CN114438254B - InDel marker for identifying perfume coconuts and application thereof - Google Patents

Abstract

The invention provides an InDel marker for identifying aromatic coconut and application thereof, wherein an InDel marker primer pair is shown as SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6 or SEQ ID No.7-8, and the InDel marker primer group consists of two or more than two of the primer pairs. The kit for identifying the coconuts can be prepared by adopting the InDel marked primer pair or the InDel marked primer group. The InDel mark primer pair or the InDel mark primer group or the kit is adopted to carry out PCR amplification electrophoresis on different varieties of coconuts, namely, the varieties of the coconuts can be identified according to electrophoresis strips or sequencing results, the coconuts can be detected at any time from seedling stage to adult big tree, the identification accuracy is high, and the method has important significance for distinguishing the coconuts.

Description

InDel marker for identifying perfume coconuts and application thereof

Technical Field

The invention relates to the fields of molecular biology and bioinformatics, in particular to an InDel marker for identifying aromatic coconuts and application thereof.

Background

Coconut (Cocos nuifem l.) is the only species in the genus coco of the palmaceae, belongs to monocotyledonous perennial evergreen arbor, is precious throughout its body, is known as "living tree" as important woody oil crops and food energy crops in tropical areas, has an economic life of 40-80 years and a natural life of more than 100 years.

Coconut can be classified into high-seed coconut, low-seed coconut, and intermediate types between high-seed and low-seed coconut according to conventional classification methods. The main characteristic of the high-seed coconuts is that the plants are tall and big, the base parts of the coconuts are expanded to form gourd heads, and cross pollination is carried out; the dwarf coconut plants are short, the base part of the coconut trunk is not provided with a gourd head, the self-pollination is carried out, and the economic life and the natural life are short; the intermediate type is between the two. In addition, coconut germplasm resources are classified into several types among varieties according to fruit size, fruit shape, fruit peel color, etc., for example, dwarf coconuts can be classified into yellow dwarf, red dwarf, and green dwarf according to fruit peel color.

The perfume coconut (Aromatic Green Dwarf, AROD) is a specific mutant of the green dwarf coconut, native to thailand celecoxib, and is most characterized by coconut leaves and coconut water smelling a specific flavor resembling the flavor of taro. Making it popular in the market place, it is statistically twice as expensive as a coconut with a flavor. The coconuts are planted for 3-4 years, then bloom and bear fruits, the fruits are early, the coconuts are planted for 8 years, then have a high yield period, about 60 years of natural life and about 35 years of economic life, the coconuts are high in yield, the average yield of each coconut is more than 80, and the highest yield can reach more than 150. The fruits are round and smaller, the diameter of the fruits is 37-45 cm, the length of the fruits is 18.2cm, the width of the fruits is 15.6cm, and the thickness of coconut meat is 9.2mm. The weight of the single fruit is 1.2kg, the outer skin accounts for 35%, the coconut shell accounts for 13%, the coconut meat accounts for 32% and the coconut water accounts for 20%. The peel is green, and the peel and the seed coat are thinner. The perfume coconut has high sugar content, soft and tender coconut meat and rich nutrition, and is one of excellent natural green foods.

InDel markers (insertional-Delions) were developed on the basis of whole genome resequencing, polymorphic variations resulting from the Insertion or Deletion of a short stretch of DNA at the allele site. In short, the difference between two parents in the whole genome is that for one parent genome, the other parent has a certain number of nucleotide insertions or deletions, and PCR primers for amplifying the positions can be designed according to the positions of the insertions or deletions in the genome, namely InDel markers.

InDels are widely distributed in the whole genome, and compared with other molecular markers, the probability of the same-sized InDel markers appearing at the same position of the genome is small, so that the ambiguity of subsequent analysis is avoided. Based on genome sequence determination, the InDel marker can be conveniently and simply obtained by a bioinformatic method. Compared with SSR markers, the amplified product of the InDel markers has clear and simple banding patterns, and has better stability and product separation effect than the SSR markers. InDel markers have the advantage of other molecular markers. InDel markers based on whole genome resequencing have the advantages of wide distribution in genome, high density, strong polymorphism, easy detection, simple typing system and stable inheritance. The InDel marker is suitable for the development of whole genome molecular markers, and is currently applied to the fields of animal and plant population genetic analysis, construction of high-density molecular marker genetic maps and gene positioning, genetic diversity analysis of organisms, molecular assisted breeding, human forensic genetics, medical diagnosis and the like.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, firstly utilizes insertion and deletion sequences obtained by coconut resequencing to develop a coconut InDel marker, uses the polymorphic InDel marker to obtain a fingerprint characteristic spectrum of part of coconut varieties, utilizes molecular marker combinations obtained by screening to carry out purity identification of the coconut varieties and early breeding of seedlings, and shortens the breeding period of new varieties of the coconuts by molecular marker assisted breeding.

