AU2010228215B2

AU2010228215B2 - Genetic marker linked to genetic sex of yellowtail, sex discrimination method for yellowtail, and primer for use in sex discrimination method

Info

Publication number: AU2010228215B2
Application number: AU2010228215A
Authority: AU
Inventors: Kazuo Araki; Kanako Fuji; Keita Hattori; Masanori Okauchi; Akiyuki Ozaki; Takashi Sakamoto; Kazunori Yoshida
Original assignee: Tokyo University of Marine Science and Technology NUC
Current assignee: Tokyo University of Marine Science and Technology NUC
Priority date: 2009-03-26
Filing date: 2010-03-25
Publication date: 2014-11-20
Anticipated expiration: 2030-03-25
Also published as: JP2010226974A; JP5452047B2; WO2010110373A1; AU2010228215A1; NZ595999A

Abstract

Genomic DNA from a yellowtail is subjected to a PCR method or a nucleic acid amplification reaction method using an yellowtail-derived nucleic acid as a template, wherein the PCR method or the nucleic acid amplification reaction method is carried out using, as primers, oligonucleotides comprising the nucleotide sequences: 5'-TTTCATTGTGGCGCTCAG-3' and 5'-GGTTGTAATGTGTCCCAG-3' both of which are produced based on a gene marker for genetic linkage group (LG) 12 in an yellowtail genetic linkage map. A product of the method is subjected to a gel electrophoresis method to analyze the product. In this manner, the genetic sex of the yellowtail can be determined. This technique enables the determination of the genetic sex of an immature yellowtail with reliability without killing the yellowtail.

