Plants of the Species Beta Vulgaris With Resistance to Cercospora

Abstract

Provided herein are Beta vulgaris plants resistant to Cercospora. Also provided herein are methods for identifying Cercospora resistant Beta vulgaris plants, methods for providing Cercospora resistant Beta vulgaris plants and means for identifying Cercospora resistant Beta vulgaris plants. Specifically, provided herein are Cercospora-resistant Beta vulgaris plants including a first Cercospora resistance providing genomic fragment on chromosome 4, wherein said first Cercospora resistance providing genomic fragment includes at least one sequence from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, and 15.

Claims

1. A Cercospora-resistant Beta vulgaris plant comprising a first Cercospora resistance providing genomic fragment on chromosome 4, wherein said first Cercospora resistance providing genomic fragment comprises at least one sequence from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, and 15.

2. The Beta vulgaris plant according to claim 1, wherein said first Cercospora resistance providing genomic fragment further comprises at least one sequence selected from the group consisting of SEQ ID Nos. 17, 19, and 21.

3. The Beta vulgaris plant according to claim 1, wherein said plant comprises a second Cercospora resistance providing genomic fragment on chromosome 9, wherein said second Cercospora resistance providing genomic fragment comprises at least one sequence selected from the group consisting of SEQ ID Nos. 23, 25, 27, 29, 31, 33, 35, and 37.

4. The Beta vulgaris plant according to claim 1, wherein the first and second Cercospora resistance providing genomic fragments are obtained, obtainable, or are from a Beta vulgaris plant deposited under deposit accession number NCIMB 43769.

5. The Beta vulgaris plant according to claim 1, comprising a first Cercospora resistance providing genomic fragment on chromosome 4, wherein said first Cercospora resistance providing genomic fragment comprises SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, and 15, and a second Cercospora resistance providing genomic fragment on chromosome 9, wherein said second Cercospora resistance providing genomic fragment comprises SEQ ID Nos. 23, 25, 27, 29, 31, 33, 35, and 37.

6. The Beta vulgaris plant according to claim 1, wherein said plant is cytoplasmic male sterile (CMS).

7. The Beta vulgaris plant according to claim 1, wherein said plant is a hybrid plant.

8. The Beta vulgaris plant according to claim 1, wherein said plant is a Beta vulgaris plant deposited under accession number NCIMB 43769.

9. The Beta vulgaris plant according to claim 1, wherein the plant is selected from the group consisting of Beta vulgaris ssp. vulgaris var. conditiva, Beta vulgaris ssp. vulgaris var. altissima, Beta vulgaris subsp. vulgaris var. flavescens, Beta vulgaris subsp. vulgaris var. cicla, and Beta vulgaris ssp. vulgaris var. crassa.

10. A method for identifying a genomically encoded resistance against the plant pathogen Cercospora as found in a Beta vulgaris plant deposited under accession number NCIMB 43769, the method comprises the step of identifying a first Cercospora resistance providing genomic fragment on chromosome 4 by detecting at least one genomic sequence selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.

11. M xl according to claim 10, wherein the method further comprises identifying a second Cercospora resistance providing genomic fragment located on chromosome 9 by detecting at least one genomic sequence selected text from the group consisting of SEQ ID Nos. 23, 25, 27, 29, 31, 33, 35, and 37.

12. A method for providing a Beta vulgaris plant according to claim 1, comprising steps of obtaining Cercospora susceptible plant material from a plant of the species Beta vulgaris, subjecting said Cercospora susceptible plant material to mutagenesis to create mutagenized plant material, and analyzing said mutagenized plant material to identify a plant having resistance to Cercospora beticola.

13. A seed or plant part of a Beta vulgaris plant according to claim 1.

14. The seed according to claim 13 wherein said seed is polished, coated, encrusted, pelleted, or primed.

15. A molecular marker selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.

Description

DESCRIPTION OF THE INVENTION

[0022] The term at least one can be used interchangeably with the term one or more. As such, the term at least one should be understood to mean at least one, two, three, four, five, six, or more, or all. Preferably, the resistance providing genomic fragment on chromosome 4 comprises SEQ ID No. 5; more preferably SEQ ID No. 5, and SEQ ID No. 7; more preferably SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7; more preferably SEQ ID No. 1. SEQ ID No. 3. SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9; and most preferably SEQ ID No. 1. SEQ ID No. 3. SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15. The resistance providing genomic fragment is not comprised of SEQ ID No. 2. SEQ ID No. 4, SEQ ID No. 6, SEQ, ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 16. Said resistance providing genomic fragment on chromosome 4 may further comprise at least one sequence from the group consisting of SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21.

