WO2005120214A1 - Markers for salinity tolerance in wheat plants and the use thereof in breeding programs - Google Patents
Markers for salinity tolerance in wheat plants and the use thereof in breeding programsInfo
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- WO2005120214A1 WO2005120214A1 PCT/AU2005/000846 AU2005000846W WO2005120214A1 WO 2005120214 A1 WO2005120214 A1 WO 2005120214A1 AU 2005000846 W AU2005000846 W AU 2005000846W WO 2005120214 A1 WO2005120214 A1 WO 2005120214A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/13—Abiotic stress
- Y02A40/135—Plants tolerant to salinity
Definitions
- the present invention relates to markers of a Nax locus of wheat plants, particularly durum wheat, linked to reduced sodium accumulation, as well as enhanced tolerance to saline and/or sodic soils.
- the present invention also relates to the use of these markers in breeding programs to produce plants with reduced sodium accumulation, as well as enhanced tolerance to saline and/or sodic soils. Furthermore, the invention relates to plants produced by these breeding programs.
- Soil salinity causes significant reductions in plant productivity, and consequent economic losses associated with reduced grain quality and yield of agricultural crops (Pitman and Lauchli 2002). Over 6% of the world's land is affected by either salinity or sodicity. A large proportion of the Australian wheat belt is at risk of salinisation due to rising water tables, and a further and larger part has soils that are sodic, and underlain with subsoil salinity (Rengasamy 2002). This subsoil salinity is formed in semi-arid zones (with annual rainfall less than 450 mm), and is transient in nature as it moves in and out of the root zone according to soil wetting and drying cycles (Rengasamy 2002).
- Cultivars of durum wheat are more salt sensitive than bread wheat (Gorham et al. 1990; Rawson et al. 1988), and may yield less when grown on saline soils (Francois et al. 1986; Maas and Grieve 1990).
- the usual high price of durum wheat on the international market can bring a better return to farmers than bread wheat and other crops, so, breeding new cultivars of durum wheat with improved salt tolerance can allow growers more options in dealing with subsoil salinity. Marker assisted selection is potentially the most efficient approach to developing cultivars that can tolerate saline soils.
- Salt tolerance in the Tritiaceae is associated with sodium exclusion, which limits the entry of sodium into the plant and its transport to leaves.
- Sodium exclusion from the transpiration stream reaching the leaves is controlled at three stages: (1) selectivity of the root cells taking up cations from the soil solution, (2) selectivity in the loading of cations into the xylem vessels in the roots, and (3) removal of sodium from the xylem in the upper part of the roots and the lower part of the shoot (Munns et al. 2002; Tester and Davenport 2003).
- the landrace had very low rates of Na + accumulation in the leaf blade, as low as bread wheat cultivars, and maintained a high rate of K + accumulation, with consequent high K + /Na + discrimination.
- the low- Na + durum landrace had a K + /Na + ratio of 17 whereas the durum cultivars Wollaroi, Tamaroi and Langdon had K + Na + ratios of 1.5, 0.7 and 0.4 respectively (Munns et al. 2000).
- the bread wheat cultivars Janz and Machete had K + /Na + ratios of 10 and 8 respectively.
- the low Na + trait was shown to confer a significant yield advantage at moderate soil salinity (Husain et al. 2003), indicating that this novel germplasm provides the opportunity to improve the salt tolerance of cultivated durum wheat
- Methods for selection of Na + excluding individuals in wheat breeding populations are time-consuming and expensive.
- the method involves growing plants in pots using a sub-irrigation system to provide a gradual and uniform exposure to NaCl to the plant, and the harvesting of a given leaf for Na + accumulation.
- this screening method is very reproducible, it is labour intensive and requires a controlled environment. It is not possible to screen plants in the field or with large numbers of individual lines using this method.
- QTL mapping and marker-assisted selection is a technique that has many advantages over phenotypic screening as a selection tool.
- Marker-assisted selection is non-destructive and can provide information on the genotype of a single plant without exposing the plant to the stress. The technology is capable of handling large numbers of samples.
- developing a QTL map is laborious, the markers identified may prove to be sufficiently robust to use as the sole selection tool for a specific trait in a breeding program.
- PCR-based molecular markers have the potential to reduce the time, effort and expense often associated with physiological screening.
- the markers In order to use marker-assisted selection in breeding programs, the markers must be closely linked to the trait, and work across different genetic backgrounds. There is a need for further markers which can be used in wheat plant breeding programs, particularly for use in durum wheat breeding programs, to produce plants with reduced sodium accumulation, as well as enhanced tolerance to saline and/or sodic soils.
- the present inventors have identified molecular markers which allow screening of plants for alleles that confer upon the plant reduced sodium accumulation which is associated with enhanced tolerance to saline and/or sodic soils.
- the present provides a method of identifying a wheat plant with enhanced tolerance to saline and/or sodic soils, the method comprising detecting a nucleic acid molecule of the plant, wherein the nucleic acid molecule is linked to a Nax locus of wheat that comprises an allele that confers enhanced tolerance to saline and/or sodic soils.
- the present invention provides a method of identifying a wheat plant having a phenotype of reduced sodium accumulation in an aerial part of the plant, the method comprising detecting a nucleic acid molecule of the plant, wherein the nucleic acid molecule is linked to a Nax locus of wheat that comprises an allele that confers reduced sodium accumulation.
- the methods can be performed on any nucleic acid of the plant which is a suitable marker for (is linked to) the desired trait.
- the Nax locus is Naxl. Any technique known in the art for detecting a nucleic acid molecule of interest can be used in the methods of the invention.
- the methods comprise: i) hybridising a second nucleic acid molecule to said nucleic acid molecule which is obtained from said plant, ii) optionally hybridising at least one other nucleic acid molecule to said nucleic acid molecule which is obtained from said plant; and iii) detecting a product of said hybridising step(s) or the absence of a product from said hybridising step(s).
- the second nucleic acid molecule is used as a primer to reverse transcribe or replicate at least a portion of the nucleic acid molecule which is obtained from the plant, or as a hybridisation probe.
- the nucleic acid is detected using a technique selected from the group consisting of: restriction fragment length polymorphism analysis, amplification fragment length polymorphism analysis, microsatellite amplification and/or nucleic acid sequencing.
- the method comprises nucleic acid amplification.
- the Nax locus is Naxl and the amplification is performed using primers which amplify a polymo ⁇ hic GA-repeat, wherein the polymo ⁇ hic GA- repeat can be amplified using the primers ATCGCATGATGCACGTAGAG (SEQ ID NO: 11) and ACATGCATGCCTACCTAATGG (SEQ ID NO: 12).
- the Nax locus is Naxl and the amplification is performed using primers which amplify a polymo ⁇ hic GA-repeat, wherein the polymo ⁇ hic GA-repeat can be amplified using the primers
- the Nax locus is Naxl and the amplification is performed using a primer comprising the sequence ATCGCATGATGCACGTAGAG
- the Nax locus is Naxl and the amplification is performed using primers which amplify a polymo ⁇ hic repeat, wherein the polymo ⁇ hic repeat can be amplified using the primer pairs selected from : i) ACATCCACGTTTATGTTGTTG (SEQ ID NO: 13) and TTGGTTGCTCAACGTTTACTT (SEQ ID NO: 14), ii) TGTGGTGCATCACAGGGCTGTTC (SEQ ID NO:21) and AGCGCTTGCATACTCGTCCGG (SEQ ID NO:22), and iii) AGCAATGAGGATGGTGCTTTCTC (SEQ ID NO:23) and TGTGAGCGACTCCTCGATTTCAG (SEQ ID NO:24).
