genetic principle, the obtained molecular markers were coded as aa x bb according to the parents. After quality filtering, high-quality molecular markers were screened, and finally 7817 SNP markers were obtained.

[0049] (5) High-density genetic linkage map construction and QTL location: 7817 molecular markers were divided into 13 linkage groups by using MSTmap software to construct a high-density genetic map; combined with the RIL population genotype, genetic linkage map and grain color data, R / qtl software was used for QTL location, and a major QTL located in the 6th linkage group LG0693.2-96.9 cM interval was identified, which was repeatedly detected in Zhumadian, Nanyang and Sanya of Henan in three environments, respectively explaining 35.68%, 35.79% and 29.46% of the phenotypic variation, and named as qSC_LG06, as shown in Figure 1

[0050] (6) Development of markers closely linked to major QTL: In the major QTL interval, there is a SNP variation site between the parents, which is closely linked to qSC_LG06; the SNP site is base G in the golden yellow grain material 'Yanzhou Erhongpi' and base A in the white grain material 'Yuzhi No. 8', and the marker SNP6804 is developed according to the SNP marker development principle, and the primer sequences are as follows: SNP6804F1: 5'-GACAAAGTTGCGATACGCCA-3', SNP6804F2: 5'-TCGACGACAAAGTTGCGATACGACG-3', SNP6804R: 5'-TGCAGGACTCAGTCTTCATGGGTG-3'.

[0051] (7) Application of primer sequences of marker SNP6804: The primer sequences of the marker were used for PCR amplification of total DNA of leaves or other tissues of single plants of white grain variety 'Yuzhi No. 8', golden yellow grain variety 'Yanzhou Erhongpi' and breeding offspring of RIL recombinant inbred line population, respectively.

[0052] The system for PCR amplification is 10 μL, containing 25-50 ng of template DNA, 0.1 μL of 5 U / μL Taq enzyme, 1 μL of 10×PCR buffer, 0.2 μL of 10 mM / μL dNTPs, 0.2 μL of 10 μM / μL forward primer, 0.2 μL of 10 μM / μL reverse primer, and the rest is supplemented with ddH2O to 10 μL.

[0053] The program for PCR amplification is as follows: pre-denaturation at 94℃ for 1 min; denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 30 s, 35 cycles, and extension at 72℃ for 10 min.

[0054] The PCR amplification product is separated by electrophoresis, that is, 9% non-denaturing polyacrylamide gel electrophoresis; the electrophoresis buffer during the electrophoresis is 0.5×TBE, and the electrophoresis is 150 V constant power electrophoresis; the determination method of the results is:​

[0055] The primer can only amplify 110bp product in golden kernel material 'Yanzhou Erhongpi', can only amplify 105bp product in white kernel material 'Yuzhi No.8', and can amplify 105bp and 110bp products in hybrid genotypes.

[0056] Example 2

[0057] Verification of the effect of the molecular marker SNP6804 on the assisted selection by using RIL population:

[0058] (1) Construction of RIL population and DNA extraction: F1 seeds were obtained by crossing 'Yuzhi No.8' as female parent and 'Yanzhou Erhongpi' as male parent, and F2 seeds were obtained by selfing. The RIL population was constructed by single-seed transmission method. The DNA extraction method was the same as that in Example 1.

[0059] (2) Genotype identification of RIL population: The DNA of single plant of RIL population was used as template, and the SNP6804 primer was used for amplification. The amplified bands were read to identify the genotypes of RIL population. The PCR reaction system, gel and amplification product observation method were the same as those in Example 1. Among the 246 RIL single plants, the amplification products of 114 single plants were 105bp fragments, and the genotypes were recorded as A; the amplification products of 129 single plants were 110bp fragments, and the genotypes were recorded as B; the amplification bands of 3 single plants were 105bp and 110bp products, and the genotypes were recorded as H. See Table 1 for details.

[0060] (3) Effect of the molecular marker SNP6804 on the assisted selection: To verify the selection effect of the marker SNP6804, the kernel color of the 246 RIL population single plants was investigated after mature harvest. See Table 1. It was found by comparing the genotypes and phenotypes that the 114 single plants with 105bp amplification products (genotype A) had white kernels; the 129 single plants with 110bp amplification products (genotype B) had golden yellow kernels; and the 3 single plants with 110bp and 105bp amplification products (genotype H) had golden yellow kernels. The above results show that the molecular marker SNP6804 has good effect on the assisted selection of sesame kernel color.

[0061] Table 1 in each step above is the genotype and flowering date of 246 RIL single plants, and Table 1 is as follows:

[0062]

[0063]

Claims

1. A method for applying the molecular marker SNP6804 and primers thereof which are closely linked to the sesame golden seed major QTL site to marker-assisted selection of sesame golden seed, characterized in that, The total DNA sample of leaf or other tissue of the breeding offspring material of the sesame is amplified by the primer PCR of the molecular marker SNP6804, if the amplification product is only one 105 bp band, the sample is the white kernel homozygous material 'Yuzhi No.8'; if the amplification product is only one 110 bp band, the sample is the golden yellow kernel homozygous material 'Yanzhou No.2 red skin'; if the amplification product is two bands of 105 bp and 110 bp, it is the golden yellow kernel heterozygous material; The primer is: F1: 5'-GACAAAGTTGCGATACGCCA-3', F2: 5'-TCGACGACAAAGTTGCGATACGACG-3', R: 5'-TGCAGGACTCAGTCTTCATGGGTG-3'.

2. The use according to claim 1, characterized in that, The reaction system of the PCR amplification is 10 μL, containing 25-50 ng of template DNA, 0.1 μL of 5U / μL Taq enzyme, 1 μL of 10×PCR buffer, 0.2 μL of 10 mM / μL dNTPs, 0.2 μL of 10 μM / μL forward primer, 0.2 μL of 10 μM / μL reverse primer, and the rest is supplemented with ddH2O to 10 μL; the PCR amplification procedure is: pre-denaturation 94℃ 1 min; denaturation 94℃ 30 s, annealing 57℃ 30 s, extension 72℃ 30 s, 35 cycles, and re-extension 72℃ 10 min.

3. The method of use of claim 2, wherein, The amplification product is separated by 9% non-denaturing polyacrylamide gel electrophoresis, the electrophoresis buffer is 0.5×TBE, and the constant power electrophoresis separation is 150 V.

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