[0023] Alternatively, this object, amongst other objects, is achieved by providing Cercospora-resistant Beta vulgaris plants comprising a combination of two resistance providing genomic fragments, wherein a first genomic fragment is located on chromosome 4 and comprises at least one sequence from the group consisting of SEQ ID No. 1. SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9. SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15, and wherein, a second genomic fragment is located on chromosome 9 and comprises at least one sequence from the group consisting of SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37. Preferably, said first genomic fragment on chromosome 4 comprises SEQ ID No. 5; more preferably SEQ ID No. 5, and SEQ ID No. 7; more preferably SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7; more preferably SEQ ID No. 1. SEQ ID No. 3. SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9; and most preferably SEQ ID No. 1. SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 15. Preferably, said second resistance providing genomic fragment on chromosome 9 comprises SEQ ID No. 35; more preferably SEQ ID No. 35, and SEQ ID No. 37; more preferably SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37; more preferably SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37; and most preferably SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37. The first resistance providing genomic fragment is not comprised of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ, ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 16, and the second resistance providing genomic fragment is not comprised of SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 36, and SEQ ID No. 38. Hence, in one preferred embodiment, the Cercospora-resistant Beta vulgaris plant comprises SEQ ID No. 5, and SEQ ID No. 35; more preferably SEQ ID No. 1. SEQ ID No. 3. SEQ ID No. 5. SEQ ID No. 7, SEQ ID No. 9. SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37; and most preferably SEQ ID No. 1. SEQ ID No. 3. SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37.

[0024] The resistance according to the invention can be introduced in a plant cell by transformation (e.g., using Agrobacterium tumefaciens). Genomic fragments can be amplified by long-range PCR amplifications, de novo synthesized, or isolated from gels or columns (e.g., after restriction digestion). The resulting fragments can be reassembled (e.g., in yeast) or introduced in an expression vector, subsequently transformed into Beta vulgaris cells and allowed to integrate or recombine with the Beta vulgaris genome. The fragment may be introduced in a single step or in a series of transformations ultimately resulting in a Beta vulgaris plant comprising the resistance of the present invention.

[0025] According to a preferred embodiment of the present invention, the resistance providing genomic fragments are obtainable, obtained, or are from a Beta vulgaris plant deposited at NCIMB (National Collections of Industrial, Food and Marine Bacteria; NCIMB Limited, Ferguson Building; Craibstone Estate, Bucksburn Aberdeen, Scotland, AB21 9YA United Kingdom) on 7 May 2021 under number NCIMB 43769.

[0026] According to a preferred embodiment, the present Beta vulgaris plants are cytoplasmic male sterile (CMS).

[0027] According to yet another preferred embodiment, the present Beta vulgaris plants are hybrid plants. The present invention also relates to hybrid Beta vulgaris plants obtainable either by crossing Cercospora-susceptible Beta vulgaris plants with Beta vulgaris plants comprising the resistance to Cercospora beticola or by crossing a Cercospora-susceptible Beta vulgaris plant with deposit number NCIMB 43769.

[0028] Within the context of the present invention the following Beta vulgaris plants are contemplated: Beta vulgaris ssp. vulgaris var. conditiva (beetroot, red beet), Beta vulgaris ssp. vulgaris var. altissima (sugar beet), Beta vulgaris subsp. vulgaris var. flavescens (chard, Swiss chard), Beta vulgaris subsp. vulgaris var. cicla (spinach beet), and Beta vulgaris ssp. vulgaris var. crassa (mangelwurzel).

[0029] The present invention further relates to methods for identifying the genomically encoded resistance against the plant pathogen Cercospora beticola as found in the Beta vulgaris plant deposited under deposit number NCIMB 43769. The method comprises the step of identifying one genomic fragment on chromosome 4 by detecting at least one genomic sequence from the group consisting of SEQ ID No. 1. SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21. Preferably the method further comprises identifying a further resistance providing genomic fragment on chromosome 9 by detecting at least one genomic sequence from the group consisting of SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37. Several common genotyping methods exist for detecting a single nuclear polymorphism (SNP) in a genomic sequence, including PCR-based methods, direct hybridization, fragment analysis, and sequencing. An example of a method suitable for detecting a genomic sequence is isolating DNA from available plant material (e.g., from a piece of a leaf from a plant, or a seed), followed by nucleic acid amplification of isolated DNA (e.g., using PCR), and detecting the presence of said genomic sequence (e.g., by sequencing, measuring fluorescence, or visualizing and analyzing PCR amplification using agarose gel electrophoresis).

