CN117683780B - Wheat stripe rust resistance gene YrLumai and application thereof - Google Patents
Wheat stripe rust resistance gene YrLumai and application thereof Download PDFInfo
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Abstract
The invention discloses a wheat stripe rust resistance gene YrLumai and application thereof, belonging to the technical field of molecular genetics. The invention firstly separates and clones from wheat Lu Mai to obtain a new stripe rust resistance gene YrLumai. It was found that YrLumai gene was linked to the wheat stripe rust resistance phenotype and that mutation of this gene resulted in loss of the wheat stripe rust resistance phenotype. Therefore, yrLumai genes can provide stripe rust resistance, and have important application value in the cultivation of wheat stripe rust resistant varieties.
Description
Technical Field
The invention relates to the technical field of molecular genetics, in particular to a wheat stripe rust resistance gene YrLumai and application thereof.
Background
Wheat stripe rust (WHEAT STRIPE rust or yellow rust) is a worldwide air-borne disease caused by a rust (Puccinia striiformis f.sp.tritici, pst) infestation. The disease has destructive harm to wheat production in China, and the epidemic year can lead to the wheat yield reduction of more than 40 percent, even the wheat yield is out of service (Ma Zhanhong, 2018).
The traditional medicament control not only increases the production cost, but also threatens the ecological environment, and the most economical, effective and environment-friendly stripe rust control measure is to cultivate and popularize disease-resistant wheat varieties. Under the disease-resistant breeding bottleneck that the mutation speed of the rust race is too high and the genetic basis of the main cultivated variety resistance is narrow, yr gene combination has very good resistance effect on rust bacteria, and the cultivation of durable disease-resistant varieties with multiple disease-resistant gene aggregation becomes the main direction of disease-resistant breeding (Feng Jing and the like, 2022). However, isolation of disease resistance genes in the huge genome of wheat is a great challenge, and of 83 stripe rust resistance genes which have been named currently, only 7 genes are cloned (Fu et al.,2009)(Krattinger et al.,2009)(Moore et al.,2015)(Clemence et al.,2018)(Klymiuk et al.,2018)(Zhang et al.,2019)(Athiyannan et al.,2022). and thus, more disease resistance genes are urgently required to be discovered to enrich the diversity of disease resistance genes.
The substitution of the short arm of the 1B chromosome of wheat with the short arm of the 1R chromosome of rye results in a wheat-rye 1BL/1RS translocation line, the 1BL/1RS variety harboring multiple disease-resistant genes from rye, including the stripe rust resistance gene Yr9 et al (magoetal, 2015). Wheat Lu Mai has high resistance to leaf rust, leaf rust and stalk rust, immunity to No. 22-28 physiological races in the strip, and high resistance to No. 29 physiological races in the strip (Sun Lanzhen et al, 1994). The variety carries the stripe rust resistance gene from the 1RS chromosome in the whole growth period, is linked with a far-end marker Iag95 (Mago et al, 2002), but the stripe rust resistance gene is successfully separated from the strain by no one, so that the sequence of the disease resistance gene and the information of the coded protein are ambiguous, and the disease resistance potential of the stripe rust resistance gene is difficult to fully develop.
Disclosure of Invention
Aiming at the prior art, the invention separates and clones the stripe rust resistance gene YrLumai from the wheat Lu Mai. YrLumai15 genes are linked to the wheat stripe rust resistance phenotype and mutation of the genes results in loss of the wheat stripe rust resistance phenotype. Therefore, yrLumai genes can provide stripe rust resistance, and have important application value in the cultivation of wheat stripe rust resistant varieties.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect of the present invention, there is provided a wheat stripe rust resistance gene YrLumai, said wheat stripe rust resistance gene YrLumai being a nucleic acid molecule as set forth in i) or ii) or iii) or iv) below:
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 3;
iii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i) or ii);
iv) a nucleic acid molecule which has 90% or more identity to the nucleotide sequence of i) or ii) and expresses the same functional protein.
Wherein, the full-length cDNA sequence of wheat stripe rust resistance gene YrLumai is shown in SEQ ID NO. 1; the amino acid sequence of the protein coded by YrLumai gene (namely YrLumai protein) is shown as SEQ ID NO. 2; yrLumai15 genome full-length expression frame includes promoter, genome coding region and terminator, its nucleotide sequence is shown in SEQ ID NO. 3.
In a second aspect of the present invention, there is provided a protein encoded by the wheat stripe rust resistance gene YrLumai described above.
The recombinant expression vector carrying the wheat stripe rust resistance gene YrLumai, a transgenic cell line or a genetically engineered bacterium are also the protection scope of the invention.
In a third aspect of the present invention, there is provided the use of wheat stripe rust resistance gene YrLumai in (1) or (2) as follows:
(1) Regulating and controlling the resistance of plants to stripe rust;
(2) Cultivating the stripe rust resistant plant variety.
In the above application, the plant is preferably a gramineous plant; further preferred are wheat plants; more preferably, the plant is wheat, barley or rye.
In a fourth aspect, the invention provides the use of a recombinant expression vector carrying wheat stripe rust resistance gene YrLumai, a transgenic cell line or a genetically engineered bacterium in the cultivation of wheat plants with increased or decreased stripe rust resistance.
Preferably, the wheat plant is wheat, barley or rye.
In a fifth aspect of the present invention, there is provided the use of a protein encoded by wheat stripe rust resistance gene YrLumai in (1) or (2) as follows;
(1) Improving the disease resistance of the plants to the stripe rust;
(2) Preparing the medicament for preventing and treating the stripe rust.
Preferably, the wheat stripe rust resistance gene YrLumai encodes a protein as shown in any one of the following (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;
(A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.
Wherein, the proteins (A1) and (A2) can be synthesized artificially or can be obtained by synthesizing the encoding genes and then biologically expressing.
Among the above proteins, the protein tag refers to a polypeptide or protein that is fusion expressed together with the target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. Wherein, in order to facilitate purification of the protein in (A1), a tag may be attached to the amino-terminal or carboxyl-terminal of the protein of (A1). The tag may be Poly-Arg (typically 6 RRRRR), poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).
In the above application, the plant is preferably a gramineous plant; further preferred are wheat plants; more preferably, the plant is wheat, barley or rye.
In a sixth aspect of the invention, there is provided a method of increasing resistance to stripe rust in a plant, comprising: transferring the stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 into a plant to obtain a plant with improved stripe rust resistance;
or up-regulating expression of stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in plant genome, and screening to obtain plant with improved stripe rust resistance.
In the above method, the method for transferring the stripe rust resistance gene into wheat or barley comprises: polyethylene glycol method, agrobacterium mediated method or gene gun bombardment method.
In the above method, the method of up-regulating the expression of a stripe rust resistance gene in a plant genome comprises: introducing a DNA fragment capable of activating or increasing the transcription level or translation level or protein activity of the stripe rust resistance gene; or controlling the synthesis of specific small RNA molecules, and up-regulating the accumulation of stripe rust resistance gene mRNA.
In the above method, the plant is preferably a gramineous plant; further preferred are wheat plants; more preferably, the plant is wheat, barley or rye.
In a seventh aspect of the invention, there is provided a method of reducing resistance to stripe rust in a plant, comprising: inhibiting expression of stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in plant genome, and screening to obtain plant with reduced stripe rust resistance.
In the above method, the method of inhibiting expression of a stripe rust resistance gene in a plant genome comprises: mutating or knocking out all or part of the sequence of the stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO. 3; or interfering RNA is used for interfering the expression of stripe rust resistance genes shown in SEQ ID NO.1 or SEQ ID NO. 3; alternatively, the anti-stripe rust gene shown in SEQ ID NO.1 or SEQ ID NO.3 may be silenced using a gene silencing system.
Preferably, the stripe rust resistance gene shown in SEQ ID NO.1 is subjected to at least one of the following mutations (1) to (6):
(1) Mutating the 211G base to an A base;
(2) Mutating the G base at position 1396 to an A base;
(3) Mutating the G base at position 2023 to an A base;
(4) Mutating the 2107G base to a T base;
(5) Mutating the G base at position 2633 to an A base;
(6) The G base at position 1503 is mutated to a C base.
In an eighth aspect of the present invention, there is provided a molecular marker YrLumai-2728 for identifying wheat stripe rust resistance gene YrLumai, wherein the molecular marker YrLumai-2728 is a PCR marker designed based on YrLumai coding region, and the nucleotide sequence is shown in SEQ ID NO. 4.
The primers used for amplifying the molecular markers YrLumai-2728 are YrLumai-FP (SEQ ID NO. 5) and YrLumai-RP (SEQ ID NO. 6). After amplification by the primer, if a 1261bp specific band appears, the wheat YrLumai gene is carried; if nonspecific amplification occurs, it is recommended that the corresponding wheat is not resistant to wheat stripe rust.
In a ninth aspect of the present invention, there is provided the use of the above molecular markers YrLumai-2728 or primers for amplifying the molecular markers YrLumai-2728 in any one of the following (1) to (3):
(1) Identification of stripe rust resistance genes;
(2) Plant breeding;
(3) Preventing and treating plant stripe rust.
