PRIMER PAIR FOR IDENTIFYING GENE SEGMENT OF MALE-SPECIFIC MOLECULAR MARKER OF AMERICAN SHAD AND USES THEREOF

Abstract

The disclosure provides a primer pair for identifying a gene segment of a male-specific molecular marker of American shad (Alosa sapidissima). The gene segment has a nucleic acid sequence represented by SEQ ID NO: 2, and the primer pair has nucleic acid sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4.

Claims

1. A primer pair for identifying a gene segment of a male-specific molecular marker of American shad (Alosa sapidissima), the gene segment having a nucleic acid sequence represented by SEQ ID NO: 2, and the primer pair having nucleic acid sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4.

2. A method for preparing a kit for determining a genetic sex of American shad, the method comprising applying the primer pair of claim 1.

3. A kit for determining a genetic sex of American shad, and the kit comprising the primer pair of claim 1.

4. The kit of claim 3, further comprising a DNA extraction reagent, a PCR amplification reagent, a DNA molecular marker, a nucleic acid stain, or a combination thereof.

5. A method for determining a genetic sex of American shad by use of a DNA marker, the method comprising: 1) extracting total DNA of American shad of unknown sex; 2) amplifying a target fragment of the total DNA through a PCR reaction with the primer pair of claim 1 or the kit of claim 3; and 3) determining, when an amplified fragment is 470 bp in length, the American shad is male; otherwise, the American shad is female.

6. The method of claim 5, wherein a PCR system for the PCR reaction comprises a 2×Taq reaction mixture II (2×Taq Plus Master Mix II (Dye Plus)), the primer pair, a template DNA and H.sub.2O; PCR cycling and reaction parameters are as follows: initial denaturation at 95° C. for 3 min, denaturation at 95° C. for 15 s, annealing at 60° C. for 30 s, elongation at 72° C. for 1 min, and final extension at 72° C. for 5 min.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a graph showing average growth rate of male and female American fish;

[0029] FIG. 2 shows the results of genetic sex determination in American shad using a primer pair Tag-5; and

[0030] FIG. 3 shows the results of genetic sex determination and validation of a male-specific molecular marker in the population of American shad.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The disclosure uses Restriction-site associated DNA (RAD) sequencing to provide a male-specific molecular marker for sex determination in American shad; the male-specific molecular marker is elongated within a reference genome of American shad to form a second nucleic acid sequence for primer design.

[0032] The RAD sequencing is a technique that uses restriction enzymes to isolate a RAD Tag that is employed as a template and analyzed through use of high-throughput DNA sequencing to generate a large volume of raw sequence reads for preparation of a library. The RAD technique is a simple, stable and efficient genotyping method that reduces the complexity of a genome.

Example 1 Identification of a Male-Specific Molecular Marker of American Shad

[0033] The method involves DNA extraction, restriction enzyme digestion, ligation, enrichment, purification, introduction of an index sequence by PCR, pooling and sequencing.

[0034] Total DNA was extracted from caudal fins of five female and five male American shad of known sex using a Marine Animals DNA Kit (Catalog number: DP324-03, TIANGEN);

