CN110373487B - InDel marker related to peppery taste character of pepper and application thereof - Google Patents
InDel marker related to peppery taste character of pepper and application thereof Download PDFInfo
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Abstract
The invention discloses an InDel marker related to hot taste character of pepper and application thereof, and discloses an insertion/deletion fragment related to hot taste character of pepper fruits, wherein the nucleotide sequence of the insertion/deletion fragment is shown as SEQ ID NO.1, and the fragment sequence is positioned at the upstream of a key MYB transcription factor Cap1 gene for regulating and controlling capsaicin biosynthesis; meanwhile, an InDel molecular marker is developed, and the content of capsaicin substances can be well typed by detecting the existence of the SEQ ID NO:1 sequence at the upstream of Cap1 gene. The invention has very important guiding significance for genetic improvement of hot pepper flavor, improvement of breeding efficiency and saving of breeding cost.
Description
Technical Field
The invention relates to the technical field of pepper breeding, in particular to an InDel marker related to pepper spicy character and application thereof.
Background
Capsaicin and dihydrocapsaicin, and analogs thereof, collectively known as capsaicinoids, are secondary metabolites specific to plants of the genus Capsicum, and are important components of the quality of the Capsicum fruits, determining the peppery taste of the fruits. Capsaicin used in food, medicine (relieving pain, resisting cancer, reducing weight, lowering blood pressure, killing bacteria, strengthening intestine and stomach), military (as tear bomb and for preventing and treating diseases) Raw materials of sanitary weapons), chemical industry and the like. At present, the worldwide market demand for capsaicin is about 7 million tons every year, the annual demand total value is up to 175 million yuan, the world total production capacity is less than 0.5 million tons every year, and the demand gap is up to more than 80%. The capsaicin is required to be extracted from pepper fruits, more than 90% of the cost in the production and extraction process is the cost of raw materials, so that the application of the variety with high capsaicin content and high disease resistance and yield becomes the technical key for extracting the capsaicin. In the currently common 5 pepper cultivarsC. chinenseThe content of capsaicinoids is obviously higher than that of other 4 kinds of capsaicinoids (i.e.)C. annuum、C. baccatum、C. frutescens、C. pubescens). However,C. chinensethe seeds have the characteristics of long growth cycle, low fruit setting rate, weak environmental adaptability and the like.C. annuumThe seed has high fruit setting rate, rich varieties, strong environment adaptability and wide cultivation area (85 percent of the cultivation area of the pepper is the seed), but the capsaicin content of the seed is low, so that the capability of improving the capsaicin content of the offspring by adopting an intraspecific hybridization strategy is very limited.C. chinenseAndC. annuumclose relationship, will be controlled by conventional cross breeding methodC. chinenseGenetic locus transfer of high capsaicin content materialC. annuumThe material obtains germplasm resources with high pungency, high yield, high quality and strong adaptability, and is an important precondition for breeding new varieties suitable for capsaicin processing and extraction. However, the traditional breeding method has the defects of unstable phenotype identification, time consumption, labor waste, high cost and the like.
In addition, the traditional method for detecting capsaicin substances mainly comprises the steps of extracting capsaicin substances in pepper and then measuring by adopting methods such as liquid chromatography, mass spectrometry and the like, so that the time and labor are wasted, the cost is high and the efficiency is low. The molecular assisted breeding by using the molecular marker of the target character coseparation has the advantages of high efficiency, rapidness, stability and the like, and the target character can be identified in advance in the seedling stage, so that the method can overcome a plurality of limitations of long period, large occupied area, high cost and the like of the conventional breeding to a certain extent. Insertion/deletion polymorphisms (indels) are length polymorphic variations due to the insertion/deletion of nucleotide fragments in the DNA sequence at an allelic site between different individuals. The polymorphism frequency of the InDel marker is second to that of the SNP marker and is far higher than that of the SSR marker in the whole genome. The InDel marker polymorphism can achieve the purpose of genotyping through simple steps of Polymerase Chain Reaction (PCR), agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresis and the like. Patent CN201811285982.8 discloses an InDel marker for pepper hybrid purity, and patents CN201810360454.8 and CN201810499212.7 both disclose an InDel marker for distinguishing five pepper cultivars; however, no relevant reports for distinguishing the molecular markers with high and low capsaicin content are found at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an InDel marker related to the peppery taste character of hot pepper.
Another object of the present invention is to provide a primer for detecting the InDel label.
The invention further aims to provide a method for screening the characters with high and low capsaicin content.