In the above application, the plant is preferably a gramineous plant; further preferred are wheat plants; more preferably, the plant is wheat, barley or rye.
The invention has the beneficial effects that:
The invention firstly separates and clones from wheat Lu Mai to obtain a new stripe rust resistance gene YrLumai. It was found that YrLumai gene was linked to the wheat stripe rust resistance phenotype and that mutation of this gene resulted in loss of the wheat stripe rust resistance phenotype. Therefore, yrLumai genes can provide stripe rust resistance, and have important application value in the cultivation of wheat stripe rust resistant varieties.
Drawings
Fig. 1: the mutant of disease-resistant YrLumai is high-sensitive wheat stripe rust at seedling stage; wherein 1-5 are different disease-inducing mutants, and 6 and 7 are mutagenesis parents Lu Mai.
Fig. 2: detecting the susceptibility mutant with an Iag95 marker; wherein 1-5 are 5 susceptibility mutants 2365, 3289, 3530, 2333 and 3222.
Fig. 3: yrLumai15 gene structure and mutation site of the wheat 15 stripe rust mutant; 2365. 3289, 3530, 2333 and 3222 represent 5 mutants, of which 2365 carries 2 mutation sites.
Fig. 4: identifying molecular markers YrLumai-2728 of wheat YrLumai gene; wherein Lu Mai is a parent material carrying a stripe rust resistance gene YrLumai, and CB037-PstS is a stripe rust resistance material; f 2 -R is Lu Mai/CB 037-PstS combined F 2 single plant carrying YrLumai gene, F 2 -S is single plant not carrying YrLumai gene.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the development of new stripe rust resistance genes is not only important but also impossible to terminate in the evolving situation of the continued pathogenic bacteria. Wheat variety Lu Mai exhibits high resistance to wheat stripe rust, in order to isolate its stripe rust resistance gene, we used Lu Mai 15 to mutagenize with ethyl methanesulfonate (Ethyl methanesulfonate, EMS), screen stripe rust type mutants, and identify the disease resistance gene in parent Lu Mai by analyzing mutant genes shared by stripe rust type mutants.
And (3) inoculating and identifying more than 3000M 4 generation Lu Mai mutants, and screening 5 stripe rust mutants. The physiological race of the adopted rust bacteria is CYR29, CYR31 and CYR32 mixed spores. The mutant phenotype is shown in FIG. 1.
RNA-seq sequencing was performed on mutagenized parent Lu Mai and 5 parts of the infected mutant material, and SNP-calling was performed to obtain single base differences between the mutant material and mutant parent Lu Mai. And then analyzing the SNP, counting the number of the SNP by taking the transcript as a unit, and finding that 6 SNP are on the transcript trans/1460, wherein the SNP corresponds to 5 mutants of the infectious disease respectively.
Then we combined with the Kenong 9204 reference genome data also containing 1BL/1RS translocation to analyze, find the corresponding gene sequence TraesKN B01HG01710, confirm the full-length coding region of the gene.
In summary, the invention performs RNA-seq sequencing on 5 randomly selected mutants, and finds that compared with the mutant parent Lu Mai, the 5 mutants identified as being infected all have single base mutation on the same gene (the base of the EMS mutation is random, the probability of mutation of a plurality of independent mutants on the same transcript is very low, and if the 5 mutants all have mutation on the same transcript, the relationship between the transcript and the disease resistance phenotype is enough proved). Thus, this gene was designated YrLumai by the present invention. The full-length cDNA sequence of YrLumai gene is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by YrLumai gene (namely YrLumai protein) is shown as SEQ ID NO. 2; yrLumai15 genome full-length expression frame includes promoter, genome coding region and terminator, its nucleotide sequence is shown in SEQ ID NO. 3. The method comprises the following steps:
Full-length cDNA sequence of YrLumai Gene:
ATGGATATTGTCACGGGTGCCATTGCCAACCTGATCCCCAAGCTGGGAGAGCTGCTTGTGGGGGAGTACAAGCTGCACAAGGGCGTCAAGAAAAATATCGAGGACCTCCTGAAAGAGCTCAAGACCATGAACGCTGCGCTCATCAAGATTGGTGAGGTGCCGCCGGACCAGCTCGACAGCCAAGACAAGCTTTGGGCCGATGAGGTCAGAGAGCTCTCCTACGTCATTGAGGATGTGGTGGACAAGTTCCTCGTACGGGTCCATGGCATTGAGCCCGATGACAACACTAACGGATTCAAGGGGCTCGTCAAGAGGACCACCAAGTTGTTGAAGAAGGTCGTGGATAAGCATGGGATAGCTCACGCGATCAAGGACATCAAGAAGGAACTCCATGAGGTGGCTGCTAGGCGTGACAGGAACAAGTTCGATGGTATTGCTTCTACTCCTACTGAAGCAATCGATCCTCGTCTTCATGCTCTCTACATAGAAGCCGCAGAGCTAGTTGGCATCTATGGGAAGAGGGATCAGGCACTCATGAGTTTGCTCTCCTTGGAGGGCGATGATGCCTCTACCAAGAAACTGAAGAAGGTCTCCATTGTTGGATTTGGAGGGTTGGGCAAGACCACTCTTGCCAGAGCAGTATACGAGAAGATTAAAGGTGATTTTGGTTGCCACGCATTTGTTCCTGTCGGGCAGAACCCTGATATCAAGAAGGTTTTCAGGGATATCCTCATTGACCTCCGAAAATCAAACTCAGATCCTAGCAACTCTAACTCAGATCTTGTGATACTGGATGCAACACAGCTTATCGACAAGCTTCGTGAATTCCTTGAGAACAAGAGGTACCTCGTAATAATTGATGATATATGGGATGAAAAATTGTGGAGATACATCAACCTTGCTTTCTCTATCAATAACAATCTAGGAAGTCGGCTAATCACCACAACCCGAGATTTCGATGTCTCCAAATCATGCTGCTCATCAGCTGATGATTCAATTTATCATATGAAACCTCTTTGTACCAATGACTCCAGAAGGCTCTTCTATAAAAGAGTATTTGCCGACGCTAGTGGATGTCCAAGTGAATTTGAACAAGTGTCTAAAGATATATTGAAGAAATGTGGCGGGGTACCACTAGCCATCATTACTATTGCAAGTGCTTTGGCTAGTGGCCAGCAAGTGAAACCAAAGCATGAGTGGGATATTCTACTCCAGTCCCTTGGCTCCGGAGTAACAAAAGATAATAGTTTGGCTGAGATGCGGAGAATACTATCTTTCAGCTATTATAATCTACCGTCTCATCTGAAAACTTGTCTACTTTACCTATGTATATATCCAGAAGATAGCACCATTGGTAGAGATAGACTGATATGGAAGTGGGTGGCCGAAGGATTTGTCCACCATGGAGATCAAGGGACCAGCCTGTTTTTGGTCGGATTAAACTACTTCAACCAGCTCATTAATAGAAGTATGATCCAGCCTATATATGATAATGCAGGCCAGGTATATGCTTGCCGTGTACATGATATGGTTCTGGACCTTATCTGCAACTTGTCACATGAAGCAAAGTTTGTTAATCTATTGGATGCCACTGGGAATAGCACATCTTCACAAAGTAATGTTCGTCGTTTGTCCCTTCAGAATAAAAATGAAGATCATCAAGCCAAGCCTCTCACAAATATCATGAGTATGTCACGAGTGAGGTCCATTACTATCTTTCCACCTGCTGTTAGTATCATGCCAAGTCTGTCAATGTTTGAAGTTCTGAGTGTACTTGATCTGTCGAACTGTGATTTGGGACAAAGTAGCAGCCTGCAGCTTAACCTAAAGGGTGTTGTACATTTAATCCACCTAAGGTACCTTGGTCTAGCAGGCACTCAAATTAGTGAACTCCCGACTGAGATAGGAAACCTGCAGTTTTTGGAGGTGTTGGATCTTGGAGATAATTATGAGCTAGATGAATTGCCTTCCACTCTTTTCAAATTGAGAAGATTAATCTACCTAAATGTTTATCTCTTTAAGGTGGTTCCAACTCCTGGTGTGTTGCAGAATCTGACATGCATAGAAGTGTTGAGGGGGATCTTGGTCTCTCTGAACATTATTGCACAAGAGCTTGGCAACCTGGCAAGGCTGAGGGAGCTTTCGATTCGCTTCAAGGATGGTAGTTTGGATTTGTATGAAGGTTTCGTGAATTCTCTGTGCAACCTACATCACATAGAATGCCTAAGTATTGGTTGGAATTCTGAAAAAACGTCTTTTGAACTGATGGATCTCTTGGGAGAACGCTGGGTGCCTCCTGTACATCTCCGCGAATTTGTGTCTAGGATGCCCAGCCAACTCTCTGCACTGCGAGGGTGGACAAAGAGAGACCCCTCGCATCTCTCCAACCTCTCCAAGTTAATCCTCTCGTCAGTGAAGGAAGTGCAGCAGGAGGACGTGGAAATCATTGGGGGGTTGCTGTCCCTTCGCCGTCTCTGGATAAAGAGCACCCACCAAACACAGCGGCTGCTAGTCATCCCTACAGATGGGTTCCGCTGTATGGTAGAATTTTACTTGAATTGTGGGTTAGCAGCGCAGATAATGTTTGAACCAGGAGCTTTGCCGAGGGCGGAAGAAGTTACGTTCAGCCTGGGCGTGCGGGTGGCAAAAGAGGATGGAAACTGTGGCTTCGAATTGGGCGTGCAGGGGAACCTGCTCTCCCTTCGGCGGCAGGTCTGGGTTAGGATGTATTGTGGTGGAGTGAGGGTTGGGGAGGCAAAGAAAGCGGAGGCTGCGGTGAGGCACGCACTCGAAGCCCATCCCAATCATCCCAGGATTTATATTGATATGTGCCCGCGTATAGCAGAAGATGCTCATGATGACGATTTGTGCGAGGACGAGGAGATCTGA.
