WO2000017341A1 - MICROSATELLITES DE $i(MYRTACEAE) - Google Patents

MICROSATELLITES DE $i(MYRTACEAE) Download PDF

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WO2000017341A1
WO2000017341A1 PCT/AU1999/000820 AU9900820W WO0017341A1 WO 2000017341 A1 WO2000017341 A1 WO 2000017341A1 AU 9900820 W AU9900820 W AU 9900820W WO 0017341 A1 WO0017341 A1 WO 0017341A1
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Prior art keywords
microsatellite
myrtaceae
isolated
rvs
fwd
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PCT/AU1999/000820
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English (en)
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Maurizio Rossetto
Anne Mclauchlan
Fiona Carole Lesley Harriss
Robert James Henry
Peter Raymond Baverstock
Leonard Slade Lee
Tina Louise Maguire
Keith Joseph Edwards
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Business And Research Management Pty. Ltd.
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Priority claimed from AUPP6099A external-priority patent/AUPP609998A0/en
Priority claimed from AUPP8718A external-priority patent/AUPP871899A0/en
Application filed by Business And Research Management Pty. Ltd. filed Critical Business And Research Management Pty. Ltd.
Priority to AU61837/99A priority Critical patent/AU6183799A/en
Publication of WO2000017341A1 publication Critical patent/WO2000017341A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • TITLE "MYRTACEAE MICROSATELLITES" FIELD OF THE INVENTION
  • Microsatellites or simple sequence repeats (SSRs), characteristically have nucleotide sequences which are variably repeated, and are widely interspersed throughout the genome.
  • SSRs simple sequence repeats
  • microsatellite includes at least a "microsatellite repeat sequence”, which for the purposes of this specification will be represented in the following general manner: (nucleotide sequence)., where subscripted n specifies the number of consecutive repeats. For example, (GCT) 8 is a "perfect" microsatellite repeat consisting of eight consecutive GCT trinucleotides.
  • microsatellite sequences may have more than one microsatellite repeat sequence and may also include non-repetitive nucleotides, e.g. (GCT) 2 (ACT)(ACC)(GC) 5 .
  • microsatellites are highly polymorphic. That is, each microsatellite "locus” may have a number of "ailelic” forms which vary largely according to the value of n. A major cause of this variation is genetic recombination, the rate of genetic exchange at mammalian microsatellite loci being very high ( ⁇ 10 "4 per kilobase genomic DNA; Lewin B, 1994, in Genes V, Oxford University
  • Polymorphism is a feature of microsatellites which contributes greatly to their usefulness in genetic mapping or "genotyping".
  • Microsatellites were initially described in humans (Litt & Luty, 1989, Am. J. Hum. Genet. 44 397), and subsequently in other mammalian species such as mice (Love et al., 1990, Nucl. Acids Res. 21 1111 ), pigs (Johansson et al., 1992, J. Hered. 83 196) and cattle (Kemp et al, 1993, Animal Genet. 24 363). Similarly, microsatellites have been identified in plants including rice (Wu & Tanksley, 1993, Mol. Gen. Genet. 241 225), barley (Saghai Maroof et al., 1994, Proc. Natl. Acad. Sci.
  • microsatellites which confer this usefulness include:
  • microsatellite-based genotyping has proven superior to other techniques such as Restriction Fragment Length Polymorphism (RFLP) and Random Amplification of Polymorphic DNA (RAPD; Powell et al., 1996b, Mol. Breed. 2225).
  • RFLP Restriction Fragment Length Polymorphism
  • RAPD Random Amplification of Polymorphic DNA
  • microsatellite-based genotyping has proven to be of great value to selective breeding practice (Gupta ef al., 1996, Curr. Sci. 70 45; Powell ef al., 1996a, Trends Plant Sci. 7 215).
  • the highly polymorphic nature of microsatellites has made them particularly useful in distinguishing between closely related cross-bred plants. Consequently, microsatellites have become the preferred markers for genotyping crop species which display minimal intra-species polymorphism (Roder et al., 1995, supra).
  • a plant species which is rapidly increasing in economic importance is Melaleuca alternifolia, commonly known as tea tree and a member of the Myrtaceae family of plants.
  • tea tree oil This species is the principal source of "tea tree oil", which oil is highly valued for its broad-spectrum germicidal properties and hence usefulness in the pharmaceutical and cosmetic industries.
  • tea tree oil production has relied on harvested material from natural Melaleuca populations, but the recent expansion of interest in tea tree oil has resulted in a shift toward harvesting leaves from Melaleuca plantations.
  • Many of the existing plantations have been established from a limited genetic pool and cultivation and breeding practices have been based largely on anecdotal knowledge. Recent evidence suggests that there is actually great genetic variation within the Melaleuca alternifolia species (Butcher ef a/., 1992, Aust. J. Bot. 40 365).