- the Nax locus is Naxl and the amplification is performed using primers pairs
- TTGGTTGCTCAACGTTTACTT (SEQ ID NO: 14), or at least one primer which is a variant of any one of said primers, ii) TGTGGTGCATCACAGGGCTGTTC (SEQ ED NO:21) and AGCGCTTGCATACTCGTCCGG (SEQ ID NO:22), or at least one primer which is a variant of any one of said primers, and iii) AGCAATGAGGATGGTGCTTTCTC (SEQ ED NO:23) and TGTGAGCGACTCCTCGATTTCAG (SEQ ID NO:24) or at least one primer which is a variant of any one of said primers.
- the molecular markers of the invention can be used in wheat breeding programs to select progeny plants which possess an allele of an Nax locus that confer upon the progeny plants reduced sodium accumulation, and/or enhanced tolerance to saline and/or sodic soils.
- the present invention provides a method of selecting a wheat plant from a population of wheat plants, the method comprising; i) crossing two wheat plants of which at least one plant comprises a Nax locus comprising an allele which confers enhanced tolerance to saline and/or sodic soils, and ii) screening progeny plants from the cross for the presence or absence of said Nax locus by a method of the invention, wherein progeny with said allele have enhanced tolerance to saline and/or sodic soils when compared to progeny lacking said allele.
- a further aspect provides a method of selecting a wheat plant from a population of wheat plants, the method comprising; i) crossing two wheat plants of which at least one plant comprises a Nax locus comprising an allele which confers reduced sodium accumulation in an aerial part of the plant, and ii) screening progeny plants from the cross for the presence or absence of said Nax locus by a method of the invention, wherein progeny with said allele have reduced sodium accumulation when compared to progeny lacking said allele.
- the method further comprises analysing the plant for other genetic markers.
- the wheat is tetraploid wheat.
- the tetraploid wheat is durum wheat.
- At least one of the wheat plants of step i) is a hexaploid wheat plant.
- the cross is between a durum wheat plant comprising said allele and a hexaploid wheat plant lacking said allele.
- one of the wheat plants is Line 149, Line 150, Line 151 or a progenitor or progeny plant thereof comprising said allele.
- the present invention provides a method of introducing a Nax locus into the genome of a wheat plant lacking said locus, the method comprising; i) crossing a first parent wheat plant with a second parent wheat plant, wherein the second plant comprises a Nax locus which comprises an allele which confers enhanced tolerance to saline and/or sodic soils, and ii) backcrossing the progeny of the cross of step i) with plants of the same genotype as the first parent plant for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising said allele, wherein progeny plants are screened for the presence or absence of said allele by a method of the invention.
- the present invention provides a method of introducing a Nax locus into the genome of a wheat plant lacking said locus, the method comprising; i) crossing a first parent wheat plant with a second parent wheat plant, wherein the second plant comprises a Nax locus which comprises an allele which confers reduced sodium accumulation in an aerial part of a wheat plant, and ii) backcrossing the progeny of the cross of step i) with plants of the same genotype as the first parent plant for a sufficient number of times to produce a plant with a majority of the genotype of the first parent but comprising said allele, wherein progeny plants are screened for the presence or absence of said allele by a method of the invention.
- the first and/or second parent wheat plant is a durum wheat plant.
- the first parent wheat plant is a hexaploid wheat plant.
- one of the wheat plants is Line 149, Line 150, Line 151 or a progenitor or progeny plant of any one of these comprising said allele.
- a wheat plant produced using a method of the invention provides a hexaploid wheat plant identified by a method according to the invention.
- the present invention provides a seed of a wheat plant of the invention.
- the present invention provides a hexaploid wheat plant comprising an allele of the Naxl gene on chromosome 2AL which confers enhanced tolerance to saline and/or sodic soils, and/or reduced sodium accumulation in an aerial part of the plant.
- the present invention provides a product produced from a wheat plant according to the invention. Also provided is a product produced from a seed of the invention and/or the plant of the invention. Such products can be food or non-food products. Methods of producing such products are well known to those skilled in the art.
- Examples of food products include, but are not limited to, flour, starch, leavened or unleavened breads, pasta, noodles, animal fodder, breakfast cereals, snack foods, cakes, pastries and foods containing flour-based sauces.
- Examples of non-food products include, but are not limited to, films, coatings, adhesives, building materials and packaging materials.
- the present invention also provides an oligonucleotide capable of being used in methods of the invention, wherein the oligonucleotide is not selected from the group consisting of: ATCGCATGATGCACGTAGAG (SEQ ID NO: 11), ACATGCATGCCTACCTAATGG (SEQ ID NO: 12), ACATCCACGTTTATGTTGTTG (SEQ ED NO: 13) TTGGTTGCTCAACGTTTACTT (SEQ ID NO: 14), CAAATGGATCGAGAAAGGGA (SEQ ID NO: 9) and CTGCCATTTTTCTGGATCTACC (SEQ ID NO: 10).
- the oligonucleotide is capable of being used as a primer for nucleic replication or reverse transcription.
- the oligonucleotide comprises the sequence
- the present invention provides a kit for identifying a wheat plant with enhanced tolerance to saline and/or sodic soils, and/or identifying a wheat plant having a phenotype of reduced sodium accumulation in an aerial part of the plant, the kit comprising at least one oligonucleotide capable of being used in a method of the invention.
- the kit may also comprise other components useful for performing the methods of the invention such as, but not limited to, a polymerase for use in DNA amplification procedures.
- preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
- FIG. 1 Relationship of salt tolerance with leaf Na + concentration in families from a cross between subspecies durum selections, Lines 141 and 149. Na + concentrations were measured on leaf 5 after 20 d in 150 mM NaCl and biomass after 24 d. Methods as described in Munns and James (2003). Open circle is the data for Line 149.
- Figure 3 Effect of different levels of salinity on chlorophyll content in leaf 6 of low sodium (•) and high sodium ( ⁇ ) genotypes grown in the presence of (a) 1 mM NaCl, (b) 75 mM NaCl, and (c) 150 mM NaCl.
- Leaf 6 emerged 21 days after the salt treatment started. Bars show the s.e.m.
- Figure 6 Genetic linkage map on chromosome 2A. One of the linkage groups containing chromosome 2A markers and a QTL for the Na + exclusion trait. The map distances are in centimorgans (cM) as determined using the Kosambi function. Numbers in brackets are LOD scores. The arrow points to the centromere location and the markers shown are on the 2AL region.
- Figure 7. Variation among parents and F 2 progeny in DNA fragments amplified by PCR reaction using microsatellite gwm312. Upper band is the A allele from Tamaroi, lower band the B allele from Line 149.
- the lanes show the results from Line 149 (lane 1), Tamaroi (lane 2), with seven F 2 progeny, which are either homozygous for B (lanes 3 and 4), for A (lanes 5 and 6) or heterozygous (lanes 7-9).
- A homozygous parental allele of Tamaroi
- B homozygous parental allele of Line 149
- H heterozygous state.
- the edges of the box closest and furthest to the x axis indicates the 25 th and 75 th percentile, respectively. Whiskers on the box indicate the 10 th and 90 th percentile.
- the line within the box indicates the median.
- Figure 9 Validation of microsatellite gwm312 as a robust marker for the Na + exclusion trait on chromosome 2AL in three populations resulting from backcrosses between Line 149 (B allele) and three different recurrent parents (A allele). 25 individuals were screened at random. The edges of the box indicate the 25 th and 75 th percentile, respectively. Whiskers on the box indicate the 10 th and 90 th percentile, while points outside that range are graphed separately. The line within the box indicates the median.