[0030] The present invention accordingly provides a method of producing a Beta vulgaris plant of the invention comprising the steps of obtaining plant material from a plant of the species Beta vulgaris, subjecting said plant material to mutagenesis to create mutagenized plant material, analyzing said mutagenized plant material to identify a plant having resistance to Cercospora beticola. Said plant material is preferably a seed.

[0031] Suitable mutagenesis methods comprise chemical mutagenesis (e.g., using ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), sodium azide (NaN3), methylnitrosoguanidine (MNNG), diethyl sulfonate (DES), TILLING, or mutagenesis by generating reactive oxygen species) and radiation mutagenesis (e.g., using UV radiation or ion beam radiation). Mutagenesis can lead to one or more mutations located in the coding sequence (mRNA, cDNA or genomic sequence) or in the associated non-coding sequence and/or regulatory sequence regulating the level of expression of the coding sequence. The presence of one or more mutations (e.g., insertion, inversion, deletion and/or replacement of one or more nucleotide(s)) may lead to the encoded protein having a new or altered functionality (gain of function), reduced functionality (reduced function) or no functionality (loss-of-function), e.g., due to the protein being truncated or having an amino acid sequence wherein one or more amino acids are deleted, inserted or replaced. Such changes may lead to the protein having a different 3D structure or conformation, being targeted to a different sub-cellular compartment, having one or more modified catalytic domains, having a modified binding activity to nucleic acids or proteins, etc.

[0032] The present invention further relates to seeds capable of providing the present plants. Seeds can be coated, colored, washed, polished, encrusted, pelleted, primed or undergo a combination of treatments. Coated seeds are covered by a relatively thin layer of polymer supplied to the seed; to this polymer fungicides or insecticides can be added to protect the seed against soil borne pathogens and insect damage. Additionally, a dye can be added. This added color gives the farmer the opportunity to check for correct drilling of the seeds. Alternatively, also other beneficial compounds can be added as micronutrients or beneficial micro-organisms promoting the growth of the young seedlings. Encrusted seeds are not only covered by a polymer with or without extra substances, as described above, but the seeds are provided with a smooth surface as well. This makes drilling easier and the added weight enables a more precise direct drilling of the seeds. Polishing removes the outermost layer of the seed, so that the seed assumes a more rounded form. Polishing and washing promotes germination of the seed. With pelleting the seeds are covered with more material, e.g., polymer bound clay, to produce a regularly shaped, round pellet. This pellet, besides having protecting substances described above, can be constructed in such a way that it will melt or split after water uptake. Priming or pre-germination is a treatment in which seeds are given enough moisture to initiate germination of the embryo inside the seed. This results in a faster emergence of the seedling, a higher emergence rate and better growth. It is believed that priming leads to a better root system and faster growth.

[0033] In addition to the Beta vulgaris plant according to the invention, the present invention also relates to a seed, a plant cell, a protoplast, a plant organ, plant tissue, edible parts, pollen, microspores, ovaries, ovules, egg cells, callus, suspension culture, somatic embryos, embryos or plant parts of the Beta vulgaris plants defined above comprising a Cercospora beticola resistance providing genomic fragment. Plant parts include, but are not limited to, the shoot, the stalk, the stem, leaves, blossoms, inflorescence, roots, fruits, and cuttings.

[0034] The present invention furthermore relates to molecular markers which markers co-segregate with a genomically encoded resistance against the plant pathogen Cercospora beticola as present in deposit number NCIMB 43769, which molecular markers are selected from the group consisting of SEQ ID No. 1. SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9. SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, and SEQ ID No. 37.

[0035] The present invention will be further detailed in the following examples.