  • microsatellite enrichment techniques have been developed in which microsatellite sequences can be enriched from preparations of plant genomic DNA. This development has offered new opportunities for large scale microsatellite characterization in species for which little or no genomic sequence information is available (Edwards ef al., 1996, BioTechniques 20 758, which is herein incorporated by reference; Powell et al., 1996a, supra).
  • hybridization refers to the formation of complementary base pairs between respective nucleic acid strands, under appropriate conditions of tonicity, temperature and the presence or absence of denaturants such as formamide or sodium dodecyl sulphate (SDS).
  • SDS sodium dodecyl sulphate
  • the invention resides in a method of isolating a microsatellite, said method comprising the steps of
  • step (i) producing a nucleic acid extract from a Myrtaceae plant; (ii) combining the nucleic acid extract produced in step (i) with one or more immobilized single-stranded oligonucleotides under conditions which allow hybridization between one or more nucleic acids present in said extract and said immobilized single- stranded oligonucleotides, each said single-stranded oligonucleotide having a consensus microsatellite repeat sequence or a nucleotide sequence complementary thereto;
  • the one or more immobilized oligonucleotides comprise a repeat sequence selected from the group consisting of: (CT) 15 ;
  • CA 20 ; (ACT) 14 ; (AGA) 14 ; (CAA) 14 ; (CTA) 14 ; (CTT) 14 ; (CTG) 10 ; (CAG) 10 ;
  • an isolated Myrtaceae microsatellite is provided.
  • the isolated Myrtaceae microsatellite comprises a microsatellite repeat sequence selected from the group consisting of the sequences shown in column 2 of Table 1.
  • the isolated Myrtaceae microsatellite comprises a nucleotide sequence selected from the group consisting of the sequences shown in FIGS 1-46.
  • the present invention resides in a Myrtaceae microsatellite primer.
  • said primer comprises a nucleotide sequence selected from the group of nucleotide sequences shown in column 4 of Table 1.
  • a method of amplifying a microsatellite which method includes the steps of:- (i) isolating DNA from a Myrtaceae plant; and (ii) using a nucleic acid sequence amplification technique together with one or more Myrtaceae microsatellite primers to amplify one or more microsatellites from the DNA isolated in (i).
  • each said primer comprises a respective nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in column 4 of Table 1.
  • primers of the invention together with the microsatellite amplification method according to the fourth aspect, for example, in genotyping Myrtaceae plants.
  • C. salignus Callistemon salignus
  • E. cloeziana Eucalyptus cloeziana
  • E. acmenoides Eucalyptus acmenoides
  • citriodora Backhousia citriodora
  • N number of individuals in the population tested
  • A number of alleles present in the population tested
  • FIG. 1 Microsatellite nucleotide sequence comparison between M. alternifolia and other Myrtaceae species, (a) nucleotide sequence of scu023TT microsatellite locus PCR amplified from all species tested; (b) nucleotide sequence of scu004TT locus PCR amplified from the three Myrtaceae species listed; (c) nucleotide sequence comparison of the scu082TT locus PCR amplified from M. alt. and fast (F) and slow (S) alleles of the scu082TT locus PCR amplified from C. sal. Species abbreviations are:
  • FIG. 2 scuOOITT isolated microsatellite sequence including flanking region sequences.
  • FIG. 3 SCU003TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 4 scu004TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 5 scuOO ⁇ TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 6 scuOIOTT isolated microsatellite sequence including flanking region sequences.
  • FIG. 7 scuOHTT isolated microsatellite sequence including flanking region sequences.
  • FIG. 8 SCU013TT isolated microsatellite sequence including flanking region sequences.
  • SCU015TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 11 scu016TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 12 SCU019TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 13 scu021TT isolated microsatellite sequence including flanking region sequences.
  • SCU023TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 15 SCU025TT isolated microsatellite sequence including flanking region sequences.
  • FIG.16 scu031TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 17 scu031TT isolated microsatellite sequence including flanking region sequences.
  • SCU036TT isolated microsatellite sequence including flanking region sequences.
  • FIG.18 scu037TT isolated microsatellite sequence including flanking region sequences.
  • SCU039TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 20 SCU041TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 21 scu044TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 22 scu044TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 23 scu049TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 24 is a diagrammatic representation of SCU048TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 23 scu049TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 24 is a diagrammatic representation of SCU048TT isolated microsatellite sequence including flanking region sequences.
  • SCU051TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 25 scu056TT isolated microsatellite sequence including flanking region sequences.
  • SCU062TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 27 SCU063TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 29 SCU064TT isolated microsatellite sequence including flanking region sequences.
  • SCU066TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 30 scu067TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 31 scu068TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 32 scu069TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 33 scu069TT isolated microsatellite sequence including flanking region sequences.