The above object of the present invention is achieved by the following technical solutions:
an insertion/deletion fragment related to hot taste of Capsici fructus, has nucleotide sequence shown as SEQ ID NO.1, has length of 66 bp, and is located atCap1Upstream of the coding region of the gene.
SEQ ID NO.1：AAACGCCTCACATCAAACATGCAATTCAAGTTCCAACA TTGACAGTGCAATTCACAACAACAAGAA。
The present inventors have found 740 inbred line with high capsaicin content (C. chinesne) And CA1 inbred line with low capsaicin content (C. annuum) Is/are as followsCap1The upstream sequence of the coding region of the gene has a large number of SNPs and 7 InDel, the most interesting of which is an insertion/deletion fragment with a length of 66 bp and sequence information as shown in SEQ No.1, which is present in the 740 inbred line and deleted in the CA1 inbred line and is typically high in capsaicin content from 47C. chinenseGenotypic material and 73 representativeC. annuumGenotype material analyzesCap1The coding region was sequenced upstream and the 66 bp fragment was found in all C. chinenseThe genotypic material is present inC. annuumThe deletion of the genotype material shows that the insertion/deletion fragment shown in SEQ ID NO.1 is related to the hot taste character of the pepper, and can be used for screening the characters of high and low capsaicin content and predicting the hot degree of the fruits of pepper plants or the hot degree of the fruits of filial generations thereof. At the same time, it was shown that the insertion/deletion fragments shown in SEQ ID NO.1 can also be used for discriminationC. annuumGenotyping materials andC. chinensea genotypic material.
Therefore, the invention also claims the application of the insertion/deletion fragment in screening the high-low character of the capsaicin or in preparing a kit for screening the high-low character of the capsaicin; and the insertion/deletion fragment is used for distinguishing pepperC. annuumGenotyping materials andC. chinenseuse in genotypic material.
The invention also provides an InDel molecular marker for screening the high-content and low-content characters of capsaicin, which comprises an upstream primer and a downstream primer, wherein the sequences of the InDel molecular marker are sequentially shown as SEQ ID NO. 3-4:
InDel-F：5’-CGCTTCCTACTGGTAGAATCA-3’（SEQ ID NO.3）；
InDel-R：5’-TTCTGCTGTGGATAATGATTAG-3’（SEQ ID NO.4）。
the invention also requests to protect the application of the InDel molecular marker in screening the high-low character of the capsaicin or in preparing a kit for screening the high-low character of the capsaicin.
A method for screening/predicting the content of capsaicin in hot pepper or its descendants comprises the following steps:
S1, extracting genome DNA of a pepper plant to be detected;
s2, detecting the genome in the step S1Cap1Whether an insertion/deletion fragment shown as SEQ ID NO.1 exists at the upstream of the gene or not;
s3, judging a result: if the insertion/deletion fragment shown in SEQ ID NO.1 exists in the genome, the content of capsaicin in the hot pepper to be detected is high, and the pungency degree is high; if not, the content of the capsaicin is low, and the pungency degree is low. Can establish a method for screening the high and low content of the capsaicin by the molecular marker in the seedling stage.
Or: if the insertion/deletion fragment shown as SEQ ID NO.1 exists in the genome, the pepper to be detected is shown asC. chinenseA genotypic material; if not, the pepper to be detected is represented asC. annuumA genotypic material.
The pepper plant is root, stem, leaf, flower, pericarp, seed, or other parts, tissues or derived tissues from which the complete genome of pepper can be extracted, including but not limited to pepper plants.
Preferably, the detection in step S2 is a PCR amplification reaction using the genomic DNA in step S1 as a template and the InDel molecular marker described above; if the 434 bp fragment is obtained, the hot degree of the capsicum or the filial generation thereof containing the capsaicin with higher content is high; if a 368 bp strip appears, the content of the capsaicin to be detected is low, and the pungency degree is low.
Preferably, the reaction system of the PCR is: 10 uL Taq enzyme PCR Mix, 1 uL each of the upstream and downstream primers, 50 ng DNA template, plus ddH 2 O to 20 mL.
Preferably, the reaction procedure of the PCR is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 10s, and extension at 72 ℃ for 30 s, and repeating 35 cycles; extension at 72 ℃ for 10 min.
The invention also provides a kit for screening/predicting capsaicin materials of hot peppers or offspring thereof, which comprises a primer pair for detecting the insertion/deletion fragment of SEQ ID NO. 1.