YrLumai15 gene encodes a protein:
MDIVTGAIANLIPKLGELLVGEYKLHKGVKKNIEDLLKELKTMNAALIKIGEVPPDQLDSQDKLWADEVRELSYVIEDVVDKFLVRVHGIEPDDNTNGFKGLVKRTTKLLKKVVDKHGIAHAIKDIKKELHEVAARRDRNKFDGIASTPTEAIDPRLHALYIEAAELVGIYGKRDQALMSLLSLEGDDASTKKLKKVSIVGFGGLGKTTLARAVYEKIKGDFGCHAFVPVGQNPDIKKVFRDILIDLRKSNSDPSNSNSDLVILDATQLIDKLREFLENKRYLVIIDDIWDEKLWRYINLAFSINNNLGSRLITTTRDFDVSKSCCSSADDSIYHMKPLCTNDSRRLFYKRVFADASGCPSEFEQVSKDILKKCGGVPLAIITIASALASGQQVKPKHEWDILLQSLGSGVTKDNSLAEMRRILSFSYYNLPSHLKTCLLYLCIYPEDSTIGRDRLIWKWVAEGFVHHGDQGTSLFLVGLNYFNQLINRSMIQPIYDNAGQVYACRVHDMVLDLICNLSHEAKFVNLLDATGNSTSSQSNVRRLSLQNKNEDHQAKPLTNIMSMSRVRSITIFPPAVSIMPSLSMFEVLSVLDLSNCDLGQSSSLQLNLKGVVHLIHLRYLGLAGTQISELPTEIGNLQFLEVLDLGDNYELDELPSTLFKLRRLIYLNVYLFKVVPTPGVLQNLTCIEVLRGILVSLNIIAQELGNLARLRELSIRFKDGSLDLYEGFVNSLCNLHHIECLSIGWNSEKTSFELMDLLGERWVPPVHLREFVSRMPSQLSALRGWTKRDPSHLSNLSKLILSSVKEVQQEDVEIIGGLLSLRRLWIKSTHQTQRLLVIPTDGFRCMVEFYLNCGLAAQIMFEPGALPRAEEVTFSLGVRVAKEDGNCGFELGVQGNLLSLRRQVWVRMYCGGVRVGEAKKAEAAVRHALEAHPNHPRIYIDMCPRIAEDAHDDDLCEDEEI.
YrLumai15 genome full-length expression framework:
CATCAATAACGATTAAGTACCTGGCAGAGAAATTAATTAATGGAAATAATATTTTACAGACTTAAAGTATGGGTAATTCAAATAGGCATGTAGGAGGATGCGCTCCTGGGTGAAAGAAATGATATTCTTGGTGTTAAAAAGTTCTGGAAGAACGTACACATGTACATGTCCGCATCCTATGTGTAGATGTGATTTTGCATGCAAAACACAATTTTTGTGGCCCGTGTGAAACCCAAAATGGGCCAGCTGGTTTTCTTTTGGCACAACCCAATTTATGTCTATGGGGCGGTCGGAGACTCGGAGTTGACGTCGGAGGGGGACGGTGTGTATGGCAGTGAGGCGTATGGTGGCATGGATCTGCTCCACCATGAGGCAGCATGCTTGGCCGCAGCCACCTCTTTCTCAGACGTCGTCCGCTTGGATGCCTCGATGACCTCCCATAATGCCTTCGCGTTTTTGCGACGACGGCCACGGGCGCCCATCTCCGTGGAGGTCAATGAACGGAGCCGGACCTAGTCGTCGTCGAAGAAAGATGGCATGCACATGATTGAAGCAGAGCCGCACCGGCTAGATCCGACCTTCTGGGTCACCTGGCAGACGCCAGCCGCTAGCGCCTTGGACTCCGGGAGCTGTTACAGGCTCAGATGGCGCGGCACGATAAGCTAGCGGCGACAAGGAGTGCATACGACAGTGATACACCGAAGCTGTATGACTGACGCCACACGACTAGGGACAGCACGAAATGCCTCCATCCTAGGATGCATTCTCCCTGGCAATCGGTGGGGGATGACACGAAGCTCGAAGAGCCTCCCTTGTCCATCCGCTTGCGGCAGAGGGCGACATAGAGGCGAGGTCCTCATCGGAGGCACAGAGCTTGAGTCCTTCGCCGCGAGCGCATCCCAATCATTCGGATAAGCCTCGCTGTCAGAGTCGGCTATGTCGGAGCAGAGGGCAACGAGGAGGGGAGATGATTGTGATGGAGCGGTTGAGAATGAGGACCCGATGTTTGGTTTGGGGTGGGGGTATATGTGGGGTTGGAGTGAGCCAGCCTGGGCCGGTCAGACGGCCCCATATCTGCCCGACATATGGGTTGGGTTTGACCACGCAACAAACACACACAACAACACCAACACAGAGACAAACAAACAACAAGGATACAATAGACTACAGAAAAAGCTAGCAGGCCACGACAAAGCACTAACACAAAGCTTCGACAAACAATGCATTCGATCGACGCCACGATCTTGTCGAAGAGGATACAAAAGCCAGGGAGGTGGCTTGCCACCGGAAGAAACCTGTGTGCCATCAAATCCCTGAGGCCACACCTGCGTCACACCAAACATCTCCGCCGCCAAAGAAGGCACCACCTGCCCTCTCGGCCTAGTTCGTAAGGCGCCGCCCCGTCGGTATGGCGGAGTTGCTCCCCTTGAGCATGCATGGATATTGAACAACCAGAGGCCCAAGGTAGGATGACAAGCACCAACACCTCATCTCACGAGACACCTCATCACTTGCACCTCTCTCCTTCAAAACCCACCACAGTGCACGACCCAACTGCCGGACGAAAGCACCACCGAAGACGCAGATAGAAGAAATGCACGACGCCATGGGGCACAACTGGAGCTCTAGCAGGAACCCTATCTACCACTGAGCACCGCACCAATGAAGAACAAATCAACCGCAAACAGCTGCGACCACCTGCGCGACAGCAATGCCTGCCCTCCCGTTGTCCAAAGCTGACGCCTTGAAGAAGGCGAACGACACCGGAGCGCCGCCGCTGCCAAAATCCCAAGCCCTAGGTCAACCCGCTACATCTGACGAACCAAAGGCCACCGAAATCCCGGTGTGGCGGGGTTAGCCAAGTTGCACGCCAAGCAACCACCTTCTTCAGAATCTTGCGCCAAGAGCATGTGGAGACGGCCCTCGGGCCATGACCCACATCCATCGCCGCCTTGCCGCGCGCGCGGCCAAGGGGACTGCTCGTAGCCACGGCGTCCGCCACCTGCTGCAAGGGAGTGACGCCCTTGCCATGGGGGCCGCTGCCTCAGATCAAGAACCCCCCCCCCCTCCACATGGACCATGGGGCGGAGAAGATCCTCGCCGCCGCCTTCCTCGGCGGCCGTGTGGGCTGCCCGGCGACCCCCTCAGGCAACGACGAGAGGGAGGAAAGGGGAGGGGATCTGGTGTGGCGGCGCTAGGGTTTCGCCATGGAGGGGCGACGCGAGGGGAGGGGAAGGGGAGCCACACACAAATCTAGAGATAACTAGCAAGGTCATTTGCCCTGTACGTGTGAGGTGGGTTCGAGGGTTCTGGTTGTAGATGGTCTTAGCGTAGTGGAATATGATTATATTTTTATGTTATTTTGATCTATGATGTTACCTCGGCATCCCTGTGAAAATATATTCAAGGAGATGGGACTAGTTAAGAGTTAAGAGTCATTTCATATTTTTGAATTGCAGAACGCAAGTAGTAAGAGAATATAACTAGCTGGTGAAGATTCCCTTTGGACGAAATTGATTAGGAAAACATGTATTATATTTAAAAGATCTTTGAATTTGTCATCAGACGAAGTTTCTAACCATATAATATTTTTGCATAGTATACCTGTTTTATTAGTCAAATCAAACATCTAAACCACCGACTTATAAACTCAATCAGAAGTATATAGTCCCTCCGTCCCATAGGACATGGAGGGAGTACTTCTTAGAGCATCTCCAGCCGTTGGCCCCAAGGACGTGTCGAAAACCGCCGTCTGGGGGCGAGCCGGCGCTAGTTTCGGCTTGGGTGCCAGTTCGTTCCCAGTCGCCGGCCCCCCAGGTCGCCCCCCAGGCGCCGATATCGGCCCATTTTCGGTGCTTTCAGCCCATTTTCGGCGCTTTTCAGCCCTTTTCTGGCGCAAAACGGCCCAGGCTCGACGTTGTCGTAAATAGTTCACATAGATAGATAGATAGTTCACATATATAGATAGATAGATAGTTCACATAATCTTAACTCTACGATAGATAGATAAATAGATAGTTCACATAGTCTTCTCCTACGACCTACTACTACTAGTCATTCTCCGACTCGGACTCTTCCTCGGTGATGTCCTCCTCCGATGTCTCAGCGTAGGTGTCAAGGAACCGCTCGTCGGTGAAGTCCCAGGAGGACGCATCTCCTAGCTCGACGTTGGACTTAGCGATCGCCCTCCGCGCTCGCTTGTCCTCGCAATAAGCCGCTCGCTCCTTCCTCCTCTTCTTCCTCTCCGCCCTCTTCTCGGCCAAGAACTGGTGCTCGTCGATGAAGTCTTGCGGTAAGTTTTGGCGCCACGTCGCCATGGCTTCCTCATCCATCTCGGCCAAGTTGAGATGATGCGCCCGCCTACGGTTCTTCCGACGATTTTCGTCGGTTCTAAGCCGCGGGGGAGGCGCCAGCTCCTGTGCCCGCTCCAGAGTCGGCACGTCCGTGAAGTTCATGTCCCAACGGGAACGCCGGAGGCGCCACGCCGCAGCGTCGTACGCGCGGGCGCCCTCCTGAGCGGTGTCGAAGGTTCCGAGGCGGAGGCGCACGTCGCCCGACTGAATCTTGGCAGAGTAGGTGCCAGACGGACGAACCCGGACGCCGCGGTAGCCCGCACCTCCCCGGCGGCGAGGCGGCATGGGGGCGCAGTGCATGTCGGCGACGAAACAGCCAGCGGCCGGAGGGGCGCAGTGCGTGGCAGTGTGGAGAGTGCGGTGGGGCGATGGGGCTGCGGTGTGGGCGTTGACCGGCGAGGTAGGCTGCTTTTATAGCCGGAGCTCGGGCGGCGAGAGGCGCATGCCAGGCGACGCGTGGCGGTCGCCCGTGGTGCCGGGCGACGCGACGGGCCGCTGGGCGGCATCAATGGCAGGCGACGGCTCGGCAGTGCGGCAGCTTTGGCATTGATTCCCGCGGGAACCGAGGCGATGAGGACGACGAAGCAGCGCGTCGCTGACAGGGCGGGCCCATCACATTCGCGCCAAAAAACGCCTCGCCCGGCGCCCCCAGGCGACCCCAGCGCCCCGGGTTCGGGTTGGGTCCGCCGGCGCCAGTTTCGGCCCAATCCGGCAATTTTCGGCGTCCTGGGGGCGCGACTGGGCCATTTTTTCAGCGCCGGCGCCTAAAAAGTGGCCTTGGGGGGGGGGCGGCTGGAGATGCTCTTAGCGTTTCTCTTTTGTAGTAGCTGGTAATAATCGAGTTCCTCCGTCCAGTTTGAAATTTTGGTTTGGTTGAGAGTATTGACTGGGATGGGGATCTTTCGATTTCGGGTGCTTGTAATTATGATTCTCGCCATCCTTCAACTCTGGATCCTCCATCCTTCAAAACAACAACTACCAAAACACGAACTCAACGGCGTCTGAATTGATTTTTTTTTTAAATTACAAATAATAATCTAAAATTGCTAATTTCTTTTGAAACACACACGATCTAGTGTTATACTCATGTGAAAAGTAAGGAATGATTTTCCTGGTATTCTGAGTGAAAGAAAACAAAATCGACATTGTATAAATGTTACTATTCGCGCTTTTTTACTCATAATATATATTTTGCCTTGAAGTCAACTTTAGTCCTTCCATTGTGAATTTTTTTATACGAGTATAACACTAGATCACATATGTTTCCAAAATGTTCCAGAAATTTTTGAACTGTATTGCAGTTTCTCAATTATTATTTCAGTTCAGGTGCATTGGGACCCAAGACCCATTGGGTATACCAACCGGCAAATGTGTGCATGAAGGAGAGAGAGAGGGGTAAATGCATGATGTGGACTGAGCAACCAACTGAGAGAGATTCAGAGAAATGAGAGAAGAGTAAATACAGTGAAGCAATGGGATATGTCATTGTTTTCTCTGAATCCCTGTCTCTCTATGACCCACTGCACTGAATGATTACATCAACGAAAAACTGTACTGTTCGGAGGGATCGTGCGGCTCTGTTATCTCTTCCAAGAGCATACTGCTAGTAATTTCCTCTCTTGATTGATTGTTGTTGTGGGTCTTCCTCGCCTCCACTGAGTGCTCCCTTCCTCTCGTCTCAGTCATCTCCATCTTTTCAAGTTGTGAACTGAAACTATGTGCTTATTCTACAGTTTAGCCTATATATGTTCATGATCCCTCAGTTTTGTAGGTATGGTTTCAGCTTCTGTTACAGTTTAACGCACGTTCATGAACTGAAACTATGCTTTCCCTAGCACTAGTACCCAAAGGCATTGAGTATTCACTACCAAACCGAAATTTTCAAACTGAACAGAGGGATCCAATTATTTACCGGTTGCAAAGATGAACGCTAGTGAGTAAAATTAGTAGTGTCATTTCCTGATAAGTGCATGAAGGATGGAGAGATGGTAGATGCCTGGCAGGTGCATGAGGGAGATTGAGAGAAATGAGAGGACGTACAATTATTGTCTCTTGAGTCACTGTCCCTGTATGACCCACTGCACTGAATGATTGCATGACCCACTGTACAATTATTCAGAGGCATCGTGGGTGCTCTCTTATCTCTTGACCTGCTGTTGTTAGTCTTCCTCTGCTCCTCATCTCCACCACCGACAGAAGAGTGCTCCCTTCCTCGCATCTCAGTCCTCTCCTCCATTGCGCGCCCTATTGGTGATCTCATCCAACCTCCGACTCCGAGCTCGCCCGTGGACATTTTTGGGTATATTTCTTGTCGATTTGTTTCCAGTTAAAGTAGCAATGCTTGCATCTAAAAGTTATTATTTCTCCTGCTTGAGATTCTGATCCATTTGTGTTGATTGAATCCAGGGTGGGATCGTGGGGCTCTGTATCTTTCTAGTTCAGAGTACTATTGTGCTTCCTCTGCTCCTCGTCTCCGCTTCCAACTAGCGGCCTGGTAAAAATTTCACTCCCATCTTTATTGCTATTCTCTTCTTATTAATCTGCTGATTTGCTAGATTGTAAAGTTCTTATTACTTATTCTTCCAGATCGAGTGAAGCGACCTCCGCTGCCGTCTTGTCCCCTAGGACGCAGTTGTGTCCTGCTCTGTCCTGCTCTGCTGGTGAAGCATCAACGGTGATGGCCATCCGGTGTTGATGCCGCCGCTGTGTGCTGCTCTGCCGTGAAGTATCAAAGGTGGGTTTAGATGGATATCGCAGTTTGGATTGGCAACTCACTTCTAGAGCTCGTTGAAATTAAAGCGTCACACTGTTTGGATTGGATTACAGCTCCCCATATTCATCTATTTTGTAGGTTTCCGGTTATTATTCAGAGCAACACGCTCACGGATCTGACCTCTGGTGCCCAGCCGCCGCTTCCGCTTTGTTGTCCCCACAACACGACCATCCGACGAAGACGCGGCCGTTGTGTTCTGCTCTACTGTGAAGCACCAAAGGTGGGCTTGGTCCAGATCCCTACATTCATCGATTTGGCTTGGCAACTCCGTTCTAGAGCTTGGATTGTGTTACAGATCCCTACATTCATCGATTTGCAGGTTTCCAGTTAATTGATCCAGAGAGCTCTCATGGATATTGTCACGGGTGCCATTGCCAACCTGATCCCCAAGCTGGGAGAGCTGCTTGTGGGGGAGTACAAGCTGCACAAGGGCGTCAAGAAAAATATCGAGGACCTCCTGAAAGAGCTCAAGACCATGAACGCTGCGCTCATCAAGATTGGTGAGGTGCCGCCGGACCAGCTCGACAGCCAAGACAAGCTTTGGGCCGATGAGGTCAGAGAGCTCTCCTACGTCATTGAGGATGTGGTGGACAAGTTCCTCGTACGGGTCCATGGCATTGAGCCCGATGACAACACTAACGGATTCAAGGGGCTCGTCAAGAGGACCACCAAGTTGTTGAAGAAGGTCGTGGATAAGCATGGGATAGCTCACGCGATCAAGGACATCAAGAAGGAACTCCATGAGGTGGCTGCTAGGCGTGACAGGAACAAGTTCGATGGTATTGCTTCTACTCCTACTGAAGCAATCGATCCTCGTCTTCATGCTCTCTACATAGAAGCCGCAGAGCTAGTTGGCATCTATGGGAAGAGGGATCAGGCACTCATGAGTTTGCTCTCCTTGGAGGGCGATGATGCCTCTACCAAGAAACTGAAGAAGGTCTCCATTGTTGGATTTGGAGGGTTGGGCAAGACCACTCTTGCCAGAGCAGTATACGAGAAGATTAAAGGTGATTTTGGTTGCCACGCATTTGTTCCTGTCGGGCAGAACCCTGATATCAAGAAGGTTTTCAGGGATATCCTCATTGACCTCCGAAAATCAAACTCAGATCCTAGCAACTCTAACTCAGATCTTGTGATACTGGATGCAACACAGCTTATCGACAAGCTTCGTGAATTCCTTGAGAACAAGAGGTATGCATCACTTACAGCAAAATTGTGCACTATTATGACATGATTATTTCATATGCTAGTTGTACAAGTAATACCGAAAGTGTCTAAACATATTGTAGCGGGAGGGTTCAGAATAATTTTTCCATTGAGGCCACCTTTACTGGCATATGCAGTTCACACTAAACTGTAGACCTTCTAGGTGATAAATTTCAAGCATGTAGAAGTTTATACTATGTTACTATGTCTGAAACTTATTGGCATTGGTCCAGGAAATCCATCTAAACATTTTGCGTTACACCAAAGTGAGTGTAATGACAACTGTAACAAGTAAGGAAAGAAAGAGGAAGATAAATGTTACAAAAATTTAAATCAAACTTATTACCATTTCTTTCTTAACCCACCTAGTTTTAAAACACATATTCTAAAGGGTGGCGATGCGTGCCAAAGGCTGCCCAATAAACTTCCTAAAATATTGTATATTGGATCCAAACAAGAAGTTAAATTGTCCCTTCACCAACTCATTATATTCACCATGTATGCCTTTTTTTCTGGAGACAAACTATCAGCAAAATATAGATGAAAAGACACTTCTTTTTCGGTACAACCCCCTTAGACACATCAATGGACCAGATCTGCCTCTACCCCTTCATAAGAAAGGGTATGATTGTCCTTTTACGAAATACGTCTCCCGGACCGTACAACAGGGAGCAGTAAACTGGGCCGGCCCAACAGCAAACGATGCACGTAGTGGACTTGTACTGAAACAGTGGAACAGCCTAAAAAATTAAAATTCATTCTAGCAAGTATCGAGCACAAGACCTCCCACATTAACACAAACCAACACAACCACTTGATTGAGGCAGCTATCTTGACTAATTTGCAGCGCGGCCTTTTAAGAACTATGACCAGTGACATATTTGACCATTATTTTGGATATTAGAACGTGACTTTTTTAAAACGTGGACATTTTTCCAAAATGTAAACAAATGTTTTAAAATTCTAACAAATTTTCCGAAAAGTGGAACACATTTATAAAGTCTCAACCAATTTAAAATAATGAGACTTTTTTTAAAATTCTGACAATTTTTTAAACACAAACTATTTTGAAACTTGAAACTTGTTTGAAAATGCGGCCATTTTTTGGTATTTTCGAACTTTATTGAGAAATATGAATAAATAAATAAATAAATAAATATGTTGAACATTTTTTGAAGTTTGCGATTTTGAAAGATAAAAAGATTCATGTACCTTTTTAAACATTCCCAAAACAGGTTAAAACCGGCCTAAAACCATCTAACAAGGTTCTCAAAACCGGCATCTGCAGAGGCAACGAACAAATTAAGATGGGCTGGATTATGTTCAATCGTATCGATTGTGTGTGTGAGAAACAACAACTGTTTGACGTAATAAATGTCAAATAGAGTTTCCCTCAACACATGCCCGATGGGATCGTTGTCGTCCGCTAGTTAAGCCAACGGCTAACCATTTTGTTTTCTATTTTTTTGTTATTACTTTTGAAACTTGTTCAAAAAATTTGAAAATTATTTGGAATACCTGCAATGTTCTTCTTTTCAAATATTTTTTAGAAATTTCAAAAAAATCTGGAATCTCAAAAAATGTTCGTGTTCTCGAATATCTTGTTCACAAAAGAAGAAATGCACGTGTTTCAAAAAATGATAATTTTATAGAAAAGTAAGAATTCCCTATTTTTGTTCAAATTTTTTATCAACTGTTCGAAATTTGTTATCAATTGTCGAAAAGGTGTTTAAACTTGTAATTTTTTCATAAATAAATTTAAATTTCAAAATTGTTCACAAATTTCAAGAGATGGTTAGTTCACAACAAAATGTTCATGTTGGTTGATAATGACAGAAGTTACAAATAACAAAAGCTTTTAAATTGGGAAAATTTACATGCTCATGGGACTCTGTCTGGCCTAGTCTAGCACACTCCTCTCAACTAGTGGCCTTTTGACTATGGATAAATGACCCCAACTTTTTTTAGCAGCCTGGTACGGACACATGAGGCATCCGTGCTAGGTTTGAACGACTTCCGACATTGTTTGCATGTTGTGACATGTCCAACCATTTATTGGTCCTTTTTCATCAAGAAAACTCTAGAAAAGGAATTCTTTTCCAAAACTCAAACAACTTGGCATGGTGTCTAGAATTGGCCATATAAGCTCATGGAATAAATTTGGGTCATATGATGGATGCCGAAAAATGGTCTCAAGCGGACCCTTCTCGCCTACTTGAACACTCCATGATAGGTGTGAATGAATTCCCAATAACATGGTCTAATTTAAGACATGCATGAAATGACCCCAACTTTTGCAAGCAGGTGGGAATGCCCGCGGTAGGCATCCATGATAGGTTTGAACGAATTCCTACACCATATGTAAGTTGCGTCACGTCTAGCCATTTATTGGTCATTTTTCATTGAGAAAACTCCAAAAAATGCAAGAGTTGTCGAAAACTCAAACAGTTGGCCTGATACCTTGAAATGGTCATGCATGGCCATGAAAACAAAATTGGGTCCATTTAAGGGATGTCGACAAACGAGTTGCTCCCAAACGGACCATTTTGGACCCAAACACTCTCTGTTGAACATGATATTTTATTGGAAATCTCAGGATCGACCTCAACTTTTGCAAGCAGGTTGGCATGCCCATGTTAGGCGTCCGACCATTTATGGGGCTATTTTCACCGAGAAAACTCTAGAAAAAGCAAAACTTGTCAAAAACCGAAATAACTTGTTATGGTGCCTTCAAATGGTGTCAATGAAAAAAATTGGGGCCAGTTCAAGGGTGTGATATGAGAATATGAAGCCTATTATTTTGGAGATGGGAATGAAGCAAATATTTAGGTCGTTGGAAACATGCTATACGGACCAATGAAGCATGCTAAATTTTGAGTTGATGTGGAGGCAAAAAACGTAGTTTTGGTGCACTGTAAATGAATAGAGACGATTTTTTTTGGTGTCATTTTAGTTGCACTGGAAGTAAATTTTGGTAACATTGGATAATTTTTTTGCTTGATCGATTATTTTCCTTTAAAATCTTGAATTAGTTTTCTAAGGTTTATTTTGCTTTCTTAAATCGGTGATTTGTTTCCCAACTCGTGTGATCTATGGGCAGTTATTAGTATCCACCAAGCACATTTAGAAAAAGCTTTGCTGGCGCACGTTCTATGCCTTCCAATCGGATCTGTGTGTCAGTTTCTACATATTGAATGATGATGAAGTGGTCTGATCTGATTACTAGCAGTTCTCTTCTTTTAGCGTCACATCTGGCGCATGAACAAGTAAAATCAAATGTTGTAGGTAGTACAGCAGCCGTAGGTCTTCCAGTTGGTTACTGCCAGTGGATCGTTACAGAGAGGTAACGCGATCGATTCTCATTACCACCATCTAATTTTGAATGTTATGGAAAAAATTAATGGGCGAGCCCAGGACGCCAGAATGTTAAAGAAAAATATTTATGGGCCTAGCCCATGACGCGTGGGGGCGACTATTCGCACAGAGTTTTTTTTTTTGCATGGCTATTCGCACAGAGTTGGGCACTCCAAATGAACAAACATATGGCGTATACGTCTCAGCGGACAACCCATCATGCATGCAAAAATGGATACGATATGTGAAATGTCTGTACTATCCTTTATACGTTTTAGAGGGGGGTTGTACCGAAGCAAAACTCATGAAAAGATAATGCCATACTATTTGAGTGAGTCTCCAAGTGGTTCATTAGCTGCCATATATTAAATTGGTGGGCCAATCTATACTAGGATCTTTTCGATATATCTACCTGACCATTTTAACTTCTGTAGTTAATTGTATTCTACATATGGTCACTGATTCAGCAATTGCTAGTTGTGTTTTACTCCCTTGAATCTTAAATATTTATTCATTTTGTTTGTCGTTTTAAGATGCATGCCTGTTACGAGTTTGTTTCTGAATATACGCCCTTATAGATTGTTAGTTCCACCCATATATTCGTATGCCCTCAGTTTTTTGATATGTGTAGACCTTACACTGATGCTCTGAACTAATGTAGGTACCTCGTAATAATTGATGATATATGGGATGAAAAATTGTGGAGATACATCAACCTTGCTTTCTCTATCAATAACAATCTAGGAAGTCGGCTAATCACCACAACCCGAGATTTCGATGTCTCCAAATCATGCTGCTCATCAGCTGATGATTCAATTTATCATATGAAACCTCTTTGTACCAATGACTCCAGAAGGCTCTTCTATAAAAGAGTATTTGCCGACGCTAGTGGATGTCCAAGTGAATTTGAACAAGTGTCTAAAGATATATTGAAGAAATGTGGCGGGGTACCACTAGCCATCATTACTATTGCAAGTGCTTTGGCTAGTGGCCAGCAAGTGAAACCAAAGCATGAGTGGGATATTCTACTCCAGTCCCTTGGCTCCGGAGTAACAAAAGATAATAGTTTGGCTGAGATGCGGAGAATACTATCTTTCAGCTATTATAATCTACCGTCTCATCTGAAAACTTGTCTACTTTACCTATGTATATATCCAGAAGATAGCACCATTGGTAGAGATAGACTGATATGGAAGTGGGTGGCCGAAGGATTTGTCCACCATGGAGATCAAGGGACCAGCCTGTTTTTGGTCGGATTAAACTACTTCAACCAGCTCATTAATAGAAGTATGATCCAGCCTATATATGATAATGCAGGCCAGGTATATGCTTGCCGTGTACATGATATGGTTCTGGACCTTATCTGCAACTTGTCACATGAAGCAAAGTTTGTTAATCTATTGGATGCCACTGGGAATAGCACATCTTCACAAAGTAATGTTCGTCGTTTGTCCCTTCAGAATAAAAATGAAGATCATCAAGCCAAGCCTCTCACAAATATCATGAGTATGTCACGAGTGAGGTCCATTACTATCTTTCCACCTGCTGTTAGTATCATGCCAAGTCTGTCAATGTTTGAAGTTCTGAGTGTACTTGATCTGTCGAACTGTGATTTGGGACAAAGTAGCAGCCTGCAGCTTAACCTAAAGGGTGTTGTACATTTAATCCACCTAAGGTACCTTGGTCTAGCAGGCACTCAAATTAGTGAACTCCCGACTGAGATAGGAAACCTGCAGTTTTTGGAGGTGTTGGATCTTGGAGATAATTATGAGCTAGATGAATTGCCTTCCACTCTTTTCAAATTGAGAAGATTAATCTACCTAAATGTTTATCTCTTTAAGGTGGTTCCAACTCCTGGTGTGTTGCAGAATCTGACATGCATAGAAGTGTTGAGGGGGATCTTGGTCTCTCTGAACATTATTGCACAAGAGCTTGGCAACCTGGCAAGGCTGAGGGAGCTTTCGATTCGCTTCAAGGATGGTAGTTTGGATTTGTATGAAGGTTTCGTGAATTCTCTGTGCAACCTACATCACATAGAATGCCTAAGTATTGGTTGGAATTCTGAAAAAACGTCTTTTGAACTGATGGATCTCTTGGGAGAACGCTGGGTGCCTCCTGTACATCTCCGCGAATTTGTGTCTAGGATGCCCAGCCAACTCTCTGCACTGCGAGGGTGGACAAAGAGAGACCCCTCGCATCTCTCCAACCTCTCCAAGTTAATCCTCTCGTCAGTGAAGGAAGTGCAGCAGGAGGACGTGGAAATCATTGGGGGGTTGCTGTCCCTTCGCCGTCTCTGGATAAAGAGCACCCACCAAACACAGCGGCTGCTAGTCATCCCTACAGATGGGTTCCGCTGTATGGTAGAATTTTACTTGAATTGTGGGTTAGCAGCGCAGATAATGTTTGAACCAGGAGCTTTGCCGAGGGCGGAAGAAGTTACGTTCAGCCTGGGCGTGCGGGTGGCAAAAGAGGATGGAAACTGTGGCTTCGAATTGGGCGTGCAGGGGAACCTGCTCTCCCTTCGGCGGCAGGTCTGGGTTAGGATGTATTGTGGTGGAGTGAGGGTTGGGGAGGCAAAGAAAGCGGAGGCTGCGGTGAGGCACGCACTCGAAGCCCATCCCAATCATCCCAGGATTTATATTGATATGTGCCCGCGTATAGCAGAAGGTACTCACGCCGCACCTAACTACTCACGCTCAACTCCCATCCCAGTCATCCCCTGATTAGGTATATGTTTTTTTCGAAATGATGGACTGACATTATTACTTTCTGCATTGATTTTGATCTCTGAATCTACCAAGATGCTCATGATGACGATTTGTGCGAGGACGAGGAGATCTGATTTCTGATCCAGAGCGTCTCACATTGCATCAGATGTGCTCTCAGGTATGTAGCAGATATTTGCATGTTATGTTTTCCATCTTTCTCCCTCGTCTAGAGCTCAGCTTTTCGTTTCATTCACCGACTATGCTATAGTGATTATACGGAAATTAGAAACAGATTAAGGTATTTACAAAAATTGCTTAGACACAAGGATCTGATCAGAAAAGTGAACTTGGCAGTGTACTGTGAAACCTGCCCAGTCTCTTTTTTGGCAAAGGGCTGATGAAAGAAAATAATTATTTTATGCAAATTTGTAGAAGGATAGTTAATAATGGGGAATTAGCCACAGATCTATCTTTATGTAAGCATATCTCTGTAGCTAGAGTACTACATGCTAATTTCTCTTTATGTAAGCATATCTCTGTAGCTAGAGTACTACACGCTAATTTCACTGCTCTAAAATTTAGGGGGGGTCTGTATGGTGAAACTGCTGAGCTATGGCATCAATTTCTTGCTGATCGTGAAGGTTTTAGTCTTGAGGAATGAGCAAGATTCATGTAGATGACCCCACAAAATAAATATTCATTTAAATGGCTTCTAGAGAAGAATGAAGTATTTTCCGTGCATCCTATGTTTATAGCTTTGAAAGCTCAACAAGTGAAATGACTAGAAATATAATTTTGGTATGTAAAAGGTCAACTTAATACCATAGTATTTTTGTGGTTAGCATTCAGAAATAGTATCAGCAGAGTTGATCTGAAGCGTCGAGGTAGTGTGGTATAGGAAAGTGTCTTTTTCGAAGTAGAAAATAGCCAGTGCATCTTCCTAAAGTGTTTGGTTGCTTGTTATATTTGCTTTTTGTGTTGACAGTCCGGATTTTGTGATTGGGTGATGAATTCCAGGGATAAAGATAGGAGTTTGATTTTTGCTGGGATAGGGTCAGTTATTTAGTCTATTTGGAAAGCGAGGAATGATAATTGCTTGAAAACTATTTTTTCCATGGATCCCAACTGAGTGTAGGTTGTAAAGTTTCATATGGTATGTTTTATTAGTCTAGCTTACAAAGGAAAGAATTTCAGGATGTGCAGGTGGCAGGGGCGCGGCGTTACTGCTGGTGGCATCGAGGATACATGTTTCACAAACATTTTGGATGGGCGCCGGACGTGCCAAGAATGGAAGTTGTGTGATGAGCTTCTTTTAAATAGCTAAGAAAGAGATATTGCTTTGTTTTGTAATGAATAAGTAAGGGCGTTGGAGTGAATTACAAGGATACCTTTGCTTTGCTTCAGTTGAGGGCCATCGTTGCTGCTCTGTTTTGCTCTGTTTTTTGTTTTTAAAGCTGAACCGGACAAACCTGAGGTTGTATTATCAGTTTCGTGATGAGTGAAATGAGGGGTTCACCTAAGCTCCTTAATTCGAATAAAACTGTGATTCACTGTTGGGATTTGTTACTTGAAAAATATTAACTAGTTTTCCTATTTTTAGTGTGTGCTTCCAACTGACAAGCAGTAGTATTTTAAAGTGTGCTTCCATCTAACAAGCATGTATACCCCATGGTAGCAAATTATTTTCGTATTTTTGGAAGTGTCGTCTCGCGAAAGGACCTAGAACGAAAAGGGCTATATATGTTTGTACAGAGAGTGGCAGAGCTACAATGAAAAAGATGAAGAGCACAGAAACTTCAAATTTTGGAGCATATAAACTATTGAGTGAATTATATATTTATACGCTACCAAATTAGAAATTATTGTTAAACCGTGAGCTAGTAACAATGGCTTTGTTCTGAACTTGTATAAACTTTAAAAGTGGCTTTTGTTGGGCAAACATCACTATTCTAATGAGCCATTGACGAAATTTTTCCACCCGGCTGACTCTTTTGTGTAGTGCCCAGCAGTTAGGTGCCACACCACACTGTGTAGCGCCTGACTGATAGGCGCTACACGCCTAGCCAGAGTCACATCCCGGGTTGTCTGAAACTTGCTAAGCCAATGTGCAAAGTCTGAGAGTTAGGCGCTACACTGTATAGGCGTTGCACTAGTGGCTACAATATTAGCCACTACATCTAGAAGCAGCCCGCTTTTGAGGGAGTACTTCATCACTGAAACTTGGTAAGAAAAAAAAAATCTCTGAATTCCGCTTTTGAGCATACTTCAACACTGAACTTCAAAAAATTGAAAACCTGAAAAAAAGGAAAGAAAAAAAAACATTAGTGCCAAACTGCCTCTCACCACTCACCTATGCTAAGAAAAATGAAAAGGAAGAAAGAAAGAAAAAAAGCAGCACCGATCGCTGATAAAAAGATATGCAGAGCTCTGAAGTATGCTACTGAACATAGATTAACACTGAAGTATAGACAGCGCGTAATGTAATCCCCACAACTACAGCGAGACAGACATCACATGGGAAAAAGATCACCGGCGAGCAGGCGACGGGCGGTGATTCCGGGCGAGCATGCTATATATCCATCCCCTCCTTCCTTCCTCCCGCTGCTCCGGTCCGCCTCCGCCTCCCTCGTCGGCGACGCCCGGACCCTCGCTGTTCCGGAGGCAGCATCGATCATCTGCTATTCGCGGGCATCAATCAAGTGAATCCCAAGCACGCCCTGACCCTCACTCTCGTCATCCGTCTCCTTCCATTAATCCTATATATAATTATATACAGTATCTCTCTCAGTGGTGATGTCTCACTCACCACACCCAAGGTGTTTGACAAAATGACCAACTAAGATAAGGGTGGCTTTCACAGCACGTACTAGTAGTATTTGTGTTTTCCTTTCTCTCTGCTAATGTAACTAATGTGGGTCTAACATTTCTAATGTCGTGGGTGGCAGTGTGAGCGGCTGCAGCTGCACATGACGCCGCTCAGTGCTGATTTGCCGCGGATGCATGCTGCCATAGTAGTGCTGGAGGACGCCGCATCAGCCTGTTTCAAGAAATCGAGGCTGCTTCCTGGACGCCCTTGAGTTACTCGACGAAATGCACAGGAGAGAGAGAGACTGCGGTCGACCACAACAACGACACTATTGGCAGGTGTGTAACAACTTGCTTGCCTTCACTCTCTTCCTATTTTTCTGCATGTAGATTAATGTACTAGTTTATGAATGCATAGTTTGTAGCATGTCCGGCTCCTCCCCACCACCCTTAGAGCGCACCCGCTGCTCTTTCCCGCCTCGACTGCAGGCGTGAGCTCCGACGACCCCTTCCTATAGCGTTAGCACTGCTCACGTCCGCCTCAAGGCACTGCCTCCATCCGTCCAGTTTGAGATTGGGACGTGTATCTTTGGGTCTCGGGTGCCGATGCATCCGAAAAGAGAGAAATAATTTAGAAATTTCAAAAATAAATCAAAATTGTGATTTGTTTAGAACAAACATGATATTGTGTTACACTCATGTCAAAAGATTCACGAATGAATGACTTTCATGTACTCTAGGCGAAAGAAACCAAAATCGACGCTATATAAAAGTTACTATTTATGCTTTTATACTCACTATATAATATCTTTTTTGCCCTCAAGTCATTCTTTCGCAAAACTTTCTATACGAGTATAACACTATATCAAGTATGCTTCCAAAATGTTCTAGAATTTTGCGACCTTTTTTGCAATTTCTAAATTATTTTTTCAGTTCAGATGCATTGGGTATTTTCGGTATCTGATTGCCTTTGAGTATTTTATAGGAGTAATATGCATGCTGTGGACTGACCAAACAACTGAGAGAGATTCAGAGAAATGAGAAGAGAGTAAATGCAGTGAAGCATGGCTGGTGCCTGGTGGACGGACCATATACAGTGGTATGTAATTATTTTCTCTGAATCCCTGTGTCTCTATGACCCACTGAATAAACACATCATCCCAAAAGCAGTTCTGAGGGAGTGTGGGGGCTTCTTTCCACGCCCACCCACCGAAAGCACTGATGAACAAACACATGAGCCGAAAGCATTACTGTTCCGAGGGATTCGTATCTTGACTTGCTGTTGTGGGTCTTCCTCTGCTCCTCGCCTCCACTGAGTGCTCCCTTCTTCTCGTCTAAGTCCTCTCCTTCTTTTCAAGTTGTTTATTCTCCTGAAACTATGTTTTTATTGTTACAGTTTAGCATTTGTTCATGATCGCCCAGTTTTTGTAGGCAAGGTTTAAGGCTCTGAACTTCCAAACCATAATTAGTTTCATATTGGGGGTCTGTGGCGCGGATTTGTTAGCTTTACACGTTCTCGATTCTTGTGCTACCTTCAATATGGTGCAGAATTTTGTGTTGTTAAGAGCATCTCCAACAGCCGCGCTATACTAGCGCCACGCCTGAAAAAACGCCATTTTAGCGCGCGCGCAACGCGTCGCACCGCTCCAGCGGGCGCCCAAAAAACGCGCGCGCGCTAAAACGAGTTGGGCGCGCTGGTGAATGCCCCATCCCGCGCTGTTGATTTGGAGCGTTCACTTCCGCGCGCGGCACACTCTCGCGCGCGCGCGAACACTCATTTCTTCCACCGCGCTCGTTGCTCCCCCGCTCTCTCCCTCCATCCGGCCGCGCCGCCGCCGCCGCTCGCGCCGCCGCCGCCGCCGCTCGCGCCGCC.
It was found that YrLumai gene was linked to the wheat stripe rust resistance phenotype and that mutation of this gene resulted in loss of the wheat stripe rust resistance phenotype. Therefore, yrLumai genes can provide stripe rust resistance, and have important application value in the cultivation of wheat stripe rust resistant varieties.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples and comparative examples of the present invention are conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers. Wherein:
The experimental materials used in the invention are 3000 parts Lu Mai EMS mutant, wild type lupulus 15 (disease-resistant control material) and CB037-PstS (disease-sensitive control material); the stripe rust races used were mixed spores (CYR 29, CYR31, CYR 32), single race CYR34.
The wheat material of the invention is planted in fields and growth chambers, and large Tian Dongji wheat is generally planted in the first 10 months of the year and harvested in the next 6 months of the year. The material to be inoculated needs to be inserted with 2 rows of disease-sensitive contrast in 20 rows to be used as an induction row, and the field is normally managed; the wheat is germinated in a culture dish in advance, transplanted when the seeds grow 2cm tender stems, and the materials are transplanted into a small square basin or a gallon basin according to the research purpose. The growth conditions of the growth chamber are as follows: the temperature in the daytime is controlled at 23 ℃, and the illumination is carried out for 16 hours; the night temperature is 18 ℃ and the darkness is 8 hours. And (3) inoculating the physiological wheat seeds of the wheat stripe rust by adopting a manual inoculation method. The physiological seed inoculation of the field stripe rust bacteria generally adopts a comprehensive inoculation method of injection inoculation and spore spraying water, and the inoculation time is generally carried out in 3 months each year; the artificial climate box inoculation generally adopts a smearing inoculation method, and if the disease is insufficient, smearing inoculation or injection inoculation is carried out again.
The PCR primers and sequences of the invention are shown in Table 1.
Table 1: PCR primer used in the present invention
Example 1: screening of susceptibility mutants
According to the invention, ethyl methylsulfonate (Ethyl methanesulfonate, EMS) is utilized to mutagenize wild type lupulus 15, when the strain is selfed to M 4 generations, 3000 parts of mutants are inoculated to mixed spores (CYR 29, CYR31, CYR 32) to identify the phenotype, and 5 parts of lupulus 15 stripe rust mutants are screened. Inoculating mixed spores (CYR 29, CYR31 and CYR 32) corresponding to 5 parts of the infected material for M 5 generations, and verifying that 4 parts of the 5 parts of the material show the infected; if the M 5 th generation of the more toxic CYR34 is replaced, 5 parts of the material all show a disease. DNA of the infectious material was extracted and verified by using 1BL/1RS linkage marker Iag95, and the detection results showed that the infectious individual plant had genetic background of Happy 15 (FIGS. 1 and 2).
Example 2: isolation of the yrluma. I15 gene using transcriptomics
The invention uses the RNA-seq sequencing result to compare and analyze transcriptomes of disease-resistant material Lu Mai and 5 disease-sensitive mutants, and uses GATK (Genome Analysis Toolkit) to carry out SNP-calling, and filters and analyzes the SNP. Total RNA was extracted using TRIzol reagent and related methods (Life Technologies, GRAND ISLAND, NY, USA). Library construction and high throughput double-ended sequencing (HiSeq 2500, illumina; paired-end, PE 150) involved in RNA sequencing was undertaken by Beijing berui and Kangbiotech Co., ltd (Berry Genomics Company, beijin, china).