  • SCU070TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 34 SCU072TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 36 scu074TT isolated microsatellite sequence including flanking region sequences.
  • SCU075TT isolated microsatellite sequence including flanking region sequences.
  • SCU077TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 39 scu078TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 42 scu081TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 43 SCU082TT isolated microsatellite sequence including flanking region sequences.
  • SCU083TT isolated microsatellite sequence including flanking region sequences.
  • SCU084TT isolated microsatellite sequence including flanking region sequences.
  • FIG. 46 SCU085TT isolated microsatellite sequence including flanking region sequences.
  • the present invention is predicated, at least in part, on the present inventors' discovery of an improved and efficient method of microsatellite isolation.
  • isolated refers to being substantially free of accompanying material usually present in a natural state (e.g. free of other cellular or genomic components).
  • an "isolated microsatellite” is an isolated nucleic acid which includes at least a microsatellite repeat sequence, which although corresponding to a microsatellite present in the Myrtaceae genome, has been removed from its genomic environment. It is also noted that an isolated microsatellite of the invention may include non-repetitive nudeotides and "flanking sequences" located 5' and 3' of the microsatellite repeat sequence.
  • the Myrtaceae plant DNA is genomic DNA or cDNA.
  • the DNA is genomic DNA.
  • cDNA reverse transcribed from isolated RNA is also contemplated, which process is well understood in the art.
  • microsatellites are more frequently present in non-coding regions of the genome, but this does not preclude isolation of microsatellites which may be present in transcribed regions, such as represented in mRNA and cDNA derived therefrom.
  • the nucleic acid extract may be obtained from any individual plant or group of plants within the Myrtaceae family of plants.
  • the extract is obtained from Melaleuca alternifolia.
  • Myrtaceae genomic DNA is subjected to restriction endonuclease digestion prior to PCR amplification. Furthermore, it is preferred that following restriction endonuclease digestion, adaptors are ligated to each "digestion product", the adaptors having a sequence complementary to primers used in PCR amplification. This assists PCR amplification in the absence of genomic sequence information, and also assists ligation of enriched microsatellites into a plasmid vector for nucleotide sequencing. A preferred method will be described in detail hereinafter.
  • microsatellite repeat sequences used to design the immobilized oligonucleotides used in step (ii). It will be understood by the skilled person that due to recent progress in elucidating microsatellite repeat sequences in a variety of organisms, certain predictions can be made as to which microsatellite repeat sequences may be present in a particular genome. For example, (AT) n is the most commonly encountered microsatellite repeat sequence in plants (Powell ef al., 1996a, supra), although the present inventors actually excluded this sequence from the preferred method of the invention due to it being a palindromic sequence.
  • the immobilized oligonucleotide has a repeat sequence selected from the group consisting of: (CT) 15 ; (CA) 20 ; (ACT) 14 ; (AGA) 14 ; (CAA) 14 ; (CTA) 14 ; (CTT) 14 ; (CTG) 10 ; (CAG) 10 ; (GAC) 14 ; (AGC) 14 ; (CAT) 14 ; and (ACA) 14 .
  • step (iii) Another factor which contributed to maximizing microsatellite isolation according to the present invention was the wash conditions employed by the present inventors at step (iii).
  • the wash conditions specified in step (iii) were surprisingly found to significantly optimize the efficiency of enriching microsatellites from Myrtaceae.
  • hybridized nucleic acids are eluted from the immobilized oligonucleotides, and thereby constitute a pool of isolated nucleic acids, which are eventually subjected to nucleotide sequencing to determine which contain a microsatellite repeat sequence.
  • the isolated nucleic acids are again subjected to hybridization and washing steps (ii) and (iii) in order to provide further microsatellite enrichment prior to elution at step (iv) and subsequent sequencing.
  • hybridize and “hybridization” refers to the formation of base pairs between antiparallel strands of DNA and/or RNA.
  • Hybridization can include formation of DNA-DNA, RNA-RNA or DNA-RNA hybrids.
  • DNA A pairs with T and G pairs with C; in RNA, A pairs with U and G pairs with C, wherein A and G are purines and C, T and U are pyrimidines.
  • base-pairing is influenced by factors such as temperature, ionic strength, the presence or absence of certain organic solvents and/or detergents during hybridization and the duration of hybridization.
  • the enriched microsatellites are eluted following either one or two rounds of hybridization and washing, it is preferable that the enriched microsatellites are PCR amplified, the amplified products preferably being size fractionated and ligated into a plasmid vector advantageous for nucleotide sequencing (such as pUC19 or pBluescript based vectors).
  • a plasmid vector advantageous for nucleotide sequencing (such as pUC19 or pBluescript based vectors).