Preferably, the primer pair comprises an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are sequentially shown in SEQ ID No. 3-4.
The application of the insertion/deletion segment shown in SEQ ID NO.1 or the InDel molecular marker shown in SEQ ID NO. 3-4 in auxiliary breeding of pepper is also within the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an insertion/deletion fragment related to hot taste character of pepper fruit, the nucleotide sequence of which is shown as SEQ ID NO.1, and the sequence of the fragment is positioned inKey MYB transcription factor for regulating and controlling capsaicin biosynthesisCap1Upstream of the gene; based on the method, an InDel molecular marker is developed, the sequence of the InDel molecular marker is shown as SEQ ID No. 3-4 in sequence, and detection is carried out Cap11 sequence of the upstream gene SEQ ID NO can well classify the content of capsaicin and can predict the content of capsaicin in the early growth stage of plants; therefore, the plants which do not meet the breeding target can be removed early in the breeding process, and unnecessary waste of labor and capital cost is avoided. The invention has very important guiding significance for genetic improvement of hot pepper flavor, improvement of breeding efficiency and saving of breeding cost.
Drawings
FIG. 1 shows major QTL sites for capsaicin class of substance contentCap1The identification of (3); FIG. 1a represents the progeny F of the 740 and CA1 cross 2 The distribution of the capsaicin content of the population; FIG. 1b represents F 2 The content distribution of the dihydrocapsaicin of the population; FIG. 1c represents F 2 The distribution of the total capsaicin content of the population; FIG. 1d represents QTL mapping for capsaicin content; FIG. 1e represents QTL mapping for dihydrocapsaicin content; FIG. 1f represents QTL mapping for total capsaicin content.
FIG. 2 isCap1And (5) fine positioning.
FIG. 3 is a drawing showingCap1Sequence analysis and marker development upstream of the coding region of the gene.
FIG. 4 shows the analysis of the capsaicin content of a material by using a marker.
FIG. 5 analysis of capsaicin content of hybrids using markers; FIG. 5a shows a photograph of a 2013 Ginis world record reporting extremely hot pepper, "Kaolina dead Pepper" (Carolina workers); FIG. 5b shows Cap1The gene upstream 66 bp sequence is detected in the Karlaine dead pepper (Carolina reactors); FIG. 5c shows the analysis of the C progeny of the Carolina reactions and CA hybrids F using the InDel marker 2 The genotype of the locus in the population; FIG. 5d shows the progeny of cross F 2 The upstream of the population Cap1 gene contains the content of capsaicin (upper) and dihydrocapsaicin (lower) corresponding to Carolina reactions or CA sequence single strains.
Detailed Description
The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. The experimental procedures in the following examples are, unless otherwise specified, conventional procedures, such as the Molecular cloning laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual, 2001) such as Sambrook, or the conditions suggested by the manufacturer's instructions for biochemical reagents. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 acquisition of InDel tags
Using 740 inbred line with high capsaicin content (C. chinesne) CA1 inbred line as female parent and with low capsaicin content (C. annuum) As male parent, hybridizing to obtain F 1 After generation, F 1 Selfing to obtain F 2 And (4) a group. 150F are added 2 And the colony is planted in a greenhouse after being cultivated by adopting a plug seedling and is subjected to conventional management. And (3) detecting single SNP of a group by adopting the SLAF technology, and developing a high-density genetic map containing 9038 markers. The extraction and determination of capsaicin and dihydrocapsaicin in the population are carried out by high performance liquid chromatography. The main effect QTL site of capsaicin substance content is mapped by using a composite interval mapping method (CIM)Cap1Mapping to chromosome 7, this QTL accounts for 37-42% of phenotypic variation (FIG. 1). According toCap1Developing corresponding InDel markers at corresponding positions of loci on chromosomes, further taking a CA1 inbred line as a recurrent parent and F 1 Generation hybridization willCap1Restricted to a region of 510 kb containing 6 genes all encoding MYB transcription factors, whereinCap1We have shown that it is directly involved in regulating capsaicin biosynthesis (figure 2),Cap1the nucleotide sequence of the gene is shown in SEQ ID NO. 2.