Examples

Example 1. Mutagenesis of Beta vulgaris Plants with the Purpose of Making a Library to Screen for Cercospora Resistance

[0036] Random mutagenesis followed by forward screening can be a useful method for identifying mutant Beta vulgaris plants with resistance to Cercospora beticola. A mutagenized library can be generated by subjecting seeds to a step of mutagenesis, preferably random mutagenesis. Such a step may comprise, but is not limited to, the treatment of a pool of 100.000 to 200.000 seeds with a chemical mutagen, or a mixture of chemical mutagens, e.g., 0.25% EMS for 16 hours at room temperature; alternatively, radiation can be used (e.g., gamma-radiation from a radioactive Cobalt-60 source). Preferably, only a mildly mutagenized library (fewer than 1% of all genes contain a mutation in a coding region) is generated. Nevertheless, the mutagenesis step will lead to the loss of germination in some seeds. In contrast to irradiation, which can lead to mutations varying from single base substitutions or deletions to large deletions, EMS produces predominantly random point mutations by nucleotide substitution; particularly by guanine alkylation.

[0037] The mutagenized seeds can be sown and propagated in a field using standard practices. The mutagenized seed will generate plants that each have a particular set of mutations. Plants can be harvested in pools and viable seeds can be sown again (F1 population). To obtain an F1S1 population seeds can be collected from the F1 plants after selfing. As the mutations will segregate in an F1S1 population, the resistance in this population can be evaluated and used to map the resistance against Cercospora beticola.

Example 2. Field Tests for Assessing Resistance Against Cercospora beticola

[0038] The inoculum of Cercospora beticola was prepared by growing the fungus in Potato Dextrose Agar for 3 weeks incubated under near-UV light (12 hours day/12 hours night) at 25? C. Spores and mycelium were scraped with water, filtered and the concentration adjusted to 105 colony forming units per ml.

[0039] Plants were sown directly in the field in mid-June in the Netherlands, and, after germination, thinned to a 5 cm distance between plants. As susceptible control varieties Wodan F1 and Pablo F1 were used. Seven-week-old plants were inoculated by spraying the spore suspension on the leaves using an ultra-low volume or droplet sprayer, preferably during rainy weather or in the evening when dew will be formed during the night (as the fungus requires a long wet leaf period for infection). The inoculation was performed once a week, until a visibly high disease pressure was achieved (usually 8-12 consecutive weeks required, until mid-September to mid-October).

[0040] The assessment of disease resistance was performed visually by the amount and density of the typical small, round to oval-shaped spots on leaves, using a classification scale from 0 (completely susceptible, severe symptoms) to 9 (highly resistant, no symptoms). A score on this classification scale is referred to as a disease score.

Example 3. Results of Assessment of Resistance Against Cercospora beticola

[0041]

TABLE-US-00001 TABLE 1 Disease score of different genotypes and varieties Disease Score Cercospora beticola inoculation test in NL Susceptible varieties 1-2 (namely Wodan and Pablo) Plants comprising CB4.1 4 Plants comprising CB4.1 + CB9.1 5

Example 4. Development of Markers to Detect Cercospora Resistance in Beta vulgaris

[0042] A segregating F1S1 mapping population was developed to map the resistance against Cercospora beticola. This population was made by crossing a resistant Beta vulgaris plant with a susceptible Beta vulgaris plant, after which the resulting F1 plant was self-pollinated. To perform a QTL mapping, 520 individual plants were tested in a Cercospora beticola disease field trial and leaf material of these plants was used for DNA isolation, genotyping and marker analysis.

[0043] To genotype the mapping population, informative genome-wide markers were developed. Using 399 SNP markers covering the entire genome a genetic map was constructed and QTL analysis performed. Two QTL peaks were found. One QTL peak was located on chromosome 4 at base pair 45501459 of the EL10.1 public sugar beet reference genome (EL10_1.0, GenBank assembly accession GCA_002917755.1), and one on chromosome 9 at base pair 24213487 of the EL10.1 reference genome. The QTLs were named CB4.1 and CB9.1.

[0044] To narrow down the required haplotype size, a fine mapping experiment was performed. After pre-selection on 3720 plants, phenotypic assessment for the level of resistance was performed on 962 plants in a Cercospora beticola field disease trial. The results showed that CB4.1 is essential for resistance, and that CB9.1 is needed to obtain the highest level of resistance as combining the two QTL peaks gives the highest average disease score. SNPs for detecting the genomically encoded resistances against Cercospora beticola can be found in Table 2 to 4. Abbreviations are according to IUPAC nucleotide code.

[0045] Seeds of the F1S1 population comprising CB4.1 and CB9.1 were deposited at the NCIMB (NCIMB Limited, Ferguson Building; Craibstone Estate, Bucksburn ABERDEEN, Scotland, AB21 9YA, United Kingdom) on 7 May 2021 under deposit number NCIMB 43769.