In a first aspect, the invention provides an InDel marker for identifying a coconut as a perfume, wherein the InDel marker primer pair is a primer pair as shown in SEQ ID No.1-2, a primer pair as shown in SEQ ID No.3-4, a primer pair as shown in SEQ ID No.5-6 or a primer pair as shown in SEQ ID No. 7-8.

The second aspect of the present invention is to provide an InDel-labeled primer set for identifying a aromatic coconut, the InDel-labeled primer set consisting of two or more of the following primer pairs: the primer pair shown in SEQ ID No.1-2, the primer pair shown in SEQ ID No.3-4, the primer pair shown in SEQ ID No.5-6 and the primer pair shown in SEQ ID No. 7-8.

In a third aspect, the invention provides a kit for identifying a aromatic coconut comprising an InDel marker primer pair according to the first aspect of the invention or an InDel marker primer set according to the second aspect of the invention.

Preferably, the kit further comprises reagents required for performing a PCR reaction and/or electrophoresis.

In a fourth aspect, the invention provides the use of an InDel-labeled primer pair according to the first aspect of the invention, or an InDel-labeled primer set according to the second aspect of the invention, or a kit according to the third aspect of the invention, in the identification of a perfume coconut.

In a fifth aspect, the invention provides a method for identifying a aromatic coconut using the InDel-labeled primer set of the first aspect, or the InDel-labeled primer set of the second aspect, or the kit of the third aspect.

Preferably, the method comprises the steps of: (1) extracting coconut leaf DNA; (2) Performing PCR amplification on the coconut leaf DNA by adopting an InDel marked primer pair or an InDel marked primer group or a kit; (3) detecting the amplification result by gel electrophoresis; (4) Identifying different varieties of coconuts according to detection results, wherein gel electrophoresis results show that insertion or deletion fragments exist to cause electrophoresis results to be larger or smaller than target bands or target fragments, perfume coconuts are adopted, and electrophoresis or sequencing results are consistent with the sizes of the target bands or target fragments, and other varieties of coconuts (such as Ma Wa coconuts, red dwarf coconuts, yellow dwarf coconuts, brown dwarf coconuts, hainan high coconuts and the like) are adopted, wherein:

when the InDel marked primer pair is a primer pair shown as SEQ ID No.1-2, the target band or target fragment is 144bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.3-4, the target band or target fragment is 123bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.5-6, the target band or target fragment is 139bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.7-8, the target band or target fragment is 109bp;

if the gel electrophoresis result needs to be verified, the gel electrophoresis result of the aromatic coconut can be recovered and sequenced, the genomic sequence of the Hainan high-seed coconut is used as a reference to be subjected to Blast comparison with the sequencing result of the aromatic coconut, and the aromatic coconut with the inserted fragment exists. In this case, the method comprises the steps of:

(1) Extracting DNA of coconut leaves of perfume;

(2) Performing PCR amplification on the DNA of the coconut leaves by adopting an InDel marked primer pair or an InDel marked primer group or a kit;

(3) Performing polyacrylamide gel electrophoresis detection on the PCR product, and recycling the PCR product by using a gel recycling kit;

(4) After the recovered product is subjected to PCR amplification, the PCR product is purified, and after connection conversion, monoclonal sequencing is selected;

(5) Blast alignment was performed on the sequence of the aromatic coconut sequences based on the sequencing results with reference to the high coconut genome, with the aromatic coconut being present as an insert.