  • a preferred method of preparing enriched microsatellites in a form suitable for nucleotide sequencing is provided hereinafter. It will be appreciated that nucleotide sequencing serves two purposes: -
  • a "primer” comprises a sequence of nudeotides, usually at least 10 but less than 30 contiguous nudeotides, which is capable of annealing to a template nucleic acid under defined conditions of ionic strength and temperature, and which can be extended in a template-dependent fashion by the action of a suitable nucleic acid polymerase.
  • Such polymerases include Ta ⁇ f polymerase, SequenaseTM and RNA-dependent DNA polymerase.
  • Primers may be labeled such as with radionuclides, biotin, phosphorothiorates, fluorochromes such as
  • Labeling is typically employed in order to assist detection of the products of amplification reactions.
  • primers of the invention are suitably designed according to flanking sequences elucidated as a result of isolating Myrtaceae microsatellites. It will be understood by the skilled person that the flanking sequences are likely to be considerably less polymorphic than microsatellite sequences per se, and therefore may be conserved throughout the Myrtaceae family. It will therefore be appreciated that the primers of the invention are particularly suitable for use in amplifying microsatellites from nucleic acid extracts obtained from Myrtaceae plants, such as according to the fourth aspect of the invention.
  • a particular application of the primers of the invention is in genotyping Myrtaceae plants.
  • This method is applicable to comparative analysis of individual Myrtaceae plants, or groups of plants.
  • individual plants or groups of plants are members of the Myrtaceae family, genera within the Myrtaceae family such as Eucalyptus, Melaleuca or Leptospermum, or particular species such as Melaleuca alternifolia.
  • the method may be useful for both inter-species and intra-species genotyping applications.
  • genotyping methods of the invention is analysis of genetic diversity within regionally-defined populations of Melaleuca alternifolia.
  • the preferred nucleic acid sequence amplification technique is polymerase chain reaction (PCR), as will be described hereinafter.
  • amplification techniques which may be useful include rolling circle amplification (RCA; for example as described in WO 97/19193 which is herein incorporated by reference); strand displacement amplification (SDA; for example as described in US patent 5455166 which is herein incorporated by reference); ligase chain amplification (LCA); nucleic acid sequence based amplification (NASBA, for example as described in Sooknanan et al., 1994, Biotechniques 17 1077 which is herein incorporated by reference); and Q- ⁇ replicase amplification (for example as described in Tyagi ef a/., 1996, Proc. Natl. Acad. Sci USA 93 5395 which is herein incorporated by reference).
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • LCA ligase chain amplification
  • NASBA nucleic acid sequence based
  • the Myrtaceae plant DNA is genomic DNA or cDNA.
  • the DNA is genomic DNA.
  • cDNA reverse transcribed from isolated RNA is also contemplated, which process is well understood in the art (reference is made, for example, to Chapter 3 of PLANT MOLECULAR BIOLOGY: A Laboratory Manual. Ed. M.S. Clark. Springer- Verlag, 1996).
  • microsatellites are more frequently present in non-coding regions of the genome, but this does not preclude amplification of microsatellites which may be present in transcribed regions, such as represented in mRNA and cDNA derived therefrom.
  • capillary electrophoresis is used to obtain measurements of product size by comparison with known standards.
  • FIG. 1 includes nucleotide sequences of microsatellites amplified by the same sets of primers from a number of different Myrtaceae species.
  • primers of the invention are not limited to those set forth in column 4 of Table 1.
  • Other primers may be designed according to the flanking regions as shown, for example, in FIGS 1-46, or by using the microsatellite amplification method of the invention to amplify microsatellites, and design primers based on flanking regions present in the amplified microsatellites (such as was used to produce the sequences of FIG. 1 ).
  • primer variants amplification methods and Myrtaceae genotyping methods which utilize such variants are also contemplated.
  • variants primers having base deletions, additions or substitutions which do not alter the ability of the primer to be useful in amplifying microsatellites.
  • primer sequences can be manipulated in terms of such variables as sequence length and base composition, without substantially affecting nucleic acid sequence amplification reactions which use such variants.
  • nucleic acid sequence amplification reaction conditions such as annealing temperature and salt concentration can be manipulated to accommodate such variants.
  • Synthetic oligonucleotides were constructed having the following nucleotide sequences which are complementary to consensus microsatellite repeat sequences: (CT) 15 ; (CA) 20 ; (ACT) 14 ; (AGA) 14 ; (CAA) 14 ;
  • CTA 14 ; (CTT) 14 ; (CTG) 10 ; (CAG) 10 ; (GAC) 14 ; (AGC) 14 ; (CAT) 14 ; and
  • genomic DNA was partially-digested with 5-20 Units Rsa i restriction endonuclease for 2 hours at 37°C in a total volume of 50 ⁇ L.