TABLE-US-00002 Symbol NucleotideBase A Adenine C Cytosine G Guanine T Thymine N AorCorGorT M AofC R AorG W AorT S CorG Y CorT K GorT V NotT H NotG D NotC B NotA

TABLE-US-00003 TABLE2 SNPsforthedetectionoftheresistance againstCercosporabeticola,QTLCB4.1 Position Allele Chromo- Chromosome linked some (bp) to Alternative SNP (EL10.1) (EL10.1) resistance allele 1 4 44276365 A G 2 4 44440329 T A 3 4 45501459 T C 4 4 47197127 T G 5 4 47371424 G T 6 4 47419640 C T 7 4 48257283 A G 8 4 48338565 A C 9 4 52795726 C T 10 4 55593004 A T 11 4 55597074 T G

TABLE-US-00004 TABLE3 SNPsforthedetectionoftheresistance againstCercosporabeticola,QTLCB9.1 Position Allele Chromo- Chromosome linked some (bp) to Alternative SNP (EL10.1) (EL10.1) resistance allele 12 9 21546628 G T 13 9 21859451 T C 14 9 22691063 T C 15 9 23659048 G A 16 9 24117497 A G 17 9 24118744 A G 18 9 24213487 C T 19 9 24369620 T C

TABLE-US-00005 TABLE4 Sequencesforthedetectionoftheresistance againstCercosporabeticola SEQ SNPpositionin IDNo. SNP EL10.1 Sequence 1 1 Chr4_44276365 ACACARGGGACGAAAAGCAGAACAKGACACCAAC ACARTAGCAATAACGACACAACAAGAGCAGCAAC ATAACAACAATTCAATGACCCACTAACAATAA[A]C CACCACAMCTCCTTTTAGCTAAACTCACAATCACT AATCAATCTGCTAATTCAAATCTTGAARTGAAATA AAATCYATTTCTRAGTAAACTCAGCAAAC 2 1 Chr4_44276365 ACACARGGGACGAAAAGCAGAACAKGACACCAAC ACARTAGCAATAACGACACAACAAGAGCAGCAAC ATAACAACAATTCAATGACCCACTAACAATAA[G]C CACCACAMCTCCTTTTAGCTAAACTCACAATCACT AATCAATCTGCTAATTCAAATCTTGAARTGAAATA AAATCYATTTCTRAGTAAACTCAGCAAAC 3 2 Chr4_44440329 TACTCAAGCACGTAAATGGTTCAACGAAATGGTTC TGGATGGCTGTTCCCCTAATGTGGTGACCTACACTG CCCTAATTCATGCATACCTTAAAGTACGC[T]GGATA GGTGAAGCCAATGAACTTTTTGAGATTATGTTGAA AGATGGATGCAAACCGAATGTTGTGACTTATACTG CTTTAATTGATGGTTATTGTAAATC 4 2 Chr4_44440329 TACTCAAGCACGTAAATGGTTCAACGAAATGGTTC TGGATGGCTGTTCCCCTAATGTGGTGACCTACACTG CCCTAATTCATGCATACCTTAAAGTACGC[A]GGAT AGGTGAAGCCAATGAACTTTTTGAGATTATGTTGA AAGATGGATGCAAACCGAATGTTGTGACTTATACT GCTTTAATTGATGGTTATTGTAAATC 5 3 Chr4_45501459 GAGCTGATCCGGTTTGTTTCAAACCATAAGCCTATT GTTGCTGCTATGAGGGTGTCTGAGAGAACTGTCAT GATTATCAAGAACCTAGTATCGTCTTCGG[T]GCCAT CTCTGGTATGGTAGTATTGACATATAGAAAATGCC AATTTTTCAGTTCTTGGTCATATTCACTTAAGGACA GTATGCTTGTACAGTCTTCTCTAT 6 3 Chr4_45501459 