The invention has the beneficial effects that:

the appearance of the coconut is easy to be confused with other green dwarfs, in cross breeding, the determination of the variety and relationship of parents is particularly important, the clear coconut variety is an important premise and guarantee for molecular breeding of coconuts, the PCR amplification is carried out on coconuts by adopting the InDel marked primer pair or the InDel marked primer group or the kit, and the amplified products are separated by polyacrylamide gel electrophoresis and dyed or sequenced, so that the coconut variety can be identified according to electrophoresis strips or sequencing results, can be detected at any time in a large adult tree at any time, has important significance for distinguishing the coconut variety from other coconut varieties, has high identification accuracy, can ensure the purity of the coconut, and has wide application prospect in the field of cross breeding of coconuts.

Drawings

FIG. 1 is an electrophoretogram of the InDel marker primer pair ID239, ID240, ID241, ID242 of the invention for 6 coconut variety amplification products. R1, R2 and R3 are red dwarf coconut; y1, Y2 and Y3 are yellow dwarf coconuts; x1, X2 and X3 are brown dwarf coconuts; g1, G2 and G3 are aromatic coconuts; m1, M2 and M3 are high species coconut; b1, B2 and B3 are high coconut species.

FIG. 2 shows the results of a sequence comparison of the InDel marker primer pair ID239 pair of the present invention with Cocois. Among which the insert is present is the aromatic coconut.

Detailed Description

The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1. DNA extraction from coconut leaves

Experimental materials: a total of 18 samples of 6 coconut varieties were selected, of which Ma Wa coconuts, hainan tall coconuts, red dwarf coconuts, yellow dwarf coconuts, perfume coconuts, brown dwarf coconuts were 3. All the samples are from coconut institute of Tropical agricultural sciences of Wenchang city, hainan province.

The DNA extraction of coconut leaves refers to a CTAB method, and comprises the following specific operations:

weighing about 1g of coconut leaves, adding liquid nitrogen into a mortar, rapidly grinding into powder, transferring into a 2mL centrifuge tube, adding 1mL extraction buffer solution, shaking uniformly, and placing on ice; centrifuging at 8000r/min at 4deg.C for 5min, discarding supernatant, adding 1mL of extraction buffer again, shaking, centrifuging at 8000r/min at 4deg.C for 5min, discarding supernatant; adding 800 μl of lysis buffer preheated to 65deg.C, shaking, standing in water bath at 65deg.C for 60min, and shaking every 10 min; adding equal volume of chloroform: extracting isoamyl alcohol (24:1) extract, fully and uniformly mixing, centrifuging at 8000r/min and 25 ℃ for 10min, and transferring the supernatant into a new centrifuge tube; then adding an equal volume of chloroform: isoamyl alcohol (24:1), fully and uniformly mixing, centrifuging at 8000r/min and 25 ℃ for 10min, and transferring the supernatant into a new centrifuge tube; adding 0.6 times volume of frozen isopropyl alcohol and 1/10 volume of 3M sodium acetate (pH 5.2), slowly shaking until flocculent polymeric precipitate is separated out, and standing at 4deg.C overnight; hooking the precipitate, transferring into a new centrifuge tube, rinsing with 75% alcohol for 2 times, centrifuging at 25deg.C at 8000r/min for 5min, and pouring dry alcohol; adding 200 mu L TE buffer solution for dissolution, detecting the concentration of DNA by using a Nanodrop nucleic acid tester, adding TE buffer solution to adjust the concentration of all the DNA to 50 ng/. Mu.L, and preserving at-20 ℃ for standby.

2. Design of InDel labeled primer

According to a large number of sequences obtained by sequencing the genomes of the coconuts with perfume and the coconuts with yellow dwarf, taking the genome of the coconuts with Hainan high species as a reference, obtaining relevant information of the InDel sequence through Blastn sequence comparison, selecting 400 sequence fragments from the sequences according to insertion and deletion sequences, and adopting software Primer Premier 5.0 to design primers, wherein the sizes of the insertion and deletion fragments are 5bp and more than 5 bp. 4 pairs of primers were obtained by screening and used to identify Cocois radiatus, as shown in Table 1.