- Figure 10 (A) Line 149 (cultivar of Triticum turgidum ssp. durum) amplicon (SEQ ID NO: l) using gwm312 primers. (B) Westonia (cultivar of T. aestivum) amplicon (SEQ ID NO:2) using gwm312 primers.
- FIG 11. Frequency distribution of plants in a BC 5 F 2 population according to leaf 3 Na + concentrations.
- the frequencies of plants for leaf 3 Na + concentrations for the parental lines Line 149 (grey bars) and Tamaroi (black bars) are shown.
- the frequencies for homozygous lines for the Line 149 allele are shown as hatched bars, for homozygotes for the Tamaroi allele as reverse-hatched bars, and for heterozygotes as cross-hatched bars.
- Figure 13 Schematic diagram showing mapping of microsatellite markers (gwm, wmc) and wheat EST markers (Ta) to defined physical telomeric deletion bins of chromosome 2 AL in Chinese Spring wheat. The percentages describe the approximate percentage of 2AL chromosome arm present in a particular deletion line.
- SEQ ID NO: l Amplicon from Line 149 using gwm312 primers ( Figure 10).
- SEQ ID NO:2 Amplicon from Westonia cultivar using gwm312 primers ( Figure 10).
- SEQ ID NO's:3 to 24 Oligonucleotide primers.
- a Nax locus comprises alleles within a population of wheat plants which confer reduced sodium accumulation and/or enhanced tolerance to saline and/or sodic soils upon a wheat plant when compared to a wheat plant lacking one of said alleles.
- the Nax locus is Naxl. It is preferred that the Nax locus is present on the A or B genomes of wheat.
- the Nax loci described herein do not include and are distinct from the Knal locus on chromosome 4D of hexaploid wheat (Gorham et al. 1997).
- the term "Naxl" refers to a region (locus) on the long arm of chromosome 2 of the genome of a wheat plant (see Figure 12).
- allelic variant (allele) of the Naxl locus has been shown herein to be linked to enhanced tolerance to saline and sodic soils as well as reduced sodium accumulation.
- markers of this region include AFLP markers AFLP42-1 and AFLP27-1; RFLP markers Xsprl02, XksuE16 and XksuD22, as well as microsatellite markers Xgwm249, Xgwm817, TaA, TaC, Xgwm312 (also referred to herein as gwm312) and Xwmcl70 (also referred to herein as wmc 170) (see the Examples section for further details).
- Xgwm312, Xgwm817 and Xwmcl70 are particularly preferred markers of alleles of the Naxl locus linked to enhanced tolerance to saline and/or sodic soils, as well as reduced sodium accumulation.
- the term “wheat” refers to any species of the Genus Triticum, including progenitors thereof, as well as progeny thereof produced by crosses with other species.
- Wheat includes "hexaploid wheat” which has genome organization of AABBDD, comprised of 42 chromosomes, and "tetraploid wheat” which has genome organization of AABB, comprised of 28 chromosomes.
- Hexaploid wheat includes 7. aestivum, T. spelta, T.
- Tetraploid wheat includes T. durum (also referred to herein as durum wheat or Triticum turgidum ssp. durum), T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross thereof.
- durum wheat also referred to herein as durum wheat or Triticum turgidum ssp. durum
- T. dicoccoides T. dicoccum, T. polonicum
- interspecies cross thereof interspecies cross thereof.
- the term "wheat” includes potential progenitors of hexaploid or tetraploid Triticum sp. such as T. uartu, T. monococcum or T. boeoticum for the A genome, Aegilops speltoides for the B genome, and T.
- leyii also known as Aegilops squarrosa or Aegilops tauschi ⁇
- Particularly preferred progenitors are those of the A genome, even more preferably the A genome progenitor is T. monococcum.
- a wheat cultivar for use in the present invention may belong to, but is not limited to, any of the above-listed species. Also encompassed are plants that are produced by conventional techniques using Triticum sp. as a parent in a sexual cross with a non-Triticum species (such as rye [Secale cereale]), including but not limited to Triticale. As used herein, the phrase "enhanced tolerance to saline and/or sodic soils" is considered as relative term.
- a saline soil is defined as having a high concentration of soluble salts, high enough to affect plant growth. Salt concentration in a soil is measured in terms of its electrical conductivity.
- a saline soil has an EC e of at least 1 dS/m, more preferably at least 2 dS/m, more preferably at least 3 dS/m, and even more preferably at least 4 dS/m.
- EC e is the electrical conductivity of the 'saturated paste extract', that is, of the solution extracted from a soil sample after being mixed with sufficient water to produce a saturated paste.
- Sodic soils have a low concentration of soluble salts, but a high percent of exchangeable Na + ; that is, Na + forms a high percent of all cations bound to the negative charges on the clay particles that make up the soil complex.
- Sodicity is defined in terms of the threshold ESP (exchangable sodium percentage) that causes degradation of soil structure.
- a sodic soil has an ESP greater than 5, more preferably an ESP greater than 7, more preferably an ESP greater than 9, more preferably an ESP greater than 11 , more preferably an ESP greater than 13, and even more preferably an ESP greater than 15.
- a wheat plant with enhanced tolerance to saline and/or sodic soils is defined as a wheat plant which comprises an allele of a Nax locus linked to this trait, where the presence of the allele means that the plant is more capable of growing, and/or reproducing, in saline and/or sodic conditions when compared to a plant with the same, or similar (such as members of the same species, more preferably members of the same subspecies), genotype but lacking said allele.
- Indicators of enhanced tolerance to saline and/or sodic soils linked to loci of the invention include, but are not limited to, reduced sodium uptake and/or lower levels of sodium in seeds (whether grown in saline and/or sodic soils or not).
- the term "reduced sodium accumulation” is considered a relative term. More specifically, the present inventors have identified markers of wheat plants linked to a low rate of Na+ accumulation in, for example, the leaf blade.
- a wheat plant with "reduced sodium accumulation” is defined as a wheat plant which comprises an allele of a Nax locus linked to this trait, where the presence of the allele means that the plant accumulates less sodium in an aerial part of the plant (any above ground part of the plant such as, for example, the stem, leaves and/or seed) when compared to a plant with the same, or similar (such as members of the same species, more preferably members of the same subspecies), genotype but lacking said allele.
- Reduced sodium accumulation can be determined using any method known in the art, such as those described in the Examples.
- the term “linked” refers to a marker locus and a second locus being sufficiently close on a chromosome that they will be inherited together in more than 50% of meioses, e.g., not randomly. This definition includes the situation where the marker locus and second locus form part of the same gene. Furthermore, this definition includes the situation where the marker locus comprises a polymo ⁇ hism that is responsible for the trait of interest (in other words the marker locus is directly "linked” to the phenotype).
- genetically linked loci may be 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1 or less cM apart on a chromosome.
- the markers are less than 5 cM apart and most preferably about 0 cM apart.
- An aspect of the invention relates to a method of introducing a Nax allele which confers enhanced tolerance of saline and/or sodic soils and/or reduced sodium accumulation into the genome of a wheat species lacking said allele.
- the aim of this aspect is to produce a plant with a majority of the genotype of the first parent but comprising said allele.
- the term "majority" means that the product of the breeding comprises greater than 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and even more preferably at least 99% of the genome of the first parent.
- An allele refers to one specific form of a genetic sequence (such as a gene) within a cell, an individual plant or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene.
- the sequences at these variant sites that differ between different alleles are termed "variances", “polymo ⁇ hisms", or "mutations”.
- the term "gene” is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the protein coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
- the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non- translated sequences.
- the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences; these sequences.
- genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
- Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
- mRNA messenger RNA
- gene includes a synthetic or fusion molecule encoding all or part of the proteins of the invention described herein and a complementary nucleotide sequence to any one of the above.