By further cloning of pepper 740 and CA1 inbred lines Cap1Upstream sequence of coding region of gene, between which a large amount of S is presentNP and 7 InDel, of which the most interesting is the 66 bp sequence shown in SEQ NO:1, which is present in the 740 inbred line, but absent in the CA1 inbred line (FIG. 3), and high capsaicin content from 47 representativesC. chinenseGenotypic material and 73 representativeC. annuumGenotype material analyzesCap1The upstream sequence of the coding region, a 66 bp fragment was found in allC. chinenseThe genotypic material is present inC. annuumThe genotypic material is missing. Based on this, a pair of InDel markers SEQ ID NO.3 and SEQ ID NO.4 was developed. The molecular marker can be applied to pepper molecular breeding. The specific method comprises the following steps: the pepper genome DNA is used, primers of SEQ ID NO.3 and SEQ ID NO.4 are adopted for PCR amplification, amplification products are electrophoresed on 2 percent agarose gel, if 434 bp fragments are obtained, the sequences with high content of capsaicin substances are proved to be contained, and the material (filial generation) contains the capsaicin substances with higher content.
Example 2
The content of capsaicin (capsaicin and dihydrocapsaicin) is determined efficiently. The collected sample was dried in an oven at 40 ℃ for 48 hours and ground while hot in a mortar. 0.1g of the ground sample was weighed and placed in a 15 mL centrifuge tube, and labels were written on the respective samples. 10 mL of the extract (methanol: tetrahydrofuran (1: 1)) was added to each centrifuge tube, the tube was sonicated in a sonicator for 30 min with a cap closed, and the sample was allowed to stand overnight at room temperature. A single syringe was used to aspirate 1 mL of the capsaicin extract, and the extract was filtered through a 0.22 μm filter and then put into a1 mL chromatography bottle for chromatography. 10 μ L of standard sample or sample to be detected was pipetted and separated by an XSelectric HSS C-18 SB column (4.6 = 250 mm, 5 μm) using a high performance liquid chromatograph Waters Alliance 2489 and the separated sample was detected by an absorption peak at 280 nm using an ultraviolet detection system 2489 UV/Visible. The chromatographic conditions are as follows: 80% methanol flow rate 1 mL/min, column box temperature 30 ℃, each sample analysis time 10 min. Capsaicin and dihydrocapsaicin contents were calculated according to a standard curve prepared for a standard, and total capsaicin content was calculated according to (capsaicin + dihydrocapsaicin)/0.9 (Bennett and Kirby, 1968).
11 representative extracts were extracted by CTAB methodC. chinenseGenotypic material and 10 representativeC. annuunDNA of genotypic material. The extracted genome DNA is used as a template, and InDel primers SEQ ID NO.3 and SEQ ID NO.4 are used for PCR amplification. The amplification system is 20 mu L, and comprises the following components: 10 uL Taq enzyme PCR Mix, 1 uL each of the upstream and downstream primers, 50 ng DNA template, and double distilled water H 2 The volume of O was made up to 20 mL. PCR amplification was performed using Bio-RadT 100. sup.PCR, and the amplification procedure was as follows: the procedure of pre-denaturation at 95 ℃ for 10 min, followed by denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 10s, and extension at 72 ℃ for 30 s was performed for 35 cycles. 5 μ L of the reaction PCR product was electrophoresed on 2% agarose for 40 min, followed by staining with EB for 15 min and photographing using Bio-RadGel Doc XR + Gel Doc XR Gel imaging system. According to comparison between the read bands and Marker, the capsaicinoid content of the material is higher if a 434 bp band exists, and the capsaicinoid content is lower if a 368 bp band exists (figure 4), which is consistent with the detection result of high performance liquid chromatography.
Example 3
The most spicy variety 'Carolina Reaper' reported by 2013 Ginis world record (C.A.) C. chinesne) CA1 inbred line as female parent and slight peppery (C. annuum) As male parent, and hybridizing to obtain F 1 Then F 1 Selfing to obtain F 2 And (5) planting the colonies and performing conventional cultivation management. F 2 Population DNA extraction, capsaicin content determination, PCR amplification and band detection were as described in example 2. From the read bands, it was found that the content of capsaicinoids in the progeny material was higher if a 434 bp band was present in the progeny, and lower if a 368 bp band was present (fig. 5).
The experiments prove that the content of capsaicin substances of hot peppers with different genotypes can be effectively predicted by detecting the identification sequence shown by SEQ ID NO. 1 in the hot pepper plant genome through InDel primers SEQ ID NO.3 and SEQ ID NO. 4. By utilizing the characteristic, the content of the capsaicin can be predicted in the early growth stage of the plant after the hybridization is finished. Therefore, the plants which do not meet the breeding target can be removed early in the breeding process, and unnecessary waste of labor and capital cost is avoided.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> southern China university of agriculture
<120> InDel marker related to hot taste character of pepper and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 66
<212> DNA
<213> Pepper (Capsicum annuum L.)