TABLE 1 InDel marker primers for screening of Cocois

Primer numbering	Forward primer (5 '-3')	Reverse primer (5 '-3')	Product length (bp)
				ID239	TGATAGTGGAGGCGAGGTC	TTTAGTGGTCAGCATCTTGTTC	144
ID240	CCTTCGTTAGTGCAGTGCTT	AGATGGGGTTGGGATGTG	123
				ID241	GAAAGTTTTGACGGAGGCG	TCATTCTAGTGAAGGGGATGG	139
ID242	TTGAGGCAGGGGAACACG	TTGGCTCGGGTACAGGCT	109

3. Identification of perfume coconut variety

1. Discrimination according to gel electrophoresis result

(1) InDel marker primer pair ID239

The InDel marker primer pair used to identify the aromatic coconut variety was ID239 (Table 1). Performing PCR amplification on the DNA of different varieties of coconut leaves obtained in the process of extracting the DNA of the first and second coconut leaves by using an ID 239; the PCR amplification system is as follows: 10 XBuffer (Mg) ²⁺ ) 2. Mu.L, 4 XdNTP mix (10 mM each) 0.5. Mu.L; 10. Mu. Mol/L forward and reverse primer 1.0. Mu.L each; 20 ng/. Mu.L of DNA 2. Mu.L; 5U/. Mu.L Taq DNA polymerase 0.2. Mu.L; adding sterile ddH ₂ O to 20. Mu.L; the PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 54℃for 45s, elongation at 72℃for 50s, and repeated 30 times; extending at 72℃for 10min. Adding 3 mu L of 6 Xloading buffer solution into the PCR amplification product, uniformly mixing, taking 1.2 mu L of sample application, and separating the strips by polyacrylamide gel electrophoresis; polyacrylamide gel 30 (mL): ddH ₂ O15.81mL;5 XTBE 6mL;30% (acrylamide: methylenebisacrylamide=29:1) stock 7.98mL;10% AP (ammonium persulfate) 0.21mL, TEMED 15. Mu.L, electrophoresis conditions of 6% PAGE,150V, 400mA, 30W, electrophoresis time of 1h 20min; staining was performed using silver nitrate and photographed. As a result, as shown in FIG. 1, the target bands were 144bp, ma Wa, red, yellow, brown and Hainan higher coconut bands were 144bp, and perfume coconut bands were 151 bp. The InDel marker primer pair ID239 can be used for specifically identifying the coconuts.

(2) InDel marker primer pair ID240

The identification of the variety of the aromatic coconut was carried out by the same method as the "(1) InDel marker primer pair ID239", and the InDel marker primer pair ID240 (Table 1) was used, and the result is shown in FIG. 1, the target bands were 123bp, ma Wa coconut, red dwarf coconut, yellow dwarf coconut, brown dwarf coconut and Hainan high-seed coconut were bands of 123bp in length, and the aromatic coconut bands were bands of 117bp in length, which indicated that the InDel marker primer pair ID240 can specifically identify the aromatic coconut.

(3) InDel marker primer pair ID241

The variety of the perfume coconuts was identified by the same method as "(1) InDel marker primer pair ID239", using InDel marker primer pair ID241 (Table 1), and the result is shown in FIG. 1, the target bands were 139bp, ma Wa coconut, red dwarf coconut, yellow dwarf coconut, brown dwarf coconut and Hainan high-seed coconut were 139bp bands, and the perfume coconuts had two bands, one of which was 133bp bands, indicating that InDel marker primer pair ID241 can specifically identify perfume coconuts.

(4) InDel marker primer pair ID242

The identification of the species of the aromatic coconut was carried out by the same method as that of the "(1) InDel marker primer pair ID239", and the InDel marker primer pair ID242 (Table 1) was used, and the result is shown in FIG. 1, the target bands were 109bp, ma Wa coconut, red dwarf coconut, yellow dwarf coconut, brown dwarf coconut and Hainan high-seed coconut were bands with a length of 109bp, and the aromatic coconut bands were bands with a length of 115bp, which indicated that the InDel marker primer pair ID242 can specifically identify the aromatic coconut.