- a “polymo ⁇ hism” as used herein denotes a variation in the nucleotide sequence between alleles of the loci of the invention, of different species, cultivars, strains or individuals of a plant.
- a “polymo ⁇ hic position” is a preselected nucleotide position within the sequence of the gene.
- genetic polymo ⁇ hisms are reflected by an amino acid sequence variation, and thus a polymo ⁇ hic position can result in location of a polymo ⁇ hism in the amino acid sequence at a predetermined position in the sequence of a polypeptide.
- Typical polymo ⁇ hisms are deletions, insertions or substitutions. These can involve a single nucleotide (single nucleotide polymo ⁇ hism or SNP) or two or more nucleotides.
- the "other genetic markers” may be any molecules which are linked to a desired trait of wheat.
- the invention also provides for the use of the molecular markers linked to the loci comprising Nax alleles in combination with the use of other markers linked to desirable genes in wheat breeding.
- markers are well known to those skilled in the art and include molecular markers linked to genes determining traits such disease resistance, yield, plant mo ⁇ hology, grain quality, flour colour and the like.
- markers of such genes are stem-rust resistance genes Sr2 or Sr38, the stripe rust resistance genes YrlO or Yrl 7, the nematode resistance genes such as Crel and Cre3, alleles at glutenin loci that determine dough strength such as Ax, Bx, Dx, Ay, By and Dy alleles, the Rht genes that determine a semi-dwarf growth habit and therefore lodging resistance (Eagles et al., 2001; Langridge et al., 2001; Sha ⁇ et al., 2001). Marker Assisted Selection and Detection of a Vox Locus Marker assisted selection is a well recognised method of selecting for heterozygous plants required when backcrossing with a recurrent parent in a classical breeding program.
- the population of plants in each backcross generation will be heterozygous for the gene of interest, for example Naxl alleles linked to enhanced salinity/sodicity tolerance and/or reduced sodium accumulation, normally present in a 1 : 1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles.
- the molecular marker can be used to distinguish the two alleles.
- embryo rescue used in combination with DNA extraction at the three leaf stage and analysis with markers linked to the Naxl locus, allows rapid selection of plants carrying the desired trait, which may be nurtured to maturity in the greenhouse or field for subsequent further backcrossing to the recurrent parent.
- Any molecular biological technique known in the art which is capable of detecting alleles of a Nax locus linked to enhanced salinity/sodicity tolerance and/or reduced sodium accumulation can be used in the methods of the present invention.
- Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with suitably labeled probes, single- strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage or a combination thereof (see, for example, Lemieux, 2000; Langridge et al., 2001).
- the invention also includes the use of molecular marker techniques to detect polymo ⁇ hisms linked to alleles of a Nax locus which confer enhanced salinity/sodicity tolerance and/or reduced sodium accumulation.
- Such methods include the detection or analysis of restriction fragment length polymo ⁇ hisms (RFLP), RAPD, amplified fragment length polymo ⁇ hisms (AFLP) and microsatellite (simple sequence repeat, SSR) polymo ⁇ hisms.
- RFLP restriction fragment length polymo ⁇ hisms
- AFLP amplified fragment length polymo ⁇ hisms
- SSR microsatellite polymo ⁇ hisms.
- the closely linked markers can be obtained readily by methods well known in the art, such as Bulked Segregant Analysis, as reviewed by Langridge et al. (2001).
- PCR polymerase chain reaction
- PCR is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or “set of primers” consisting of "upstream” and a “downstream” primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme.
- Methods for PCR are known in the art, and are taught, for example, in “PCR” (Ed. M.J. McPherson and S.G Moller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can be performed on cDNA obtained from reverse transcribing mRNA isolated from plant cells expressing a gene from a Nax locus.
- a primer is an oligonucleotide sequence that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR.
- primers useful for the methods of the invention include those used for markers Xgwm312, Xgwm817 and Xwmcl70 described herein.
- Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences.
- Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon.
- Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can, for example, result in the induction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences. Primers may also contain additional sequences and/or contain modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence.
- target or target sequence or template refer to nucleic acid sequences which are amplified.
- Methods for direct sequencing of nucleotide sequences are well known to those skilled in the art and can be found for example in Ausubel et al. (supra) and Sambrook et al. (supra). Sequencing can be carried out by any suitable method, for example, dideoxy sequencing, chemical sequencing or variations thereof. Direct sequencing has the advantage of determining variation in any base pair of a particular sequence.
- Hybridization based detection systems include, but are not limited to, the TaqMan assay and molecular beacons.
- the TaqMan assay (US 5,962,233) uses allele specific (ASO) probes with a donor dye on one end and an acceptor dye on the other end such that the dye pair interact via fluorescence resonance energy transfer (FRET).
- a target sequence is amplified by PCR modified to include the addition of the labeled ASO probe.
- the PCR conditions are adjusted so that a single nucleotide difference will effect binding of the probe. Due to the 5' nuclease activity of the Taq polymerase enzyme, a perfectly complementary probe is cleaved during PCR while a probe with a single mismatched base is not cleaved. Cleavage of the probe dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
- An alternative to the TaqMan assay is the molecular beacon assay (US
- the ASO probes contain complementary sequences flanking the target specific species so that a hai ⁇ in structure is formed.
- the loop of the hai ⁇ in is complimentary to the target sequence while each arm of the hai ⁇ in contains either donor or acceptor dyes.
- the hai ⁇ in structure brings the donor and acceptor dye close together thereby extinguishing the donor fluorescence.
- the donor and acceptor dyes are separated with an increase in fluorescence of up to 900 fold.
- Molecular beacons can be used in conjunction with amplification of the target sequence by PCR and provide a method for real time detection of the presence of target sequences or can be used after amplification.
- Nucleic Acids and Oligonucleotides hybridize to a Nax locus of wheat plants, or a region of the genome of said plant genetically linked thereto, under stringent conditions.
- stringent hybridization conditions refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra).
- stringent hybridization conditions can refer to hybridization at 65°C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH 2 PO 4 (pH7), 0.5% SDS, 2 mM EDTA).
- the nucleic acid and/or oligonucleotides (which may also be referred to as "primers” or "probes”) hybridize to the region of the wheat plant genome of interest under conditions used in nucleic acid amplification techniques such as PCR.
- Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
- oligonucleotide are typically relatively short single stranded molecules.
- the minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a target nucleic acid molecule.
- the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
- oligonucleotides ranging in size from a relatively short monomeric units, e.g., 12-18, to several hundreds of monomeric units.
- Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate.
- the present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules.
- Oligonucleotide of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.
- Oligonucleotides of the invention are useful in methods of detecting an allele of a Nax locus linked to enhanced tolerance to saline or sodic soils and/or reduced sodium accumulation. Such methods, for example, employ nucleic acid hybridization and in many instances include oligonucleotide primer extension by a suitable polymerase (as used in PCR).
- a variant of an oligonucleotide of the invention includes molecules of varying sizes of, and/or are capable of hybridising to the wheat genome close to that of, the specific oligonucleotide molecules defined herein.
- variants may comprise additional nucleotides (such as 1, 2, 3, 4, or more), or less nucleotides as long as they still hybridise to the target region.
- additional nucleotides such as 1, 2, 3, 4, or more
- a few nucleotides may be substituted without influencing the ability of the oligonucleotide to hybridise the target region.
- variants may readily be designed which hybridise close (for example, but not limited to, within 50 nucleotides) to the region of wheat genome where the specific oligonucleotides defined herein hybridise.