GAGCTGATCCGGTTTGTTTCAAACCATAAGCCTATT GTTGCTGCTATGAGGGTGTCTGAGAGAACTGTCAT GATTATCAAGAACCTAGTATCGTCTTCGG[C]GCCAT CTCTGGTATGGTAGTATTGACATATAGAAAATGCC AATTTTTCAGTTCTTGGTCATATTCACTTAAGGACA GTATGCTTGTACAGTCTTCTCTAT 7 4 Chr4_47197127 CAAAGTAAGAAGTCAGAAAATTTATATTCCTAGCC TCTCTTCCTCACCCCTTTTCCTGTATGTTGTGTGAGC TTTGATTAGTTTTAACGAATATAATTCT[T]TCGCTG CAAACCTGCAAGTGTGTATATACTCCATTTTGTATA TATATGAAATCATGGAGTATTGCAGTGAGGAATCT TTGTAAGACTTTCTCTGAGAATG 8 4 Chr4_47197127 CAAAGTAAGAAGTCAGAAAATTTATATTCCTAGCC TCTCTTCCTCACCCCTTTTCCTGTATGTTGTGTGAGC TTTGATTAGTTTTAACGAATATAATTCT[G]TCGCTG CAAACCTGCAAGTGTGTATATACTCCATTTTGTATA TATATGAAATCATGGAGTATTGCAGTGAGGAATCT TTGTAAGACTTTCTCTGAGAATG 9 5 Chr4_47371424 TTTCAGAATCTTCAGCTTCATGGATAGAACCAATGT GTTGAGTATTCATCGCTTGTCGATTGATAACTGACC AACTTGTCTTTCGAGTTTGCGGGAGTTG[G]ACTGGA GGGCCCAAGGTTACAGGTTTAAGGGCTTGTGCAGC TAAGGTTTTCATGATGAAGCAGAACAATCTCTGGC AGATTCCAAGAGATTGAAGCTGCT 10 5 Chr4_47371424 TTTCAGAATCTTCAGCTTCATGGATAGAACCAATGT GTTGAGTATTCATCGCTTGTCGATTGATAACTGACC AACTTGTCTTTCGAGTTTGCGGGAGTTG[T]ACTGGA GGGCCCAAGGTTACAGGTTTAAGGGCTTGTGCAGC TAAGGTTTTCATGATGAAGCAGAACAATCTCTGGC AGATTCCAAGAGATTGAAGCTGCT 11 6 Chr4_47419640 CCAACAACTTRTTTCTCCAACTCTTCCTCTTAGCTTC CATATYCATGCCTTGTGCCAAATCCATTAATTTCAA CTATCCTGCAGTTTTCAACTTTGGCGA[C]TCGAATT CTGATACCGGTGCTCTTGTGGCTTCTGGACTCGAGG GGATYACTGATCCCTACGGACARACTTACTTCANA AAACCATCAGGAAGATACAGTG 12 6 Chr4_47419640 CCAACAACTTRTTTCTCCAACTCTTCCTCTTAGCTTC CATATYCATGCCTTGTGCCAAATCCATTAATTTCAA CTATCCTGCAGTTTTCAACTTTGGCGA[T]TCGAATT CTGATACCGGTGCTCTTGTGGCTTCTGGACTCGAGG GGATYACTGATCCCTACGGACARACTTACTTCANA AAACCATCAGGAAGATACAGTG 13 7 Chr4_48257283 TTACTTTTGCCAAGAATATACTTTGTTTGCATGGTT CAACGTAAAGTTGATTGGTGCGAGAARTGTTATAT TTTCTGGTKTGGGCCACCAGAAACGYAGC[A]CTTTT CCACAKWACACTAAATGGATCCTTTCTGGACCTGG CAAAACTAGGTTGTACACCCAAATAACTCTATATA GGTAGGAATTTACTGTATTCCAAAT 14 7 Chr4_48257283 TTACTTTTGCCAAGAATATACTTTGTTTGCATGGTT CAACGTAAAGTTGATTGGTGCGAGAARTGTTATAT TTTCTGGTKTGGGCCACCAGAAACGYAGC[G]CTTT TCCACAKWACACTAAATGGATCCTTTCTGGACCTG GCAAAACTAGGTTGTACACCCAAATAACTCTATAT AGGTAGGAATTTACTGTATTCCAAAT 15 8 Chr4_48338565 AACGGTTGGAATTTTTCTGAAGACATTACTGTTTTG TAATTGTCCATGAAGGTGACTAGCTTCGGGAATGG TCATTTTAGGTGCAAGCGATGATCTTGTT[A]TNTGA