Description

GENETIC MARKER LINKED TO GENETIC SEX OF YELLOWTAIL, SEX DISCRIMINATION METHOD FOR YELLOWTAIL, AND PRIMER FOR USE IN SEX DISCRIMINATION METHOD 5 FIELD OF THE INVENTION The present invention relates to a genetic marker linked to the genetic sex of yellowtail, a sex discrimination method for yellowtail, and a primer for use in the sex discrimination method. BACKGROUND 10 There are nine species of yellowtails in the world and four species (Seriola quinqueradiata, Seriola lalandi, Seriola dumerili, and Seriola rivoliana) are distributed around Japan. Almost all of them are important as marine products and also play an important role as fishing targets. In Japan, yellowtail is highly valued as food fish. In Japan, there are 15 conducted both catching of natural yellowtail (approximately 70,000 tons/year) and farming (approximately 150,000 tons/year). Farming of yellowtail is being conducted particularly in western Japan and yields approximately 120 billion yen, occupying more than half of the total yield of seawater finfish farming. Yellowtail is also farmed in foreign countries such as South Korea (approximately 20 57 tons/year). In the yellowtail farming industry, the mainstream is aquaculture which consists in growing natural fry called "Mojako" (1.5-15 cm) caught as seedlings. For resource management of natural "Mojako", its catch in each prefecture is strictly limited and seedling production is also performed for release. 25 To develop the aquaculture industry in the future, it is necessary to produce yellowtail that is more disease-resistant, flavorful, and has more valuable (superior economic) traits than natural yellowtail, without relying on natural resources. What becomes important then is the breeding of yellowtail by retaining yellowtail lineages having valuable traits and utilizing genetic 30 information related to the valuable traits possessed by parent fish. For efficient mating for fish breeding, it is necessary to grow males and females so that the numbers of male and female parents are balanced. Collection of good quality eggs from a particular matured female yellowtail for intended mating is difficult because the period during which good quality eggs can be 35 collected from each yellowtail is short. On the other hand, collection of sperms 1 from a matured male yellowtail is relatively easy because spermiation is always found in every matured male yellowtail. For breeding yellowtail, therefore, it is necessary to grow more females than males. Since male yellowtail and female yellowtail have no external features 5 indicating their secondary sex characteristics (maturation), the only way to discriminate their sex is to insert a tube through their gonopore and find eggs or sperms within their bodies in the spawning season. It takes one or two years for the yellowtail to mature and produce eggs or sperms. It has been necessary therefore to grow a large number of young yellowtail with the numbers of males 10 and females uncontrolled, which has required a large cost. Since the sex discrimination mechanism of the yellowtail has not been known and there is a possibility of sex reversal (phenomenon that the sex is changed) under stress, temperature or other environmental conditions as confirmed among many fishes, it has been difficult to balance the numbers of 15 males and females. It is important therefore in the artificial seedling production and aquaculture of yellowtail to discriminate the sex of young yellowtail without killing them, and it is also necessary to clarify the sex discrimination mechanism. To solve these problems, the development of a sex management technique is 20 urgently needed. In a conventionally known method, the sex of fish is discriminated by detecting the expression pattern of the mRNA of a gene that is differently expressed between male and female (see Japanese Unexamined Patent Application Publication No. 2000-60569). 25 This method is targeted, however, to pleuronectiformes, and not to yellowtail. Since this method is only applicable to the fish in or after the sex differentiation stage (approximately 60 days after hatching) and uses genes that are genetically identical but different in expression, it cannot discriminate the sex of younger fish and can only discriminate the expressed sex and not the genetic 30 sex. Furthermore, since a certain amount of RNA should be extracted, immature fish may be killed. SUMMARY OF THE INVENTION The present invention provides an isolated genetic marker comprising: a base sequence indicated by sequence No. 1 in a sequence listing linked to a 35 genetic sex of yellowtail. 2 The problem addressed by the present invention is to provide a genetic marker linked to the genetic sex of yellowtail and thus available to reliably discriminate the genetic sex of immature yellowtail without killing them, a sex discriminating method for yellowtail using this genetic marker, and a primer for 5 use in this sex discrimination method. The applicant created a F1 population by mating matured male and female yellowtail that were caught from the ocean and reared in the Goto Station, National 2a Center for Stock Enhancement, Fisheries Research Agency. Linkage analysis was performed on the created F1 population and their parents using DNA markers and its results were compared to the yellowtail genetic linkage map that had already been found by the inventor et al. (Ohara et al., 2005). It was found that the genetic marker formed of the base sequence of sequence No. 1 in the sequence listing of genetic linkage group (LG) 12 on the yellowtail genetic linkage map is linked to the sex determinant locus. The applicant named this genetic marker Sequ21. It was also found that the sex determinant factor of the yellowtail is determined by a genetic factor and is of the ZW type, meaning that the sex is determined by gene/genes inherited from the female parent. The genetic marker of the present invention is formed of the base sequence of sequence No. 1 in the sequence listing linked to the genetic sex of the yellowtail. The yellowtail includes here any one of the species Seriola quinqueradiata, Seriola lalandi, Seriola dumerili, and Seriola rivoliana. The sex discrimination method for yellowtail uses a nucleic acid amplification reaction method using a yellowtail-derived nucleic acid as a template by using a primer produced from the genetic marker formed of the base sequence of sequence No. 1 in the sequence listing, and discriminates the genetic sex of yellowtail by analyzing the amplified product. Nucleic acid amplification reaction methods include PCR method and LAMP method. The PCR method is the most popular method. The products obtained by the PCR method are electrophoresed in a gel and analyzed to discriminate the genetic sex of the yellowtail. The primer used in the yellowtail sex discrimination method of the present invention is preferably a primer containing the oligonucleotide part of 10-50 bases created from the genetic marker formed of the base sequence of sequence No. 1 in the sequence listing. For example, the primer includes the oligonucleotide part formed of at least ten bases in 5'-TTTCATTGTGGCGCTCAG-3' (sequence No. 2 in the sequence listing) and the oligonucleotide part formed of at least ten bases in 5'-GGTTGTAATGTGTCCCAG-3' (sequence No. 3 in the sequence listing). According to the present invention, since the genetic sex of immature yellowtail can be discriminated without killing them, the numbers of male and female parents can be adjusted at the stage when they are still immature. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows gel electrophoresis images of PCR products produced by using a primer in Example 1; 3 FIG. 2 schematically shows the gel electrophoresis images of the PCR products produced by using the primer in Example 1; FIG. 3 shows gel electrophoresis images of PCR products produced by using a primer in Example 2; and FIG. 4 schematically shows the gel electrophoresis images of the PCR products produced by using the primer in Example 2. BEST MODE FOR CARRYING OUT THE INVENTION The genetic sex of the yellowtail is discriminated by using a genetic marker formed of a base sequence of sequence No. 1 in the sequence listing located on genetic linkage group LG12. Examples of the present invention will now be described in detail. The yellowtails used in the Examples described below are called in Japan by the Japanese standard name "Buri" and its scientific name is Seriola quinqueradiata. Analyzed family 1: Matured male yellowtail and female yellowtail reared in Goto Station, National Center for Stock Enhancement, Fisheries Research Agency (which were caught as fry from the ocean and preserved by a farmer for two years and then reared for one year in the Goto Station) were mated and the fertilized eggs of Fl population obtained were transferred to a 100 L water tank and reared in 100 L seawater at a constant water temperature of 20*C until they hatched. One day after hatching (one-day old), the fry were transferred to two 500 L water tanks and reared in flowing water at 22*C. From the third day to 30th day, they were fed with Rotatoria, from the 21st day to 39th day, fed with Artemia in addition to Rotatoria, and from 32nd day, fed with commercially available formula feed. From 41st day to 107th day, the fry were reared in a pen net installed in a 60 ton land water tank. On and after 108th day, the fish were reared in a net cage floating on the sea surface. As a result, 64 fish of F1 population were obtained. A half year after the rearing started, the bellies of the 64 fish of F1 population were dissected to check their gonads to discriminate their sex (phenotype). The male to female ratio of the 64 fish was 28 males to 36 females. Analyzed family 2: The same male used in Analyzed family 1 and a different female were mated and 55 fry of F1 population were obtained. They were reared in the same way as for Analyzed family 1. Similarly to Analyzed family 1, a half year after the rearing started, the bellies 4 of the 55 fish of F1 population were dissected and their gonads were checked to discriminate their sex. The male to female ratio of the 55 fish was 24 males to 31 females. Example 1: A PCR method using a PCR primer related to Sequ21 was applied to all the 64 yellowtail of F1 population in Analyzed family 1 as follows and the obtained PCR products were electrophoresed in a gel to check the marker type (genotype). First, DNA was extracted from each yellowtail in the following manner: One centimeter square of tail fin was collected, 600 l digestive solution containing 100 mM NaCl, 20 mM Tris-HCl (pH8.0), 100 mM EDTA, 1.0% SDS, and 100 jig/ml proteinase K was added thereto, and was kept still overnight at 37 0 C. Then, phenol/chloroform (1:1) extraction was performed once, and ethanol precipitation was used to separate chromosomal DNA. The collected DNA was washed with 70% ethanol, dried, and dissolved into 50 g1 TE solution (0.01 M Tris-HCl pH7.4, and 2.5 mM EDTA pH8.0). Then, a pair of primers F;5'-TTTCATTGTGGCGCTCAG-3' (sequence No. 2 in sequence listing) and R;5'-GGTTGTAATGTGTCCCAG-3' (sequence No. 3 in sequence listing) were synthesized, and a PCR method was conducted using a 13 p1 solution containing 0.2 pmol F primer, [y.3 3 P] ATP, 0.32 pmol R primer labeled with T4 polynucleotide kinase, 0.2 mM each dNTP, 2.0 mM MgCl2, 1% BSA, 0.02 U Ex Taq DNA polymerase (Takara Bio), and 50 ng template DNA, using a Gene Amp PCR system 9700 thermal cycler (PerkinElmer). Table 1 lists the PCR reaction composition (for one sample) using these primers, and Table 2 lists the liquid composition for RI labeling of R primer (for 100 samples). Table 1 10 x Ex Taq m buffer (TAKARA) 1.3 [jll 10 [pmol/il] F primer 0.26 [jll 10 [pmol/jl] RI labeled R primer 0.65 [ill BSA 0.13 [pll 50 [ng/pl] template DNA (for each yellowtail) 1.0 [jll 2.5 [mM] dNTP Mixture (TAKARA) 1.04 [j1] 5 [U/jill Recombinant Taq DNA polymerase (TAKARA) 0.052 [jill H20 8.568 [pl] Total 13 [pl] 5 Table 2 10 x protruding end kinase buffer (TOYOBO) 6.5 [g1] 10 [pmol/pl] R primer 2.08 [pl] 10 [U/ill T4 polynucleotide kinase (TOYOBO) 1.5 [ill [y-33P] ATP 3.3 [ptl] H20 51.62 [jll Total 65.0 [jll The conditions for PCR were two minutes at 95*C - (30 seconds at 95*C - one minute at 52*C - one minute at 72*C) x 35 cycles - (three minutes at 72*C) x one cycle. After reaction, the solution was mixed well with the same amount of loading dye (95% formamide, 10 mM EDTA, 0.05% bromophenol blue, and xylene cyanol), then the PCR product was thermally denatured into a single-stranded DNA and electrophoresed in a 6% denatured polyacrylamide gel. Subsequently, the gel was dried for one hour, and exposed to an imaging plate (IP) for 3-12 hours. This IP with a radiographic image thereon was read by a Bio-imaging Analyzer (BAS1000, Fuji Photo Films) and visualized by a computer. These results are shown in FIGs. 1 and 2. As can be seen from FIGs. 1 and 2, the female parent holds the bands of 168 bp and 152 bp. Thirty five out of the 36 female offsprings retain the band of 168 bp inherited from their female parent, while 27 out of the 28 male offsprings retain the band of 152 bp inherited from their female parent. Accordingly, in this lineage, the sex can be discriminated by checking the types of the bands inherited from the female parent and amplified by the primer of the present invention. The probability of linkage between the sex determinant trait (female sex) and these bands is indicated by a Lod score. A Lod score 3 or more indicates a risk rate 1/103 or lower. The situation with a risk rate 1/103 or lower is referred to as being linked (Lathrop et al., 1984). Contradiction between the bands of 168/152 bp and the sex determinant trait (female sex) was found in two out of the 64 offsprings with a Lod score being 2log(2/64) + (64-2)log(1-2/64) - 641og0.5, indicating a reliable linkage with a risk rate of 1/101 5' 40. The marker of sequence No. 1 in the sequence listing is therefore linked to the genetic sex of the yellowtail. Accordingly, it can be said that, in Example 1, the yellowtail having in the PCR 6 product the band of 152 bp inherited from the female parent are male, while the yellowtail having the band of 168 bp inherited from the female parent are female. Example 2: The 55 offsprings of F1 population obtained from the Analyzed family 2 were subjected to the same PCR method as in Example 1. FIGs. 3 and 4 show gel electrophoresis images of the PCR products in Example 2. As can be seen from FIGs. 3 and 4, the female parent has bands of 174 bp and 162 bp. Thirty out of the 31 female offsprings had the band of 162 bp inherited from the female parent, while all of the 24 male offsprings had the band of 174 bp inherited from the female parent. In this case, the Lod score was 14.39, indicating a reliable linkage with a risk rate of 1/101 4 . 9 . The marker of sequence No. 1 in the sequence listing is accordingly linked to the genetic sex of the yellowtail. It can be said therefore that, in Example 2, the yellowtail having the band of 174 inherited from the female parent in the PCR product are male, while those having the band of 162 bp inherited from the female parent are female. In the present invention, the genetic information transferred from parents to offsprings is analyzed using a DNA marker. As proposed in the present invention, the sex of yellowtail can be discriminated for all the yellowtail species by analyzing the genetic information transfer by using the PCR primer related to Sequ21. The point is that the genetic information transfer from parents to offsprings can be analyzed and thus the sex of offsprings can be discriminated. In the Examples 1 and 2 described above, when the genetic information transfer from parents to offsprings was analyzed, the bands linked to the sex determinant traits (female sex) had different values. This is due to the DNA marker used in the development of the method of the present invention, because the PCR primers of sequence Nos. 2 and 3 in the sequence listing were designed including CA repeat sequences (microsatellite). The PCR primer including a microsatellite can locate one genetic locus by analyzing the transferred genetic information and detect many bands among individuals due to its polymorphism (DNA Markers: Protocols, Applications, and Overview, edited by Gustavo Caetano-Anolles and Peter M. Gresshoff) differing among analyzed families. The marker locus located by Sequ21 is the place where the sex determinant gene exists and thus enables the sex discrimination. The sizes of the bands indicated in these Examples are not limited to the above values but may differ depending on the lineages. 7 The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in 5 the field relevant to the present invention as it existed before the priority date of each claim of this application. Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but 10 not precluding the presence of one or more other features, integers, steps or components, or group thereof. 7a