- a variant of ATCGCATGATGCACGTAGAG is
- Leaf injury as measured by membrane damage (leakage of ions from leaf discs), premature loss of chlorophyll (using a hand-held meter), or damage to the photosynthetic apparatus (using chlorophyll fluorescence) can identify genotypes able to germinate in, or survive, very high salinities (over 200 mM NaCl), but do not discriminate between genotypes in ability to tolerate the low or moderate salinities typical of many saline fields (50-100 M NaCl). Other studies have used measurements of leaf or root elongation rate to identify genetic differences in response to moderate salinity.
- Short term growth experiments have revealed little difference between genotypes that differ in long term biomass production or yield and so longer term biomass production or yield measurements are preferred, for example for at least two weeks to several months (Nicolas et al., 1993; Rivelli et al., 2002; Shah et al., 1987; Weimberg, 1987; Munns et al., 1995).
- long term growth experiments are demanding of labour and resources, even when limited to a relatively few number of genotypes, and so other techniques such as trait- based selection are preferred.
- Traits used for screening germplasm for salinity tolerance have included Na + exclusion (Garcia et al.
- Seeds were selected by weight, surface sterilised with 1% hypochlorite for 15 min, and germinated in Petrie dishes for 3 days. Germinated seeds were planted into 6.5 x 15.8 cm pots containing quartz gravel, one plant per alternate pot, in 90 L plastic moulded trays containing 144 pots. Trays were subirrigated with either saline or non- saline nutrient solution, as described in Munns et al. (1995).
- This gravel culture was preferred to other forms of hydroponic culture, as roots were supported, each plant was a separate replicate, the frequent subirrigation and drainage avoided hypoxia, and there was no breakage of lateral roots as occurs in unsupported hydroponics when the solution was changed (Miller 1987).
- the nutrient solution at full strength was Hoagland and Arnon solution No 2, containing 4 mM Ca 2+ and 1 mM P. Seedlings were watered initially with tap water, then half strength nutrient solution was introduced 2 days after emergence (DAE), and increased to full strength at 3 DAE. Commencing at 4-6 DAE, 25 mM NaCl was added to the irrigation solution twice daily over three days to a final concentration of 150 mM.
- Supplemental Ca was added (as CaCl 2 ) to bring the total concentration of Ca 2+ to 10 mM, and the molar ratio of Na + :Ca 2+ to 15: 1.
- Control treatments always had 1 mM NaCl added to the nutrient solution.
- the pH was measured twice weekly and adjusted as needed to pH 6.0 with HCl. Root temperature was controlled using condensers in the solution reservoirs and monitored every 5 min using thermocouples. Experiments were conducted in a glasshouse with natural light and controlled air temperature. Salinity tolerance was calculated as shoot dry weight as a percentage of control shoot dry weight.
- Na + levels alone did not correlate with salt tolerance in the other tetraploid subspecies.
- the lower sodium accumulation seen here is presumed to be due to an Na + exclusion mechanism from at least the aerial parts of the plants such as Line 149, and the terms "low sodium accumulation” and "high Na + exclusion” are treated as synonymous.
- K ⁇ Na + ratios correlated well with salt tolerance across all subspecies, but the regression coefficient was not as high as with Na + .
- the correlation of K + /Na + with salt tolerance was also driven by the durum selections.
- Na + exclusion or KYNa + discrimination did not correlate with salinity tolerance in the non-durum tetraploid genotypes, yet some had high salinity tolerance (Figure IB). Polonicums as a subspecies performed well, and the carthlicums and turgidums poorly. This suggests that some of these tetraploids have a greater capacity to handle the salt accumulated in the leaf, the trait of tissue tolerance.
- Salt tolerance as measured by shoot biomass after 24 d in saline solution, correlated negatively with leaf sodium concentration ( Figure 2). This confirmed the relationship between sodium accumulation/Na + exclusion and biomass production in a common genetic background.
- Sodium exclusion reduces leaf injury and death under saline growth conditions When plants grow in saline soil, some of the salt is taken up by roots and transported to the leaves, where it may increase to toxic high levels. Sodium ion is thought to be the toxic element, not chloride ion. If toxicity occurs, old leaves die, and there are not enough functional leaves to support plant growth at its optimal rate. Plant growth and grain yields are reduced. The relationship of sodium exclusion and leaf injury or death was assessed as follows.
- Leaves of the three low sodium lines lived longer than leaves of the three high sodium lines, the start of leaf senescence being prolonged by a week or more in the low sodium lines.
- the high sodium lines lost chlorophyll more rapidly and died earlier than the high sodium lines (Figure 3).
- Other leaves showed similar results.
- the high sodium lines had an average of 77 ⁇ 5% area of dead leaf, whereas the low sodium lines had only 9 ⁇ 3%.
- Phenotyping Plants were grown in gravel culture using an automatic sub-irrigation system as described in Example 1. Pots were sub-irrigated with half-strength Hoagland's solution and 150 mM NaCl, and phosphate was reduced to 50 ⁇ M. At 6 d after seedling emergence, when leaf 2 was half-expanded, NaCl salt solution was added to the irrigation solution twice-daily over three days in 25 mM increments to make up the final concentration of 150 mM NaCl. CaCl 2 was added to bring the Ca 2+ concentration to 8 mM. The electrical conductivity and pH were monitored twice-weekly.
- Genotyping Genomic DNA Plants that were grown in salt tanks for phenotyping were transplanted into soil and allowed to grow for approximately 4 weeks prior to DNA extraction. Leaf material from plants was harvested and DNA extracted as described by Lagudah et al. (1991). For this Example, DNA was extracted from plants in five populations, as follows: (1) Tamaroi x Line 149 population: 100 F 2 individuals, 60 F families comprising the 30 extremes for Na + concentration (the 15 with highest Na + in the F 2 generation, the 15 with lowest Na + ) and 30 F 3 families representing the distribution range of Na in the remaining 70 lines. (2) Wollaroi x Line 149 population: 30 extreme F 2 individuals. (3) line 141 x Line 149 population: 30 extreme F 2 individuals, and 30 extreme F 3 families.
- AFLP markers AFLP analysis was performed according to the method of Vos et al. (1995) using Pstl and Msel restriction enzymes and adapted primers.
- the sequences of AFLP adapters and primers are listed in Table 2.
- the selective primer set (Mse+3 and Pst+3) contained 144 primer combinations.
- Msel and Pstl digested genomic DNA was amplified with Mse+1 and Pst+1 primers to produce a secondary template.
- Mse+3 and Pst+3 primers were used to selectively amplify AFLP fragments.
- Pst+3 primers 50 ng were labeled with 33 P-ATP (10 mCi/ ⁇ l) using T4 polynucleotide kinase (lOU/ml) and PNK buffer. Samples were incubated at 37°C for 1 h followed by 70°C for 10 min to inactivate the kinase. Table 2. Mse and Pst primers and adapters for AFLP pre-amplification and selective amplification reactions.
- Pst adapter CACGATGGATCCAGTGCA 3' 3' GACGTGCTACCTAGGTC (SEQ ID NO's: 6 and 7 respectively) 5' GATGGATCCAGTGCAGAG
- PCR touchdown cycle was: 94°C/30 s, 65°C/30 s, 72°C/1 min, followed by 12 cycles where the annealing temperature dropped to 57°C over 12 cycles, followed by 23 cycles with an annealing temperature of 57°C.
- Microsatellite markers A group of 103 wheat microsatellite markers were used to screen the parental lines, Tamaroi and Line 149 (Table 3). Microsatellites were chosen on the basis of their map location in hexaploid wheat in an attempt to establish complete A- and B-genome coverage. The microsatellites that were polymo ⁇ hic between the parents were used in mapping. All amplifications were performed in 20 ul aliquots containing 1.5 mM MgCl 2 , 200 ⁇ M dNTP, 200 ⁇ M 1 X PCR buffer (Boehringer Mannheim), 2 U Taq DNA polymerase and 100 ng genomic DNA.