  • PCR was performed using a Perkin-Elmer 9600 Thermal Cycler.
  • the 50.4 ⁇ L reaction volume was made up as follows: 1 ⁇ L ligated genomic DNA; 5 ⁇ L 10 x PCR Buffer; 2 ⁇ L (200 ng) 21 mer primer; 8 ⁇ L dNTPs (1.25 mM stock) and 34 ⁇ L distilled water. Cycling parameters were: 20 cycles of 94°C for 15 seconds; 60°C for 60 seconds; 72°C for 3 minutes.
  • fragments were isolated by phenol hloroform extraction and ethanol precipitation, dried and resuspended in 25 ⁇ l distilled water.
  • the isolated PCR fragments were denatured at 95°C for 5 minutes and then added to 500 ⁇ L hybridization buffer containing 1 ⁇ g of the 21 mer oligonucleotide together with the membrane-immobilized oligonucleotides of section 1.1. Hybridization was then carried out for -18-48 hours at 50°C. Washing involved 5 x 5 minute washes in 2.0 x SSC/0.01 % SDS at 65°C, followed by 3 x 5 minute washes in 0.5 x SSC/0.01% SDS at 65°C. Hybridized DNA was then eluted in 200 ⁇ L distilled water by boiling for 5 minutes. The eluted DNA was then retrieved by ethanol precipitation, dried and resuspended in 25 ⁇ L distilled water.
  • further enrichment was performed by repeating the hybridization with immobilized oligonucleotides, followed by washing and elution. In either case, the eluted enriched microsatellites were subjected to an additional PCR amplification step. In such cases, each PCR was performed essentially as described in section 1.4, except that the number of amplification cycles was 25.
  • the enriched DNA (25 ⁇ L) was partially-digested with 30 Units Mlu I, 15 ⁇ L AP buffer, and 32 ⁇ L distilled water for 2 hours at 37°C.
  • the digested DNA was then isolated by passing the digest (75 ⁇ L) through a Pharmacia s-300 spin column, followed by ethanol precipitation and resuspension in 20 ⁇ L distilled water.
  • the isolated digested DNA was then ligated into the vector pUC19 which had been digested with BssHII restriction endonuclease and dephosphorylated.
  • the ligation reaction was as follows: 1.0 ⁇ L digested and dephosphorylated pUC19; 1.0 ⁇ L digested DNA; 2.0 ⁇ L 10 x AP buffer; 2.0 ⁇ L 10 mM ATP; 0.5 ⁇ L T4 DNA ligase; 0.5 ⁇ L Mlu I restriction endonuclease and 13 ⁇ L distilled water incubated at room temperature for 16 hours.
  • FIGS. 2-46 provide examples of complete microsatellite sequences inclusive of flanking sequences. It should be noted that where repeat sequences differ between column 2 of Table 1 and FIGS. 2-46, the skilled person will be aware that the repeat sequences shown in Table 1 are correct. The purpose of FIGS. 2-46 is primarily to give examples of flanking region sequences.
  • washing procedure specified in Edwards ef al., 1996, supra detracted from enrichment efficiency. According to Edwards et al., 1996, supra, washing including twenty washes each of five minutes duration in 0.5 x SSC at
  • the present inventors surprisingly found that by including 0.01% SDS and reducing the total number of washes to eight, and by virtue of the particular consensus microsatellite sequences used in designing the immobilized oligonucleotides, a marked improvement in enrichment efficiency was achieved.
  • Table 2 also provides a quantitative breakdown of the different classes of microsatellite repeat sequences obtained. Of the total microsatellite repeat sequences identified, 76% were unique (i.e. were not duplicated).
  • Example 2 Interspecies genotyping using Myrtaceae microsatellite markers
  • Oligonucleotide primers were designed according to nucleotide sequences flanking the microsatellite repeat sequences as set forth in FIG. 2.
  • the nucleotide sequences of the primers are shown in Table 1 column 4.
  • the primers were designed for trinucleotide repeats where n>8, and dinucleotide repeats where ⁇ >12, using specialized computer software (MacVector 6.0).
  • the general characteristics of the primers were that size was kept between 18 and 25 bases and that G+C content was >45%.
  • the primers were tested for their ability to amplify the genomic DNA sample from which the primer sequences were derived, and for their ability to amplify DNA samples from five individual plants. 2.2.
  • Cycling parameters were: 94°C for 10 minutes, then 30 cycles of 94°C for 0.5 minutes; optimal annealing temperature according to Table 1 column 5 for 0.5 minutes; 72°C for 1 minute. A further elongation step at 72°C was performed for 5 minutes following which samples were cooled to 4°C.