<400> 1
aaacgcctca catcaaacat gcaattcaag ttccaacatt gacagtgcaa ttcacaacaa 60
<210> 2
<211> 750
<212> DNA
<213> Pepper (Capsicum annuum L.)
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cccaagtatg ctggactagc aaggtgtgga aagagctgca gacttcgatg gatgagtcac 180
ttacggccaa atgttaaaag agggaattat accaaagaag aagatgaaat catcttgaac 240
ctccatgctc aacttggaaa taggtggtcg gcgattgctg ctcacttgcc aggaagatca 300
gacaatgaga taaagaatca ttggcacaca aaacttaaga agcgcggtac taattatgcg 360
acaaactcaa gtgatgaatc aagcaagaaa tgtaagaata atactaagaa gaggtatact 420
gaaagtaata ccaataaaaa tacaagtcat aataatatgc aggaaaatat agtactggaa 480
agtttagaat ggtcaccaaa ggaatcatca agtgaagaac tctcctctta cagtaccact 540
aattatcaac agcaacataa agtgtttcaa gaggaaataa ctagtggaag cttttggaca 600
gaaccatttg tagtagaaag tttcaatact actagaactg attttctagc tccttcaatt 660
gattactgtg gacttgtgtg tccaccttca ccatatatag gtcatgaatt tctttcctcc 720
tttgactttg atcattataa ttattggtaa 750
<210> 3
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<213> Pepper (Capsicum annuum L.)
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cgcttcctac tggtagaatc a 21
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<213> Pepper (Capsicum annuum L.)
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Claims (4)
1. The application of a primer for detecting whether an insertion/deletion fragment shown in SEQ ID NO.1 exists at the upstream of a coding region of a pepper Cap1 gene in screening of high and low capsaicin content traits or in preparation of a kit for screening of high and low capsaicin content traits is characterized in that the primer comprises an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are sequentially shown in SEQ ID NO. 3-4; if the insertion/deletion fragment shown in SEQ ID NO.1 exists, the capsaicin content in the hot pepper to be detected is high; if the insertion/deletion fragment shown in SEQ ID NO.1 does not exist, the capsaicin content is low; the hot pepper is filial generation obtained by hybridizing C.chinensis as a female parent and C.annuum as a male parent and a filial generation group obtained by selfing the filial generation.
2. A method for screening/predicting the high-low capsaicin content character of hot pepper is characterized by comprising the following steps:
s1, extracting genome DNA of hot pepper to be detected;
s2, detecting whether an insertion/deletion fragment shown as SEQ ID NO.1 exists at the upstream of the Cap1 gene coding region in the genome DNA obtained in the step S1 by using a primer; the primers comprise an upstream primer and a downstream primer, and the sequences of the primers are sequentially shown as SEQ ID NO. 3-4;
S3, judging a result: if the insertion/deletion fragment shown in SEQ ID NO.1 exists, the capsaicin content in the hot pepper to be detected is high; if the insertion/deletion fragment shown in SEQ ID NO.1 does not exist, the capsaicin content is low;
the hot pepper is filial generation obtained by hybridizing C.chinensis as a female parent and C.annuum as a male parent and a filial generation group obtained by selfing the filial generation.
3. The method of claim 2, wherein the step S2 is performed by performing a PCR amplification reaction using the genomic DNA of the step S1 as a template; if 434bp fragments are obtained, the capsaicin content of the pepper to be detected is high, and if 368bp bands are obtained, the capsaicin content of the pepper to be detected is low.
4. The method of claim 3, wherein the reaction sequence of the PCR is: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 10s, and extension at 72 ℃ for 30s, and repeating 35 cycles; extension at 72 ℃ for 10 min.
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| KR100671213B1 (en) * | 2005-11-23 | 2007-01-19 | (주)고추와 육종 | Development of molecular markers linked to the stability gene of pepper cgms and its use for determining the types of allele |
| CN107151709A (en) * | 2017-07-13 | 2017-09-12 | 中国农业科学院蔬菜花卉研究所 | The SNP marker related to capsicum pungent and its application |
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| CN108384875A (en) * | 2018-03-23 | 2018-08-10 | 湖南省蔬菜研究所 | A kind of and the relevant insertion/deletion site of capsicum mellow fruit color gene, molecular labeling, molecular labeling primer and application |
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