- Genomic DNA was amplified using a step- down PCR program: 95°C/4 min, 15 cycles of 94°C/30 s, 65°C-50°C/30 s decreasing by l°C/cycle, 72°C/80 s, 30 cycles of 94°C/15 s, 72°C/45 s, followed by a 4°C holding step.
- the PCR products were separated using 1.8% metaphor agarose gel. Microsatellites that produced polymo ⁇ hisms between the parental lines were used to screen individuals in three different crosses.
- RFLP markers Restriction endonuclease digestion and Southern hybridization were performed according to standard methods (Sambrook et al., supra; Seah et al., 1998). Parental lines were digested with restriction enzymes Dral, EcoRV, EcoRI, Hindlll, Ncol, Xbal,
- the last two markers showed strong association with the Na + exclusion trait in the 60 F 2 individuals from the Tamaroi x Line 149 population. These RFLP markers were used to genotype the 30 extreme F 2 individuals from the Wollaroi x Line 149 population, and 30 extreme individuals from the line 141 x Line 149 population.
- microsatellite markers used to map the sodium exclusion locus to chromosome 2AL markers that were polymo ⁇ hic for Tamaroi and Line 149 are highlighted in bold type).
- the first approach used was an AFLP analysis of the parents, Tamaroi and Line 149, and the bulked F 2 segregants from the cross. Of the 144 primer combinations used, many revealed polymo ⁇ hisms between the parents and 22 combinations each produced at least 5 polymo ⁇ hisms. Individual F 2 progeny within the bulked segregants were then tested to validate the association of the putatively linked AFLP markers.
- One AFLP primer combination (AFLP 42) had a high association with the Na + distribution. Table 4.
- Na + concentration ( ⁇ mol g "1 DW) in parental lines and F 2 generation of the Tamaroi x Line 149 population (low and high Na + selections are the 15 F 2 individuals from the 100 F 2 progeny with the lowest and highest Na + concentration, respectively, that were used for the bulked segregant analysis. Parental means were obtained in the same experiment ( 10 replicates)).
- the second approach used was a QTL analysis based on 60 F 2 individuals representing the full range of Na + concentrations, and including all lines used in the bulked segregants.
- This group of markers was used to screen the 60 F 2 individuals.
- the genotypic data from the microsatellite and AFLP screens were imported into MapManager, and using a high stringency mapping approach (LOD score of 3), several putative linkage groups were identified.
- ACATGCATGCCTACCTAATGG SEQ ID NO: 12
- Xwmcl 70 ACATCCACGTTTATGTTGTTG (SEQ ID NO: 13) TTGGTTGCTCAACGTTTACTT (SEQ ID NO: 14)
- AFLP markers were linked to the microsatellite markers on chromosome 2AL.
- RFLP markers that had previously been mapped to a similar interval in hexaploid wheat were selected.
- 10 were polymo ⁇ hic between the parents and were tested on the 60 F 2 segregants.
- Three RFLP loci namely Xpsrl02, XksuEl ⁇ and XksuD22 were tightly linked to the Na + exclusion trait and had LOD scores of 6.4, 6.9 and 6.8 respectively ( Figure 6).
- Xpsrl02 maps to a position 6.0 cM from the centromere
- Xgwm312 maps 14.0 cM from the centromere
- XksuEl ⁇ maps 15.5 cM from the centromere
- XksuD22 maps 17.6 cM from the centromere (Roder et al. 1998; www://wheat.pw.usda.gov/).
- a specific genetic interval for Xwmcl 70 is not currently available, however public information about this microsatellite is available from the Australian National Wheat Molecular Marker Database (http://www.scu.edu.au).
- the markers were first tested on the 30 extreme individuals from the Tamaroi x Line 149 population - the F 2 individuals with the 15 highest and 15 lowest concentrations of leaf Na + . There was a strong relationship between the presence of the alleles inherited from the low Na parent, designated gwm312B (abbreviated as 'B'), and the low Na + uptake trait (Figure 8A). Individuals with high Na + uptake were associated with the alternative allele (abbreviated as 'A') from the high Na + parent, Tamaroi. The markers also identified several heterozygous F 2 individuals ('H') in both low and high Na + uptake groups (Figure 8 A).
- the microsatellite wmc 170 gave identical results as gwm312 (data not shown), because no recombinants between the markers were identified among the extreme subset of low and high Na + uptake F 2 individuals.
- the markers were tested on a different population, resulting from a cross between Line 149 and a durum landrace with exceptionally high leaf Na + , Line 141. Tamaroi and Line 141 are genetically unrelated (Husain et al. 2003; Munns et al. 2003), therefore, this was a stringent test of the ability of the marker to identify individuals with the Na + exclusion trait in different genetic backgrounds.
- Genotype means + se Line 149 B 169 ⁇ 9 Tamaroi A 895 ⁇ 46 BL960273 A 1278 ⁇ 39 BL9611 1 1 A 1245 ⁇ 62 1 means ⁇ s.e. ns - not significant
- Table 5 All of the lowest Na + individuals in each population were homozygous for the B allele, and all of the highest Na + individuals were homozygous for the A allele ( Figure 9).
- Figure 9 As was observed in the initial verification of the marker from the Tamaroi x Line 149 cross, several heterozygous individuals were identified in both the high and low Na + classes ( Figure 9). However, the most salt-tolerant individuals carried only the B allele.
- a locus for the Na + exclusion/ low sodium accumulation trait in Line 149 was successfully mapped using a QTL approach.
- Several AFLP, RFLP and microsatellite markers were linked to the gene(s) at the QTL, designated Naxl.
- the markers that mapped to the Naxl locus have previously been mapped to chromosome 2A in hexaploid wheat.
- Roder et al. (1998) and Harker et al. (2001) Xgwm312 and Xwmcl 70, the two microsatellites most closely linked to the locus, map to the long arm of chromosome 2 A in hexaploid wheat.
- the Naxl locus identified in this study maps to a position between approximately 6.0 and 14.0 cM from the centromere on chromosome 2AL.
- the Naxl locus is not homeologous to the only QTL previously mapped in hexaploid wheat for Na + exclusion and its associated K + Na + discrimination - the Knal locus. This has been mapped on chromosome 4DL (Dubcovsky et al. 1996) and is therefore different to Naxl.
- a second gene or genomic region independent of Naxl may provide the full expression of the Na + exclusion trait.
- control of Na + transport to the shoots may have multiple sites of operation such as the root cortex, the root stele, and the cells lining the xylem in the upper roots and lower shoot (Munns et al.
- the second gene or genomic region may be associated with a different tissue in the plant, or a different set of transport proteins.
- the mechanism in tetraploid wheat may be different from that in hexaploid wheat, in particular from the mechanism controlled by the Knal locus, as it leads to lower accumulation of Na + at higher external salt concentrations than bread wheat (Husain 2002), and is associated with retention of Na + in the base of the shoots (James et al. 2002).
- the locus has been designated Naxl.
- This study indicates that marker-assisted selection using gwm312 or wmc 170 is an effective means of identifying Na + excluding individuals without the need for a phenotypic screen.
- the robust markers developed have proven useful for the selection of the Na + exclusion trait in durum wheat populations from a range of genetic backgrounds.
- the Naxl QTL accounts for approximately 40% of the phenotypic variation, and may denote just one of the two major genes, the markers for this locus can be useful even in the absence of a marker for the second gene.