[0035] absolute ethanol was added to a buffer GD and a rinse solution PW (see the bottle label for volume); specifically, the DNA extraction method comprises the following steps: [0036] (1) no more than 30 mg of tissue samples was taken, frozen in liquid nitrogen, ground into a fine powder, and transferred to a centrifuge tube containing 200 .Math.L of a GA buffer; the mixture was vortexed for 15 s; to remove RNA from DNA extraction, 4 .Math.L of 100 mg/mL ribonuclease (RNase) A solution was added, shaken for 15 s, and allowed to stand at room temperature for 5 min; [0037] (2) 20 .Math.L of 20 mg/mL proteinase K solution was added and vortexed; the centrifuge tube was centrifuged to remove drops from the inside of the cover; the mixture was allowed to stand at 56° C. until the tissue is completely dissolved; the mixture was centrifuged to remove water droplets on the inner wall of the centrifuge tube cover; [0038] (3) 200 .Math.L of a buffer GB was added, mixed thoroughly by inversion, allowed to stand at 70° C. for 10 min; when the mixture turned clear, the centrifuge tube is centrifuged to remove drops from the inside of the cover; [0039] (4) 200 .Math.L of absolute ethanol was added, mixed thoroughly by inversion, and a flocculent precipitate may form; the centrifuge tube was centrifuged to remove drops from the inside of the cover; [0040] (5) the resulting mixture containing the flocculent precipitate in 4) were added into an adsorption column CB3 (placed in a collection tube), centrifuged at 12,000 rpm (-13,400×g) for 30 s; the waste liquid was discarded; and the adsorption column CB3 is placed back into the collection tube; [0041] (6) 500 .Math.L of the buffer GD was added to the adsorption column CB3 (make sure the absolute ethanol has been added before use), centrifuged at 12,000 rpm (-13,400×g) for 30 s; the waste liquid was discarded, and the adsorption column CB3 placed back into the collection tube; [0042] (7) 600 .Math.L of the rinse solution PW was added to the adsorption column CB3 (make sure the absolute ethanol has been added before use), centrifuged at 12,000 rpm (-13,400×g) for 30 s; the waste liquid was discarded, and the adsorption column CB3 placed back into the collection tube; [0043] (8) the step 7) was repeated; [0044] (9) the adsorption column CB3 was placed back into the collection tube, centrifuged at 12,000 rpm (-13,400xg) for 2 min; and the waste liquid was discarded; the adsorption column CB3 was left at room temperature for several minutes, allowing the residual rinse solution PW to evaporate from the adsorption membrane of the adsorption column CB3, with no effect on the subsequent enzyme reaction experiments (such as enzyme digestion and PCR reaction); and [0045] (10) the adsorption column CB3 was placed into a new centrifuge tube; 50-200 .Math.L of an elution buffer TE was suspended in the middle of the adsorption membrane, allowed to stand at room temperature for 2-5 min, and centrifuged at 12,000 rpm (-13,400xg) for 2 min; and the solution was collected in the new centrifuge tube.

[0046] Note: less than 50 .Math.L of the elution buffer TE added to the adsorption membrane results in low DNA recovered yields. For larger DNA yields, after centrifugation, the solution was added back into the adsorption column CB3, allowed to stand at room temperature for 2 min, and centrifuged at 12,000 rpm (-13,400xg) for 2 min; the pH value of the elution buffer TE was an important influence on the elution efficiency; for example, the elution efficiency is reduced when the DNA product was eluted with ddH.sub.2O with a pH value of 7.0-8.5; and the DNA product was stored at -20° C. to prevent DNA degradation.

2. Restriction Enzyme Digestion

[0047] In the laboratory, five female and five male shad of known sex were used to prepare a library using the 2b-RAD method. Specifically, the restriction enzyme digestion reaction contained the components listed in Table 1 and took place at 37° C. for 45 min.

TABLE-US-00001 Component Volume (.Math.L/sample) DNA (200 ng/.Math.L) 1 10×CutSmart buffer 1.5 BsaXI restriction endonuclease (2U/.Math.L) 0.5 Pure water 12 Total volume 15

[0048] 4 .Math.L of the digested product was loaded on 1% (wt/vol) agarose gel and electrophoresed at 100 V for 10-15 min; and the bands were visualized under ultraviolet light.

3 Ligation

[0049] The components for ligation reaction was listed in Table 2; five samples were labeled as 1-5, respectively. According to Table 3, two adaptors (A and B) were added to a corresponding sample (Ten adaptors (Ada 1-10) were prepared by annealing two DNA strands, for example, Ada 1 is prepared from Ada1a and Ada1b, Ada 2 is prepared from Ada2a and Ada2b; the specific sequences of adapters were shown in Table 4); 0.8 .Math.L of each adaptor was added and ligated to the corresponding end of the digested product at 16° C. for 1 h; and the five ligated products were labeled as 1-5, respectively.