  • Amplification products were resolved on either 20 cm x 20 cm 10% polyacrylamide/0.5 x TBE gels run at 250 V for 3.5 hours and visualized following silver staining (Silver Staining Kit; Promega), or 3% agarose gels stained with ethidium bromide.
  • a 50 bp DNA ladder was used a size marker. The distance migrated by each marker was directly measured from the gels prior to photography of the gels.
  • a control sample having the DNA used for the enrichment process was used to check that the PCR fragments were of the expected size.
  • primers were labeled with ABI fluorescent dyes (TET, HEX and FAM) to allow visualization of PCR fragments following separation by capillary electrophoresis on an ABI 310 sequencer using dedicated software (Genescan or Genotyper). This latter method was used in generating the data in Table 2.
  • ABI fluorescent dyes TAT, HEX and FAM
  • TAT, HEX and FAM ABI fluorescent dyes
  • FAM FAM
  • Clean amplification products were purified using the Promega "Wizard” kit. Amplification products which contained non-specific bands required additional purification in order to obtain a clean template for DNA sequencing. In such cases, the target band was excised from the agarose gel to provide a template for further amplification. Further amplification products were then purified using the Promega "Wizard” kit. Sequencing reactions were performed using the ABI Prism Dye terminator cycle sequencing kit (Perkin Elmer) according to the manufacturer's instructions. Visualization of sequence data was carried out on an ABI 377 sequencer (Australian Genome Research Facility, Brisbane). Results
  • microsatellites provided sequences which were potentially useful for genotyping by virtue of their containing either dinudeotide repeats where n>12, or trinucleotide repeats where n>8. However, of these sequences, 162 (28.9%) did not have sufficient flanking sequence to permit design of primer pairs. From the 201 sequences considered potentially suitable for primer design, 138 primer pairs were designed, of which 102 produced a fragment of the expected size, 93 of which showed polymorphism. The sequences of these 102 primer pairs, the microsatellite sequence to which each relates, and the expected PCR amplification product lengths are shown respectively in columns 4, 2 and 6 of Table 1.
  • 77 were derived from flanking sequences corresponding to dinudeotide repeat microsatellites and 25 from flanking sequences corresponding to trinucleotide repeats.
  • the maximum number of alleles corresponding to dinudeotide microsatellite loci was 7 with an average of 4.2.
  • the maximum number of alleles for trinucleotide repeats was 5 with an average of 2.9.
  • Table 3 shows the results obtained for seven Myrtaceae species (in addition to M. alternifolia) tested using thirty-five microsatellite primer pairs selected from those listed in Table 1. A total of 234 (47.8%) PCR amplifications were interpreted successfully. Of these, 31 primer pairs (88.6%) amplified useful markers from at least one of the two Melaleuca species tested. C. salignus had similar success (74.3%), followed by the eucalypts (45.7% overall) and B. citriodora (25.7%). Polymorphism varied between species, with only B. citriodora giving monomorphic patterns for all except one locus. Alleles were shared across species for a number of loci tested (Table 3) and the C. salignus individuals tested were the most heterozygous (average of 1.3 alleles per individual). Thus the level of success varied between species.
  • primers tested worked for just over three test species each. Thirteen (37.1 %) primer pairs successfully amplified in at least four test species. Four (11.4%) primer pairs amplified in more distantly-related species.
  • Primer pairs derived from microsatellite trimer repeats were more generally successful across species than were primers originating from dimer repeats (an average of 3.7 and 3.0 species respectively). However, the average number of alleles amplified was similar for dimer and trimer repeats (an average of 2.1 and 2.0 alleles respectively). Primer pairs originating from microsatellites having imperfect repeats were more generally successful than those originating from microsatellites having perfect and compound repeats (an average of 4.1 , 3.0 and 1.3 species respectively). The average number of ailelic forms of imperfect, perfect and compound microsatellites also varied (2.2, 2.0 and 1.75 respectively).
  • Locus scu023TT was sequenced for one allele in each of the test species (FIG. 1A).
  • M. alternifolia the microsatellite contains a (GCC) 8 repeat, whereas in all other species tested the allele sequenced contained a (GCC) 5 repeat.
  • the comparative reduction in n from 8 to 5 was the result of a transition at the first base in repeats one and two (GCC to ACC) and a deletion of repeat eight.
  • Only E. cloeziana showed sequence variation (two transversions) within the flanking regions (FIG. 1A).
  • the M. alternifolia microsatellite contains a (GGC) 9 repeat in contrast to the (GGC) 5 repeat in M.
  • All plants were collected from natural populations, the sampling strategy being aimed at genotyping Melaleuca alternifolia plants throughout their known geographical distribution.
  • the selected populations were large enough to collect up to 15 individual plants, each individual plant taken from a location separated by at least 100 metres from the locations of other individual plants.
  • the individual plants were grouped into 40 populations that reflected natural groupings throughout the known geographic range of the species.
  • PCR primers used were selected from the primer pairs listed in column 4 of Table 1 , selection being based on previously identified polymorphism in terms of the microsatellite locus amplified by each primer pair. PCR reactions were multiplexed and preliminary optimization resulted in two separate multiplex reactions being employed. In the first reaction, 2.0 ⁇ M primer pair SCU031TT and 1.5 ⁇ M primer pair SCU023TT were employed in order to amplify microsatellite loci having the trinucleotide repeats (GCC) n (ACC) (GCC) n and (GCC)n respectively.
  • GCC trinucleotide repeats
  • PCR reactions were performed in 12.5 ⁇ L volumes comprising 10 mM Tris-HCI pH 8.3; 50 mM KCI; 0.01 % gelatin; 25 mM MgCI 2 ; 0.5 Units AmpliTaq Gold (Perkin Elmer); 0.2 mM each dNTP;
  • 2nd reaction The annealing temperature was 51 °C, otherwise conditions were identical to the first reaction.
  • Primers were labeled with fluorescent dyes (TET, HEX and FAM) and the PCR products visualized following separation by capillary electrophoresis on an ABI 310 sequencer using dedicated software (Genescan and Genotyper).
  • the number of microsatellite alleles per population, the overall number of alleles and observed and expected heterozygosity were calculated from the PCR data using the freely available program GDA version 1.0 available over the Internet (http://chee.unm.edu/gda/).
  • F was calculated using the program FSTAT version 1.2; the probability of F /s being greater than zero was determined after 10000 permutations and the 95% bootstrap confidence interval was determined after 15000 bootstraps (Goudet, 1995, J. Hered. 86 485, which is herein incorporated by reference).
  • Microsatellite-based genotyping was performed to assess genetic diversity within and between regionally-defined populations of Melaleuca alternifolia plants, a total of 98 different microsatellite alleles were amplified from 500 individual plants within the 40 different population groupings. Allele size varied from 7 to 41 dimeric repeats and from 3 to 12 trime c repeats. There were a total of 84 dimer repeat alleles identified, and 14 trimer repeat alleles. With reference to Table 4, the total number of alleles detected in a single population varied from 21 for CN to 53 for FT, with an overall average of approximately 39 per population. In general, populations from the Severn catchment produced a lower number of alleles (26.3) than the Richmond and Clarence catchments (40.3 and 39.6 respectively).
  • Example 4 General Discussion The microsatellite enrichment and isolation method of the invention achieved an unexpectedly high yield of enriched microsatellites having at least one microsatellite repeat sequence. This yield (86%) was considerably higher than that reported by Edwards et al., 1996, supra, and appeared to be at least partly attributable to the particular washing steps employed by the present inventors.
  • the present inventors have reasoned that by decreasing the number of wash steps and adding 0.01 % SDS, the washing conditions specified by the present invention produced an improved "trade-off" between retaining weakly-hybridized polynucleotide sequences having microsatellite repeats, and losing weakly-hybridized polynucleotide sequences not having microsatellite repeats during washing.
  • dinudeotide repeats The frequency of dinudeotide repeats (52.3%) was only slightly higher than that of trinucleotide repeats (46.0%) but the near absence of longer repeats was unexpected particularly since the enrichment procedure was expected to select for longer repeat motifs. It is difficult to determine whether this result reflects the true abundance of microsatellites in tea tree or whether it is a consequence of the isolation procedure.
  • dinudeotide repeats represented approximately half of the microsatellite repeats identified, although a considerable number of longer repeats were detected.
  • Two major factors affected the success rate for identifying useful microsatellites in tea tree. First, sequence redundancy and second, lack of flanking sequence.
  • microsatellites were size-selected (100- 500 bp), and increasing the size distribution may have improved the chances of finding flanking sequences. However, this would require more sequencing, thereby adding to overall cost. Furthermore, the average dinudeotide repeat was only 42 bp long, and size did not appear to correlate with the absence of flanking sequence. In this regard, it should be noted that the longest enriched microsatellite (491 bp) did not contain suitable flanking sequence from which to design a primer pair.
  • microsatellite loci having dinudeotide repeats were generally more polymorphic (an average 4.2 alleles per locus) than were microsatellites having trinucleotide repeats (an average 2.9 alleles per locus).
  • primer pairs were designed based on microsatellite sequences identified in Melaleuca alternifolia, a significant subset of the primer pairs also amplified products when tested in a variety of species within the Myrtaceae family. Thus the primer pairs and methods of genotyping of the present invention are clearly applicable to the Myrtaceae family in general.
  • Example 3 The expected efficacy of the present invention in analyzing genetic diversity was confirmed in the study presented in Example 3.