- the gwm312B allele is a reliable indicator of low Na + . It does not ensure selection of all individuals with the low Na + trait, as some low Na + individuals were heterozygous, selection of the homozygous B allele enriches back-crossed derived lines with the low Na + phenotype.
- EXAMPLE 3 Use of the Xgwm312 marker to identify wheat accessions with the Naxl locus
- the wheat accession Line 149 had the Xgwm312 marker associated with the low sodium accumulation phenotype. Further wheat accessions including some durum plants were tested for the presence of the Naxl locus using the PCR method described above with the Xgwm312 primers.
- Durum Line 150 Australian Winter Cereals Collection, Tamworth, NSW, Australia - Accession No. AUS# 17050
- Line 151 Australian Winter Cereals Collection, Tamworth, NSW, Australia - Accession No.
- AUS# 17051 which was a low sodium accumulator, together with the three Triticum monococcum lines (C68-101 (Australian Winter Cereals Collection, Tamworth, NSW, Australia - Accession No. AUS# 90382), C68- 124 (Australian Winter Cereals Collection, Tamworth, NSW, Australia - Accession No. AUS# 90393), C68-125 (Australian Winter Cereals Collection, Tamworth, NSW, Australia - Accession No. AUS# 90394)) which were parents of durum lines 149, 150 and 151, showed the presence of the Xgwm312 marker. The durum parent of Line 151, Glossy Huguenot was negative for the marker.
- durum lines 149, 150 and 151 were conferred by the Naxl gene linked with the Xgwm312 marker and that this gene had originated from the T. monococcum parents of these durum lines.
- EXAMPLE 4 Use of Xgwm312 marker in hexaploid wheat and development of improved marker As described above, the Xgwm312 marker was used as a closely linked marker to distinguish alleles conferring high and low Na + exclusion trait in durum (tetraploid) wheat. In initial experiments, this marker was used to test for the presence of Na + exclusion alleles in hexaploid wheat cultivars, for example Camm, Chara, Carnamah and Westonia.
- PCR reactions using total leaf DNA from plants of these cultivars and the primers Xgwm312R (5'-ACATGCATGCCTACCTAATGG-3') (SEQ ID NO: 12) and Xgwm312F (5'-ATCGCATGATGCACGTAGAG-3') (SEQ ID NO: 1 1) were carried out using standard PCR protocols with Qiagen Hotstar enzyme and buffer, and temperature cycles of 95°C, 15 min for 1 cycle, followed by 94°C, 30 sec; 58°C, 30 sec; 72°C, 2 min for 35 cycles.
- PCR products were electrophoresed on 2% agarose gels. The main products were fragments of approximately 200 basepairs.
- the nucleotide sequences of the inserts in three independent clones were determined using plasmid DNA extracted from transformed colonies ( Figure 10).
- the PCR fragment size from Westonia was 193 basepairs (bp) and from Line 149 187 bp, a difference of 6 bp. Based on the sequence differences, a new reverse primer was designed: 5'-
- EXAMPLE 5 Crossing of sodium-exclusion alleles into hexaploid wheat
- the hexaploid varieties were chosen as representative of the genetic backgrounds of bread wheats currently grown across the Australian wheat belt.
- Bread wheats have lower Na + uptake than durum wheats and generally have superior salt tolerance.
- Carnamah and Westonia and the tetraploid line P01819 (CSIRO Plant Industry seed catalogue no. P01819) which was a backcrossed (BCiF 3 ) selection having a low Na + phenotype identical to that of Line 149 and presumed to contain both Na + exclusion genes.
- Fi pentaploids from one of these direct crosses (P02901) were backcrossed with Westonia (male) to produce BCiFi plants, and the progeny then selfed.
- the BC ⁇ F seedlings were screened for the presence of the Na + exclusion allele from the tetraploid parent using the Xgwm312 marker.
- BC 3 F 3 selections containing the tetraploid Na + exclusion allele were backcrossed again into Westonia and also top-crossed with the hexaploid cultivars Sunstate, Carnamah and Chara. Further backcross/top-crosses were completed without selection using BC 2 F) plants, and additional top crosses performed into the hexaploid cultivars Janz and Yitpi. BC 3 F 2 populations of these crosses were screened using the Xgwm312 marker and selections made, thus generating BC 3 F 3 homozygous families containing the tetraploid Na + exclusion allele in 6 different hexaploid backgrounds.
- the homozygous lines are tested in both greenhouse and field trials under saline and non-saline conditions for Na accumulation in leaf and grain, growth rate, biomass accumulation and grain yield.
- the lines are grown in salt-affected fields.
- Na + uptake in cultivars is substantially decreased in the presence of the Na exclusion alleles from the tetraploid parent, and associated with improved salt tolerance and yield.
- EXAMPLE 6 - Detection of sodium exclusion trait by grain sodium levels Grain sodium levels were measured by atomic abso ⁇ tion spectrophotometry for grain from both low sodium (Line 149) and high sodium lines, to test whether the trait could be detected in grain. The plants had been grown in the presence of 1, 75 or 150 mM NaCl (Table 6). The data tabulated below shows that plants having the Na + exclusion trait had low sodium levels in the grain.
- Grain sodium analysis was therefore a sensitive indicator of the presence of sodium excluding genes, working even when plants were watered with tap water.
- EXAMPLE 7 Further genetic and physical mapping of the Naxl gene
- a BC 5 F 2 mapping population of 41 phenotyped F 2 lines was generated from a backcross between a BC 4 F 2 plant (Example 1) having a low salt accumulation phenotype (Naxl) and Tamaroi, followed by selfing of the BC 5 plants.
- the source of the gene controlling the low salt (high Na + exclusion) trait in the population was therefore Line 149 and, of the high salt trait, Tamaroi.
- the 41 lines were phenotyped by growing F2 plants in a glasshouse in gravel culture using the automatic sub- irrigation system as described in Example 1.
- PCR amplifications for microsatellite and other markers were performed in 20 ⁇ L reactions containing 1.5 mM MgCl 2 , 2 ⁇ M of each primer, 200 ⁇ M deoxynucleotide tri-phosphates (dNTPs), 200 ⁇ M lx PCR buffer, 2 units Taq DNA polymerase and 100 ng genomic DNA.
- Primer sequences for flanking microsatellite markers Xgwm312 were as described in Table 7 and for Xgwm817 as supplied by the supplier
- Xgwm817 One marker (Xgwm817) was identified which cosegregated with Naxl without any recombinants in the population of 41 lines. Based on the map location of Xgwm817, it was predicted that this marker was on the centromere-proximal side of Naxl. Therefore, it was predicted that Xgwm312 and Xgwm817 flanked the Naxl gene. To identify further molecular markers linked to Naxl, wheat EST libraries
- RFLP polymo ⁇ hisms in Line 149 and Tamaroi corresponding to the wheat ESTs were identified by Southern blot hybridisations using the wheat EST inserts as probes. Eleven polymo ⁇ hic ESTs were identified including wheat ESTs with Accession Nos. BF474590, BE471258, BG262791 and BE403217 (designated TaP, TaQ, TaA, TaC, respectively). These had homology to rice genes on BAC Nos. OSJNBbO 103108, OSJNBa0017B10, OSJNBa0013K16 and OSJNBa0058K23, designated OsP, OsQ, OsA, OsC, respectively.
- the discrepancy was probably due to inaccurate reports of the map position of Xpsrl02 or XksuD22 on which Figure 6 was based.
- a series of deletion lines in Chinese Spring wheat (Endo and Gill 1996) were used.
- the wheat ESTs were used as probes in Southern blot hybridisations on DNA from the Chinese Spring deletion lines that had been digested with restriction enzymes (Table 7) that produced a polymo ⁇ hic RFLP pattern.