TABLE-US-00002 Component Volume (.Math.L/ sample) Digested product 10 10× T4 ligase buffer 1 10 mM adenosine triphosphate (ATP) 1 Adaptor A (5 .Math.M) 0.8 Adaptor B (5 .Math.M) 0.8 T4 DNA ligase (400 U/.Math.L) 0.5 Pure water 5.9 Total volume 20

TABLE-US-00003 Five groups of adaptors Label Adaptor A (5 .Math.M) Adaptor B (5 .Math.M) 1 Ada 1 Ada 2 2 Ada 3 Ada 4 3 Ada 5 Ada 6 4 Ada 7 Ada 8 5 Ada 9 Ada 10

TABLE-US-00004 Oligonucleotide sequences of adapters and primers Sequence (5′ - 3′) Adaptor SEQ ID NO: 5 Ada1a ACACTCTTTCCCTACACGACGCTGTTCCGATCTN SEQ ID NO: 6 Ada1b AGATCGGAACAGC SEQ ID NO: 7 Ada2a GTGACTGGAGTTCAGACGTGTGCTCTTCACGAN SEQ ID NO: 8 Ada2b TCGTGAAGAGCAC SEQ ID NO: 9 Ada3a ACACTCTTTCCCTACACGACGCTCTTCATCGNNN SEQ ID NO: 10 Ada3b CGATGAAGAGCGT SEQ ID NO: 11 Ada4a GTGACTGGAGTTCAGACGTGTGCTCTTCAGCAN SEQ ID NO: 12 Ada4b TGCTGAAGAGCAC SEQ ID NO: 13 Ada5a ACACTCTTTCCCTACACGACGCTCTTCATGCNNN SEQ ID NO: 14 Ada5b GCATGAAGAGCGT SEQ ID NO: 15 Ada6a GTGACTGGAGTTCAGACGTGTGCTCTTCAGACN SEQ ID NO: 16 Ada6b GTCTGAAGAGCAC SEQ ID NO: 17 Ada7a ACACTCTTTCCCTACACGACGCTCTTCAGTCNNN SEQ ID NO: 18 Ada7b GACTGAAGAGCGT SEQ ID NO: 19 Ada8a GTGACTGGAGTTCAGACGTGTGCTCTTCACAGN SEQ ID NO: 20 Ada8b CTGTGAAGAGCAC SEQ ID NO: 21 Ada9a ACACTCTTTCCCTACACGACGCTCTTCACTGNNN SEQ ID NO: 22 Ada9b CAGTGAAGAGCGT SEQ ID NO: 23 Ada10a GTGACTGGAGTTCAGACGTGTGCTGTTCCGATC SEQ ID NO: 24 Ada10b AGATCGGAACAGC SEQ ID NO: 25 Prim1 ACACTCTTTCCCTACACGACGCT SEQ ID NO: 26 Prim2 GTGACTGGAGTTCAGACGTGTGCT SEQ ID NO: 27 BioPrim1 (biotin)-ACACTCTTTCCCTACACGACGCT SEQ ID NO: 28 BioPrim2 (biotin)-GTGACTGGAGTTCAGACGTGTGCT SEQ ID NO: 29 I5 index primer AATGATACGGCGACCACCGAGATCTACACNNN NNNACACTCTTTCCCTACACGACGCTCTTCCGAT SEQ ID NO: 30 I7 index primer CAAGCAGAAGACGGCATACGAGATNNNNNNGT GACTGGAGTTCAGACGTGTGCTCTTCCGATCT

4. Enrichment and Purification

[0050] (1) The components for PCR reaction were listed in Table 5; referring to Table 6, the ligated products were amplified using a corresponding primer pair labeled with the same number (the specific sequences of primers were shown in Table 4);

TABLE-US-00005 Component Volume (.Math.L/ sample) Ligated product 18 5× Phusion HF buffer 10 10 Mm deoxyribonucleoside triphosphate (dNTP) 1.2 Primer A (10 .Math.M ) 0.8 Primer B (10 .Math.M ) 0.8 Phusion high-fidelity DNA polymerase (2 U/.Math. adaptor) 0.4 Pure water 18.8 Total volume 50