GTGGTCAATCTTGATGGAAAGAAATGCTTAGGGAT GGTGTTTGTNTGCTAAGAATGGAGAAGAAACCAAT AAGCTAAGATCAGTTCATGTCTCGT 16 8 Chr4_48338565 AACGGTTGGAATTTTTCTGAAGACATTACTGTTTTG TAATTGTCCATGAAGGTGACTAGCTTCGGGAATGG TCATTTTAGGTGCAAGCGATGATCTTGTT[C]TNTGA GTGGTCAATCTTGATGGAAAGAAATGCTTAGGGAT GGTGTTTGTNTGCTAAGAATGGAGAAGAAACCAAT AAGCTAAGATCAGTTCATGTCTCGT 17 9 Chr4_52795726 CTATTAGTTTTGATGTGTCATTTTATGGTGCTGATTT TGTTTATGGTATTCCTGAACATGCTACTAGTCTTGC ACTTAAGCCTACTAGAGGCCCCGGTAT[C]GAACAT TCGGAACCTTATAGGCTTTTTAACTTGGATGTGTTT GAATATCTTCATGAGTCGCCTTTCGGGTTGTATGGA TCNATTCCGTTCATGCTTGGTC 18 9 Chr4_52795726 CTATTAGTTTTGATGTGTCATTTTATGGTGCTGATTT TGTTTATGGTATTCCTGAACATGCTACTAGTCTTGC ACTTAAGCCTACTAGAGGCCCCGGTAT[T]GAACAT TCGGAACCTTATAGGCTTTTTAACTTGGATGTGTTT GAATATCTTCATGAGTCGCCTTTCGGGTTGTATGGA TCNATTCCGTTCATGCTTGGTC 19 10 Chr4_55593004 TGCGCCAAATTATCAACCTGTGGTGCATGAACTTAT ACATGAAAAAACAAAACGAACTCATACATGAAAA CCAAAAGAGGTGGACCCTAAATGTAATAAC[A]AAC CTGGTGAGTGAGCCACATGAGCAAGATATCAACAG CAGGGACCAAGACACTTGAAGAAGCATCACCATCA ACATCCCCATCATCTTGTTGGATTACC 20 10 Chr4_55593004 TGCGCCAAATTATCAACCTGTGGTGCATGAACTTAT ACATGAAAAAACAAAACGAACTCATACATGAAAA CCAAAAGAGGTGGACCCTAAATGTAATAAC[T]AAC CTGGTGAGTGAGCCACATGAGCAAGATATCAACAG CAGGGACCAAGACACTTGAAGAAGCATCACCATCA ACATCCCCATCATCTTGTTGGATTACC 21 11 Chr4_55597074 AGAATGTGAGGGGAAGAAACMGAAGATGCATTGC ATGAAGAAGGATGRAGTATAATAGAACCCCAATCC CTCTCTCCATACTTGGCCTCTTTCAGTCTTC[T]CCTT ACTAACAATCCTCCCACATCTTCTTTTCTCATCTCA GAAACACTCCTCAGTATTGCAAGGCAYAATAACAG CACGAAAaCAGCRTCTTCTTCTGGC 22 11 Chr4_55597074 AGAATGTGAGGGGAAGAAACMGAAGATGCATTGC ATGAAGAAGGATGRAGTATAATAGAACCCCAATCC CTCTCTCCATACTTGGCCTCTTTCAGTCTTC[G]CCTT ACTAACAATCCTCCCACATCTTCTTTTCTCATCTCA GAAACACTCCTCAGTATTGCAAGGCAYAATAACAG CACGAAAaCAGCRTCTTCTTCTGGC 23 12 Chr9_21546628 AAAGAAATACTTTGACATGGTGGCGTAARTATTTTC CCGTGCTTAGACTGATAAATCCAACATCAATTCTAT TATCATGAAAAAGCAAATTTACCTCTTT[G]GATATT TGCTGCAGAATAGGTATGATCTCAGCGAAGTCAGG TCTCATTGCAGGATCTTGCTGCCAGCATCTTTCAAG AAGCTCGGTAAGCTTGGGATGAC 24 12 Chr9_21546628 AAAGAAATACTTTGACATGGTGGCGTAARTATTTTC CCGTGCTTAGACTGATAAATCCAACATCAATTCTAT TATCATGAAAAAGCAAATTTACCTCTTT[T]GATATT TGCTGCAGAATAGGTATGATCTCAGCGAAGTCAGG TCTCATTGCAGGATCTTGCTGCCAGCATCTTTCAAG AAGCTCGGTAAGCTTGGGATGAC 25 13 Chr9_21859451 