- RFLP marker bands corresponding to the ESTs were thereby located to the physical region delineated by breakpoints 0.27 (CS 2AL-04) representing a telomeric deletion line that retains approx 27% of the 2AL chromosome arm and 0.85 (CS 2AL-1) representing a telomeric deletion line that retains approx 85% of the 2AL chromosome arm ( Figure 13).
- Naxl locus was located within a physical segment of the wheat 2AL chromosome between positions 0.27 and 0.85 (with position "0" at the centromere and "1.0" at the tip of 2AL), representing approximately 58% of the chromosome arm.
- Further fine mapping of Naxl will use a population of 12000 BC 5 F seeds from the backcross between Line 149 and Tamaroi as described above. DNA isolated from part of each seed will be examined with the markers Xgwm312 and Xgwm817 as described above to identify recombinants between these two markers. The plant corresponding to each recombinant will be recovered from the remainder of each seed and tested for its Naxl phenotype. Such recombinants will closely define the map position of Naxl.
- EXAMPLE 8 Identification of further polymorphisms for use as molecular markers
- a Line 149 genomic library is made in a ⁇ vector and plaques obtained by plating 10 6 or more ⁇ infectious particles at high density in a suitable E. coli host.
- a similar library is made from a wheat plant lacking, for example, the Naxl allele of interest of Line 149, such as Tamaroi (durum wheat) or Westonia (hexaploid wheat).
- the plaques are transferred to nylon filters.
- a Naxl linked specific probe, such as the PCR fragment amplified from these plants (see, for example, Figure 10), is labelled with radioactive label and used to hybridise to the nylon filters.
- Plaques corresponding to spots of hybridisation are isolated and confirmed to be positive for Naxl linked sequences by second or third rounds of hybridisation.
- DNA sequencing of the Naxl gene segments in the ⁇ clones is carried out by standard methods to determine the nucleotide sequence of the surrounding regions amplified by the gwn321 primers. The genomic sequences are compared and further molecular techniques for distinguishing between a Naxl allele which confers reduced sodium accumulation, as well as enhanced tolerance to saline and/or sodic soils, and a Naxl allele which does not confer this phenotype can be devised.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2005251389A AU2005251389A1 (en) | 2004-06-14 | 2005-06-14 | Markers for salinity tolerance in wheat plants and the use thereof in breeding programs |
US11/629,835 US20080028480A1 (en) | 2004-06-14 | 2005-06-14 | Markers for Salinity Tolerance in Wheat Plants and the Use Thereof in Breeding Programs |
EP05749299A EP1765056A4 (en) | 2004-06-14 | 2005-06-14 | Markers for salinity tolerance in wheat plants and the use thereof in breeding programs |
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US57949604P | 2004-06-14 | 2004-06-14 | |
US60/579,496 | 2004-06-14 |
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PCT/AU2005/000846 WO2005120214A1 (en) | 2004-06-14 | 2005-06-14 | Markers for salinity tolerance in wheat plants and the use thereof in breeding programs |
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US (1) | US20080028480A1 (en) |
EP (1) | EP1765056A4 (en) |
AU (1) | AU2005251389A1 (en) |
WO (1) | WO2005120214A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008025097A1 (en) * | 2006-08-31 | 2008-03-06 | Commonwealth Scientific And Industrial Research Organisation | Salt tolerant plants |
EP2046959A1 (en) * | 2006-07-12 | 2009-04-15 | Commonwealth Scientific and Industrial Research Organisation | Polynucleotides and methods for enhancing salinity tolerance in plants |
CN110512021A (en) * | 2019-08-26 | 2019-11-29 | 江苏省农业科学院 | One molecular labeling and its application with wheat base rot disease Resistance QTL close linkage |
CN114317812A (en) * | 2022-03-03 | 2022-04-12 | 中国农业科学院生物技术研究所 | Rice salt tolerance related molecular marker and application thereof |
CN114381547A (en) * | 2022-01-24 | 2022-04-22 | 青岛农业大学 | Molecular marker closely linked with ethylene insensitivity and salt tolerance related characters of peanuts and application |
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US9943095B2 (en) | 2012-10-23 | 2018-04-17 | Montana State University | Production of high quality durum wheat having increased amylose content |
CN110527742B (en) * | 2019-09-19 | 2022-08-30 | 中国科学院遗传与发育生物学研究所 | KASP marker related to wheat spike length under low salt condition and application thereof |
CN113462806B (en) * | 2021-08-05 | 2023-05-30 | 浙江大学中原研究院 | SNP molecular marker related to barley salt tolerance and application thereof |
CN114295592B (en) * | 2021-12-01 | 2023-06-27 | 齐鲁工业大学 | Rapid detection method for salinity tolerance of algae |
CN116121431B (en) * | 2022-08-11 | 2024-02-06 | 青岛农业大学 | Molecular marker closely linked with lateral root number gene locus in wheat seedling stage and application thereof |
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US5925517A (en) * | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
DE19525284A1 (en) * | 1995-06-28 | 1997-01-02 | Inst Pflanzengenetik & Kultur | Microsatellite markers for plants of the species Triticum aestivum and of the Tribus Triticeae and their use |
-
2005
- 2005-06-14 US US11/629,835 patent/US20080028480A1/en not_active Abandoned
- 2005-06-14 AU AU2005251389A patent/AU2005251389A1/en not_active Abandoned
- 2005-06-14 WO PCT/AU2005/000846 patent/WO2005120214A1/en active Application Filing
- 2005-06-14 EP EP05749299A patent/EP1765056A4/en not_active Withdrawn
Non-Patent Citations (7)
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Cited By (9)
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EP2046959A1 (en) * | 2006-07-12 | 2009-04-15 | Commonwealth Scientific and Industrial Research Organisation | Polynucleotides and methods for enhancing salinity tolerance in plants |
EP2046959A4 (en) * | 2006-07-12 | 2009-09-16 | Commw Scient Ind Res Org | Polynucleotides and methods for enhancing salinity tolerance in plants |
US8536429B2 (en) | 2006-07-12 | 2013-09-17 | Commonwealth Scientific And Industrial Research Organisation | Polynucleotides encoding a NAX2 polypeptide and methods for enhancing salinity tolerance in plants |
AU2007272314B2 (en) * | 2006-07-12 | 2014-05-01 | Commonwealth Scientific And Industrial Research Organisation | Polynucleotides and methods for enhancing salinity tolerance in plants |
WO2008025097A1 (en) * | 2006-08-31 | 2008-03-06 | Commonwealth Scientific And Industrial Research Organisation | Salt tolerant plants |
CN110512021A (en) * | 2019-08-26 | 2019-11-29 | 江苏省农业科学院 | One molecular labeling and its application with wheat base rot disease Resistance QTL close linkage |
CN114381547A (en) * | 2022-01-24 | 2022-04-22 | 青岛农业大学 | Molecular marker closely linked with ethylene insensitivity and salt tolerance related characters of peanuts and application |
CN114381547B (en) * | 2022-01-24 | 2023-05-02 | 青岛农业大学 | Molecular marker closely linked with peanut ethylene insensitivity and salt tolerance related characters and application thereof |
CN114317812A (en) * | 2022-03-03 | 2022-04-12 | 中国农业科学院生物技术研究所 | Rice salt tolerance related molecular marker and application thereof |
Also Published As
Publication number | Publication date |
---|---|
US20080028480A1 (en) | 2008-01-31 |
EP1765056A1 (en) | 2007-03-28 |
AU2005251389A1 (en) | 2005-12-22 |
EP1765056A4 (en) | 2008-05-28 |
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