TABLE-US-00006 Primer pairs for five indexes Index position Primer A (10 .Math.M ) Primer B (10 .Math.M ) 1 Prim 1 BioPrim2 2 BioPrim1 BioPrim2 3 BioPrim1 BioPrim2 4 BioPrim1 BioPrim2 5 BioPrim1 Prim 2

[0051] (2) PCR cycling and reaction parameters:

TABLE-US-00007 Cycles Denaturation Annealing Elongation 1-16 98°C, 5 s 60° C., 20 s 72° C., 10 s

[0052] (3) 50 .Math.L of PCR product and 1 .Math.L of 100-bp DNA ladder (Takara, cat. no. 3427) were loaded on 8% polyacrylamide gel and electrophoresed at 400V for 35 min; [0053] (4) the 8% polyacrylamide gel was then stained with a nucleic acid stain (SYBR Safe) for 3 min; and the brightness of the target band (with a size of 100 bp) was observed; [0054] (5) an agarose slice with the band of interest was cut, placed into a 1.5 mL centrifuge tube, ground with a grinding rod; 30-40 .Math.L of pure water was added and allowed to stand at 37° C. for 30 min; [0055] (6) the mixture was centrifuged at 14000 g at room temperature for 2 min, and a PCR product was recovered; to increase the PCR product yield, 12 .Math.L of each recovered product was used as a template for use in PCR amplification; the PCR procedure was the same as in 2), and the number of cycles was reduced to 4-6; [0056] (7) the enriched products obtained in 6) was mixed and then purified with MinElute PCR purification kit; 15 .Math.L of pure water was added to elute the enriched products; the purified product was quantified with a NanoVue spectrophotometer; preferably, the concentration of the purified product was 10-30 ng/.Math.L;

[0057] (8) the enzyme digestion reaction was prepared according to Table 8 and took place at 37° C. for 30 min;

TABLE-US-00008 Component Volume (.Math.L) Purified product 10 10×CutSmart buffer 3 10 Mm adenosine triphosphate (ATP) 3 SapI restriction endonuclease (10 U/.Math.L) 0.2 Pure water 13.8 Total volume 30

[0058] (9) 30 .Math.L of the digested product obtained in 8) was added to 10 .Math.L of magnetic beads prepared in advance, and pipetted up and down at room temperature for 5 min; [0059] (10) the centrifuge tube was placed on the magnetic stand; when the digested product was clarified, the supernatant was transferred to a new centrifuge tube; 0.5.Math.L of 400 U/.Math.L T4 DNA ligase was added and allowed to react at 16° C. for 45 min; [0060] (11) the ligated product obtained in 10) was electrophoresed; an agarose slice with the band of interest (with a size of 244 bp) was cut and centrifuged at 14000 g at room temperature for 2 min, so that a pure ligated product was recovered.

5. Introduction of an Index Sequence by PCR and Pooling

[0061] According to Table 9, the index sequence was introduced by PCR and the specific primer sequences were shown in Table 4.

[0062] (1) Components of PCR reaction mixture

TABLE-US-00009 Components Volume (.Math.L) Ligated product 15 5× Phusion HF buffer 20 10 Mm deoxyribonucleoside triphosphate (dNTP) 2.4 5 .Math.M I5 index primer 2 5 .Math.M I7 index primer 2 Phusion high-fidelity DNA polymerase (2 U/.Math.L) 0.8 Pure water 59.8 Total volume 100

[0063] (2) PCR cycling and reaction parameters:

TABLE-US-00010 Cycles Denaturation Annealing Elongation 1-16 98°C, 5 s 60° C., 20 s 72° C., 10 s