The invention discovers a new gene, which is named YrLumai gene, and the sequence of the gene is shown as SEQ ID NO.1 and SEQ ID NO. 3. Single base mutations were made in the gene at all 5 of the susceptibility mutants subjected to RNA-seq sequencing.
Example 3: sequencing confirmation of susceptibility mutants
The sequence of the amplification primer is shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 according to the gene design. The 5 mutants of example 2 were amplified and sequenced, and the sequencing data were aligned with the gene region of the mutant parent YrLumai, and it was found that the 5 susceptible mutants had single base mutations in the gene (Table 2), all consistent with the results of transcriptome sequencing in example 2.
Table 2: mutation of YrLumai Gene in a disease-susceptible mutant 1
1 The table describes the mutation of the 5 mutants in the YrLumai region.
2 The left letter is the base of the disease resistance YrLumai gene, the middle number represents the base position of the cDNA level relative to the ATG of the initiation codon, and the right letter is the base after mutation.
3 The left letter is the corresponding amino acid in the disease-resistant YrLumai protein, the middle number represents the position relative to the first amino acid, and the right letter is the mutated amino acid.
Example 4: yrLumai15-2728 marker detection YrLumai gene
YrLumai15-2728 marker is PCR marker designed based on YrLumai coding region, and its nucleotide sequence is shown in SEQ ID NO. 4.
The primers used for amplification YrLumai-2728 were YrLumai-FP (SEQ ID NO. 5) and YrLumai-RP (SEQ ID NO. 6). After amplification by the primer, if a 1261bp specific band appears, the wheat YrLumai gene is carried; if nonspecific amplification occurs, it is recommended that the corresponding wheat is not resistant to wheat stripe rust.
The specific method comprises the following steps:
1. material selection
The plant material used in this example was wheat variety Lu Mai15 containing YrLumai gene, and the susceptible wheat variety CB037-PstS containing no YrLumai gene.
2. The specific method for extracting the wheat genome DNA is as follows:
(1) The leaves are taken and put into a centrifuge tube with steel balls, and the centrifuge tube is put into liquid nitrogen to be frozen for 30 to 60 seconds.
(2) The centrifuge tube was removed and placed in an adapter and placed in a tissue grinder for shaking grinding to powder (18 hz,40 s).
(3) 800. Mu.L of the DNA extract containing RNase (100. Mu.g/mL) was rapidly added, and the mixture was placed in a water bath at 37-42℃for 30 minutes, and inverted 5-6 times every 10 minutes.
(4) The water bath sample was placed at 4℃or on ice for 10min. After a short centrifugation, 400. Mu.L of 6M ammonium acetate pre-cooled at 4℃was added, mixed upside down, and then placed on 4℃or ice for 15min.
(5) Centrifuge at 12,000rpm for 10min, aspirate 450. Mu.L of supernatant into a fresh centrifuge tube containing 450. Mu.L of isopropanol and 45. Mu.L of sodium acetate, mix upside down and then place at-20℃for 30min.
(6) Centrifuge at 12,000rpm for 10min, pour out supernatant, then add 500. Mu.L of 75% ethanol, centrifuge at 12,000rpm for 10min, then pour out supernatant.
(7) The above step 6 is repeated.
(8) And sucking out redundant liquid by using a pipetting gun with the measuring range of 200 mu L, and drying on an ultra-clean bench until no alcohol smell volatilizes in the centrifuge tube.
(9) Add ddH 2 O100. Mu.L and dissolve DNA overnight at 4 ℃. After DNA is dissolved, the DNA is preserved at-20 ℃ for standby.
DNA quality detection
Detecting the purity of DNA: after the DNA is completely dissolved, the spectrophotometer is calibrated, 1 mu L of DNA solution is taken and placed in the spectrophotometer to detect the purity and the concentration.
Detecting the DNA quality: 2. Mu.L of DNA was dispensed into a 200. Mu.L centrifuge tube, 3. Mu.L of 6X Loading Buffer and 15. Mu.L of ddH 2 O were added thereto, and the DNA mass was detected by agarose gel electrophoresis at 0.8%.
DNA purity requirement: the theoretical value of A260/A280 is about 1.8 when detecting the purity of DNA. An impurity such as protein may be present when the concentration is lower than 1.6, and contamination with RNA may be present when the concentration is higher than 2.0.
DNA quality requirements: the complete DNA electrophoresis result shows that a bright and clear band exists, and if a tail or a small fragment exists, certain degradation can exist.
The PCR amplification system and procedure were as follows:
The system was formulated on ice according to table 3.
TABLE 3 composition of the reaction system
After the reaction system is prepared, PCR amplification is carried out on a PCR instrument, and the PCR reaction procedure is as follows:
Pre-denaturation at 95℃for 3min;
Denaturation at 95℃for 15s;
annealing at 55 ℃ for 15s;
Extending at 72 ℃ for 30s;
extending for 5min at 72 ℃;
Preserving at 15 ℃.
The PCR amplified products were subjected to gel electrophoresis analysis, and the results are shown in FIG. 4.
In summary, the mutant mutation site analysis results show that the YrLumai gene mutation results in loss of wheat stripe rust resistance phenotype, and the disease resistance gene can be detected through the marks YrLumai-2728. Thus, expression of the YrLumai genome full-length expression frame (SEQ ID NO. 3) provides wheat with resistance to stripe rust, and mutation of this gene results in loss of wheat stripe rust resistance trait.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A wheat stripe rust resistance gene YrLumai, wherein the wheat stripe rust resistance gene YrLumai is a nucleic acid molecule as set forth in i) or ii) or iii) below:
i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;
ii) the nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 3;
iii) A nucleic acid molecule other than i) or ii) encoding the amino acid sequence shown in SEQ ID NO. 2.
2. A protein encoded by the wheat stripe rust resistance gene YrLumai of claim 1; the amino acid sequence of the protein is shown as SEQ ID NO. 2.
3. A recombinant expression vector or genetically engineered bacterium carrying the wheat stripe rust resistance gene YrLumai of claim 1.
4. Use of the wheat stripe rust resistance gene YrLumai of claim 1 in (1) or (2) as follows:
(1) Improving the resistance of plants to stripe rust;
(2) Cultivating a stripe rust resistant plant variety;
The plant is wheat plant.
5. The use according to claim 4, wherein the plant is wheat, barley or rye.
6. Use of a recombinant expression vector or genetically engineered bacterium carrying the wheat stripe rust resistance gene YrLumai of claim 1 in cultivating a wheat plant of the genus wheat with increased stripe rust resistance;
the wheat plant is wheat, barley or rye.
7. Application of protein coded by wheat stripe rust resistance gene YrLumai in improving stripe rust resistance of plants;
The wheat stripe rust resistance gene YrLumai encodes a protein shown in any one of the following (A1) or (A2):
(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;
(A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag;
the plant is wheat, barley or rye.
8. A method of increasing resistance to stripe rust in a plant, comprising: transferring the stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 into a plant to obtain a plant with improved stripe rust resistance;
or up-regulating expression of stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 in plant genome, and screening to obtain plant with improved stripe rust resistance;
the plant is wheat.
9. A method of reducing resistance to stripe rust in a plant, comprising: inhibiting the expression of stripe rust resistance genes shown in SEQ ID NO.1 or SEQ ID NO.3 in a plant genome, and screening to obtain plants with reduced stripe rust resistance;
the method of inhibiting expression of a stripe rust resistance gene in a plant genome comprises: mutating or knocking out all or part of the sequence of the stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO. 3; or interfering RNA is used for interfering the expression of stripe rust resistance genes shown in SEQ ID NO.1 or SEQ ID NO. 3; or silencing the stripe rust resistance gene shown in SEQ ID NO.1 or SEQ ID NO.3 by using a gene silencing system;
the plant is wheat.
10. The method according to claim 9, wherein the stripe rust resistance gene shown in SEQ ID No.1 is subjected to at least one of the following mutations (1) to (5):
(1) Mutating the 211G base to an A base;
(2) Mutating the G base at position 1396 to an A base;
(3) Mutating the G base at position 2023 to an A base;
(4) Mutating the G base at position 2633 to an A base;
(5) The G base at position 1503 is mutated to a C base.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102618638A (en) * | 2012-01-13 | 2012-08-01 | 山西省农业科学院作物科学研究所 | Molecular marker for wheat stripe rust disease-resistant new gene Yr50 assisted selection and using method thereof |
| CN109321582A (en) * | 2018-06-01 | 2019-02-12 | 山东农业大学 | Application of the aegilops tauschii Yr4DS gene in the plant stripe rust resisting breeding of wheat race |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618638A (en) * | 2012-01-13 | 2012-08-01 | 山西省农业科学院作物科学研究所 | Molecular marker for wheat stripe rust disease-resistant new gene Yr50 assisted selection and using method thereof |
| CN109321582A (en) * | 2018-06-01 | 2019-02-12 | 山东农业大学 | Application of the aegilops tauschii Yr4DS gene in the plant stripe rust resisting breeding of wheat race |
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