2. Identifying or verifying gel detection results according to sequencing results

The same method as the method of 'InDel marked primer pair ID 239' in the upper section (1), PCR amplification is carried out by using the perfume coconut DNA as a template and the primer pair ID239, glue recovery is carried out by using a PCR product as a template, PCR amplification is carried out by using the recovered product as a template, connection conversion is carried out on the purified product after the PCR product is purified, colony PCR detection is carried out by selecting a monoclonal (the primer used for PCR is M13 universal primer), and sequencing analysis is carried out by selecting a positive clone. Blast alignment of sequenced sequences was performed with reference to the high coconut genome, with the insert being of Cocois auca.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Sequence listing

<110> Cocois institute of Tropical agricultural sciences of China

<120> InDel marker for identifying Cocois in perfume and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

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<211> 19

<212> DNA

<213> Artificial

<400> 1

tgatagtgga ggcgaggtc 19

<210> 2

<211> 22

<212> DNA

<213> Artificial

<400> 2

tttagtggtc agcatcttgt tc 22

<210> 3

<211> 20

<212> DNA

<213> Artificial

<400> 3

ccttcgttag tgcagtgctt 20

<210> 4

<211> 18

<212> DNA

<213> Artificial

<400> 4

agatggggtt gggatgtg 18

<210> 5

<211> 19

<212> DNA

<213> Artificial

<400> 5

gaaagttttg acggaggcg 19

<210> 6

<211> 21

<212> DNA

<213> Artificial

<400> 6

tcattctagt gaaggggatg g 21

<210> 7

<211> 18

<212> DNA

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<400> 7

ttgaggcagg ggaacacg 18

<210> 8

<211> 18

<212> DNA

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ttggctcggg tacaggct 18

Claims (8)

1. The primer pair for identifying the InDel mark of the coconuts is characterized in that the primer pair of the InDel mark is shown as a primer pair of SEQ ID No.1-2, a primer pair of SEQ ID No.3-4, a primer pair of SEQ ID No.5-6 or a primer pair of SEQ ID No. 7-8.

2. An InDel-labeled primer set for identifying aromatic coconut, characterized in that the InDel-labeled primer set consists of two or more of the following primer pairs: the primer pair shown in SEQ ID No.1-2, the primer pair shown in SEQ ID No.3-4, the primer pair shown in SEQ ID No.5-6 and the primer pair shown in SEQ ID No. 7-8.

3. A kit for identifying a perfume coconut, comprising the InDel marker primer pair of claim 1 or the InDel marker primer set of claim 2.

4. A kit according to claim 3, further comprising reagents required for performing a PCR reaction and/or electrophoresis.

5. Use of the InDel-labeled primer pair of claim 1 or the InDel-labeled primer set of claim 2 or the kit of claim 3 or 4 for identifying a aromatic coconut.

6. A method of identifying a perfume coco, using the InDel marker primer set of claim 1 or the InDel marker primer set of claim 2 or the kit of claim 3 or 4.

7. The method according to claim 6, comprising the steps of:

(1) Extracting coconut leaf DNA;

(2) Performing PCR amplification on the coconut leaf DNA by adopting an InDel marked primer pair or an InDel marked primer group or a kit;

(3) Gel electrophoresis detection is carried out on the amplification result;

(4) Identifying different varieties of coconuts according to detection results, wherein gel electrophoresis results show that the existence of insertion or deletion fragments leads to electrophoresis or sequencing results which are larger or smaller than a target band or a target fragment to be obtained by perfume coconuts, and electrophoresis or sequencing results which are consistent with the size of the target band or the target fragment to be obtained by electrophoresis or sequencing are obtained by other varieties of coconuts, wherein:

when the InDel marked primer pair is a primer pair shown as SEQ ID No.1-2, the target band or target fragment is 144bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.3-4, the target band or target fragment is 123bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.5-6, the target band or target fragment is 139bp;

when the InDel marked primer pair is a primer pair shown as SEQ ID No.7-8, the target band or target fragment is 109bp.

8. The method according to claim 6, comprising the steps of:

(1) Extracting DNA of coconut leaves of perfume;

(2) Performing PCR amplification on the DNA of the coconut leaves by adopting an InDel marked primer pair or an InDel marked primer group or a kit;

(3) Performing polyacrylamide gel electrophoresis detection on the PCR product, and recycling the PCR product by using a gel recycling kit;

(4) After the recovered product is subjected to PCR amplification, the PCR product is purified, and after connection conversion, monoclonal sequencing is selected;

(5) Blast alignment was performed on the sequence of the aromatic coconut sequences based on the sequencing results with reference to the high coconut genome, with the aromatic coconut being present as an insert.