[0064] (3) 4.Math.L of PCR products and 1 .Math.L of 100-bp DNA ladder were separately loaded on 1% agarose gel and electrophoresed at 135 V for 10-15 min; and the brightness of the target band (with a size of 299 bp) was observed under ultraviolet light; [0065] (4) the PCR product was purified with MinElute PCR purification kit, eluted with 15 .Math.L pure water and quantified with Qubit; preferably, the concentration of the purified product was 10-30 ng/uL; [0066] (5) if multiple libraries have been built, libraries with different Index numbers were mixed according to the amount of data for sequencing; preferably, the final concentration of the mixed library was 5-10 ng/.Math.L. [0067] (6) the mixed library was sequenced by Illumina PE platform for 100-150 bp paired-end sequencing; [0068] (7) the resulting raw data was presented in fastq format, cleaned and clustered to build a reference sequence using Ustacks software (version 1.34) in Stacks; the sequencing data was aligned to the reference sequence using SOAP software (version 2.2), and SNP calling was performed using the maximum likelihood (ML); SNP markers were identified for sex identification; a short sequence was detected in the five male American shad but not in the five female Ametaican shad; and the short sequence was the male-specific molecular marker comprising a first nucleic acid sequence of SEQ ID NO: 1.

Example 2 Elongation of a Male-Specific Molecular Marker of American Male Shad

[0069] 1. The first nucleic acid sequence obtained in Example 1 was aligned to a genome sequence (JAHTKL010000000 in NCBI) of American shad using Blast to obtain a second nucleic acid sequence of SEQ ID NO: 2.

Example 3 Primer Design and Verification of a Male-Specific Molecular Marker

[0070] The second nucleic acid sequence was used as a template sequence to design a primer pair Tag-5 comprising two short nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

[0071] Primer sequences for the male-specific molecular marker were listed in Table 11

TABLE-US-00011 Primer sequences for the male-specific molecular marker Primer name Primer sequence (5′-3′) Tag-5 F: GTTCATTAGTTCCCCTGTGCTGAC (SEQ ID NO: 31) R: TCATTATTGGGTTGATAGCAGGCT (SEQ ID NO: 32)

[0072] In the laboratory, total DNA was extracted from 20 American shads (including 10 males and 10 females) of known sex; the DNA extraction method was the same as in Example 1, followed by PCR verification; and the PCR amplification was performed under the conditions: [0073] a 20 .Math.L PCR reaction contained 10 .Math.L of 2 × Taq Plus Master Mix II (Dye Plus), 1 .Math.L of Tag-5-F, 1 .Math.L of Tag-5-R, 1 .Math.L of template DNA, and 7 .Math.L of H.sub.2O; [0074] PCR cycling and reaction parameters were detailed as follows: initial denaturation at 95° C. for 3 min, denaturation at 95° C. for 15 s, annealing at 60° C. for 30 s, elongation at 72° C. for 1 min, and final extension at 72° C. for 5 min.

[0075] Preferably, the cycle number was set to 35 cycles.

[0076] As shown in FIG. 2, the genetic sex of the 20 American shads (comprising 10 males and 10 females) was determined by using the primer pair Tag-5.

Example 4 Genetic Sex Determination in American Shad by Using the Primer Pair and Verification Thereof

[0077] 20 female shad and 20 male shad were randomly collected from four shad culture ponds, and subjected to sex verification by using the primer pair for the male-specific molecular marker; the DNA extraction method was the same as in Example 1, followed by PCR verification; the primer sequences were shown in Table 10; and the PCR amplification was performed under the conditions: [0078] a 20 .Math.L PCR reaction contained 10 .Math.L of 2 × Taq Plus Master Mix II (Dye Plus), 1 .Math.L of Tag-5-F, 1 .Math.L of Tag-5-R, 1 .Math.L of template DNA, and 7 .Math.L of H.sub.2O; [0079] PCR cycling and reaction parameters were detailed as follows: initial denaturation at 95° C. for 3 min, denaturation at 95° C. for 15 s, annealing at 60° C. for 30 s, elongation at 72° C. for 1 min, and final extension at 72° C. for 5 min.

[0080] Preferably, the cycle number was set to 35 cycles.

[0081] As shown in FIG. 3, the amplified products were electrophoresed on agarose gel; the results showed that the male-specific molecular marker of 470 bp was amplified from the male American shad but not from the female American shad; and the genetic sex of the American shad was verified using the primer pair for the male-specific molecular marker, which was consistent with the results by anatomical gonads, indicating that the disclosed method achieved an accuracy rate of 100%.

[0082] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.