  • One of the principal aims of this study was to define the existence and boundaries of Melaleuca alternifolia genetic provenances within its natural range. The definition of such provenances will, for example, assist the selection of superior genetic lines for enhanced production of tea tree oil.
  • the present invention has provided an improved method of isolating Myrtaceae microsatellites, provided primers based on microsatellite flanking sequences, and thereby provided a powerful method of microsatellite-based genotyping.
  • the particular usefulness of the method has been shown in genotyping tea tree populations of hitherto unknown genetic diversity.
  • the present invention will therefore be extremely useful in genotyping a variety of genera and species within the Myrtaceae family, particularly for the purpose of correlating genotype with phenotype, such as for mapping QTLs.
  • H scu083TT (CT)38 fwd TTC TCC GAT TCT GAG TGC TCG TCT O H rvs GAA GGT GCC CAA CAT GCT CAA A 53 207 Y 5 t 3 SCU084TT (TC)20 fwd CAG TTG CAG GAG GTA ACC CTA CTT rvs

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Abstract

Cette invention, qui a trait à l'isolement de microsatellites à partir de végétaux de l'espèce Myrtaceae, concerne également des microsatellites isolés, les amorces dérivées des régions flanquantes des microsatellites isolés ainsi qu'une technique permettant de génotyper des végétaux de l'espèce Myrtaceae par l'analyse des marqueurs microsatellites.
PCT/AU1999/000820 1998-09-23 1999-09-23 MICROSATELLITES DE $i(MYRTACEAE) WO2000017341A1 (fr)

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AU61837/99A AU6183799A (en) 1998-09-23 1999-09-23 Myrtaceae microsatellites

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AUPP6099A AUPP609998A0 (en) 1998-09-23 1998-09-23 Microsatellites
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107190082A (zh) * 2017-07-14 2017-09-22 中国农业科学院茶叶研究所 用于鉴别国家级茶树良种‘迎霜’的分子特异性标记引物及其鉴别方法
CN112410454A (zh) * 2020-12-02 2021-02-26 东北师范大学 鉴定兴安杜鹃与迎红杜鹃的引物及方法
CN118147276A (zh) * 2024-03-26 2024-06-07 云南省农业科学院茶叶研究所 一种鉴定茶树种质的分子标记方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997001567A2 (fr) * 1995-06-28 1997-01-16 Institut für Pflanzengenetik und Kulturpflanzenforschung Marqueurs de microsatellites pour vegetaux de l'espece triticum aestivum et de la tribu triticeae et leur utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997001567A2 (fr) * 1995-06-28 1997-01-16 Institut für Pflanzengenetik und Kulturpflanzenforschung Marqueurs de microsatellites pour vegetaux de l'espece triticum aestivum et de la tribu triticeae et leur utilisation

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BYRNE M. ET AL.: "Conservation and genetic diversity of microsatellite loci in the genus Eucalyptus", AUST. J. BOTANY,, vol. 44, 1996, pages 331 - 341 *
ECHT C.S. ET AL.: "Survey of microsatellite DNA in pine", GENOME,, vol. 40, 1997, pages 9 - 17 *
MARQUES C.M.P.: "Genetic architecture of vegetative propagation traits in eucalyptus hybrids", DISSERTATION ABSTRACTS INTERNATIONAL,, vol. 59/12-B, 1998, pages 6202 *
MATSUDA M. ET AL.: "Novel primers designed for microsatellite loci in eucalyptus and identification in PCR fingerprints", NUCLEIC ACIDS SYMPOSIUM SERIES,, no. 37, 1997, pages 169 - 170 *
PFEIFFER A. ET AL.: "Identification and characterization of microsatellites in Norway spruce (Picea abies K.)", GENOME,, vol. 40, 1997, pages 411 - 419 *
ROSSETTO M. ET AL.: "Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)", THEOR. APPL. GENET.,, vol. 98, 1999, pages 1091 - 1098 *
ROSSETTO M. ET AL.: "Microsatelitte variation and assessment of genetic structure in tea tree (Melaleuca alternifolia- Myrtaceae)", MOLECULAR ECOLOGY,, vol. 8, 1999, pages 633 - 643 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107190082A (zh) * 2017-07-14 2017-09-22 中国农业科学院茶叶研究所 用于鉴别国家级茶树良种‘迎霜’的分子特异性标记引物及其鉴别方法
CN112410454A (zh) * 2020-12-02 2021-02-26 东北师范大学 鉴定兴安杜鹃与迎红杜鹃的引物及方法
CN112410454B (zh) * 2020-12-02 2022-04-01 东北师范大学 鉴定兴安杜鹃与迎红杜鹃的引物及方法
CN118147276A (zh) * 2024-03-26 2024-06-07 云南省农业科学院茶叶研究所 一种鉴定茶树种质的分子标记方法

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