Claims

1. An isolated genetic marker comprising: a base sequence indicated by sequence No. 1 in a sequence listing linked to a genetic sex of yellowtail. 5

2. The genetic marker according to claim 1, wherein the yellowtail is any one of species Seriola quinqueradiata, Seriola 1alandi, Seriola dumerili, and Seriola rivoliana. 10

3. A sex discrimination method for yellowtail, the method comprising: applying a nucleic acid amplification reaction method using a nucleic acid derived from the yellowtail as a template by using a primer produced from the genetic marker according to claim 1, and analyzing the amplified product to discriminate the genetic sex of the yellowtail. 15

4. A sex discrimination method for yellowtail, the method comprising: applying a polymerase chain reaction (PCR) method to a genome DNA of the yellowtail by using a primer produced from the genetic marker according to claim 1, and electrophoresing an obtained product in a gel and analyzing an 20 electrophoresis image to discriminate the genetic sex of the yellowtail.

5. A primer pair for use in a sex discrimination method for yellowtail, the primer comprising: an oligonucleotide part containing at least ten bases in 5' TTTCATTGTGGCGCTCAG-3' (sequence No. 2 in sequence listing) produced from 25 the genetic marker according to claim 1 and an oligonucleotide part containing at least ten bases in 5'-GGTTGTAATGTGTCCCAG-3' (sequence No. 3 in sequence listing).

6. A sex discrimination method for yellowtail, the method comprising: 30 applying a polymerase chain reaction (PCR) method to a genome DNA of the yellowtail by using the primer according to claim 5, electrophoresing an obtained product in a gel, and analyzing an electrophoresis image to discriminate the genetic sex of the yellowtail. 8

7. A genetic marker according to claim 1, substantially as herein described with reference to any of the Examples and/or Figures. 9