AAGCAGATCCTTAACTTCCCTTCTTAACTGACGCAG TTTTGAGTCTTAAGAATGACAACCCTATGCTTTCAT TTGGATATTCAATGTCTACTGAAGCCAC[T]ACTACA GGCTTGATTACTGTTAAATTTGTACCAAAATGGGTA AATTACAGTTTATTGTTCTACTGATAATGATTTTTG TAAACATTCAAGTGCTTTGTAT 26 13 Chr9_21859451 AAGCAGATCCTTAACTTCCCTTCTTAACTGACGCAG TTTTGAGTCTTAAGAATGACAACCCTATGCTTTCAT TTGGATATTCAATGTCTACTGAAGCCAC[C]ACTACA GGCTTGATTACTGTTAAATTTGTACCAAAATGGGTA AATTACAGTTTATTGTTCTACTGATAATGATTTTTG TAAACATTCAAGTGCTTTGTAT 27 14 Chr9_22691063 CAAAAGACTATAAGGGTCAAGTTGAAGCAAACTTG AGAGTGGTCCTCCACTGGATCATTTGTCTCTATTGT CTTAATGCAACAAACGACTTGAAACCATC[T]AAAA ACAATAGATAGGATGATTCCACTAGAATCATCATT GCGAATAAGCAACCAAGGATCTTTATTGTTTACCA CATAAACATTCATATTGATCACCATG 28 14 Chr9_22691063 CAAAAGACTATAAGGGTCAAGTTGAAGCAAACTTG AGAGTGGTCCTCCACTGGATCATTTGTCTCTATTGT CTTAATGCAACAAACGACTTGAAACCATC[C]AAAA ACAATAGATAGGATGATTCCACTAGAATCATCATT GCGAATAAGCAACCAAGGATCTTTATTGTTTACCA CATAAACATTCATATTGATCACCATG 29 15 Chr9_23659048 ATATCATTGAAAAAATAATATTGAGGAAATAAAGT TCAACCTGTTTAGCACCTAGACCAAAGGTTGCATTG CCAGATAGATAAGGTTTCGAGCGATCAGC[G]GTTT TGCAGAGCATAGAAAGTGCTGCAGTAGCTCCCACT TGTATAGCCTGCATGTCCTCCAAAATGGTGAGAAG CTGAATAGCTTAAGATTCATAATTCT 30 15 Chr9_23659048 ATATCATTGAAAAAATAATATTGAGGAAATAAAGT TCAACCTGTTTAGCACCTAGACCAAAGGTTGCATTG CCAGATAGATAAGGTTTCGAGCGATCAGC[A]GTTT TGCAGAGCATAGAAAGTGCTGCAGTAGCTCCCACT TGTATAGCCTGCATGTCCTCCAAAATGGTGAGAAG CTGAATAGCTTAAGATTCATAATTCT 31 16 Chr9_24117497 AGGTAATATATAAAACAGAGGCAATCTCACAATAA AACAGTTATCATGGSGATCAGATTCACAAACCCGT AACAGCAACATGAGGGTCAAAACAGCCACC[A]GAT AAACTTCAAAAAGCCCGACCCGACCCCAACTACTG GGCAGCAACAGCCAACTTAAAGCTTACCAGCCCAA GCTACGAYCAATCAACATKGTAACATC 32 16 Chr9_24117497 AGGTAATATATAAAACAGAGGCAATCTCACAATAA AACAGTTATCATGGSGATCAGATTCACAAACCCGT AACAGCAACATGAGGGTCAAAACAGCCACC[G]GA TAAACTTCAAAAAGCCCGACCCGACCCCAACTACT GGGCAGCAACAGCCAACTTAAAGCTTACCAGCCCA AGCTACGAYCAATCAACATKGTAACATC 33 17 Chr9_24118744 GACACCACCTAATCAAATAATGCTTTCTTCATTAGT AGGGAACCCTATCCAAGAACAAGAACTTACCTCAA GTGATTTTCGAGGTGAGGGGCTACGAGAG[A]TTCC AGCTTCCCCACGCGGAGAAACYGCCTGRGGAGAAG CCAAACGATGAGGAATTGGTGCWCGTTCATCRAGG CCGYGCTTCTCTGGGCTACGACTCCT 34 17 Chr9_24118744 GACACCACCTAATCAAATAATGCTTTCTTCATTAGT AGGGAACCCTATCCAAGAACAAGAACTTACCTCAA GTGATTTTCGAGGTGAGGGGCTACGAGAG[G]TTCC AGCTTCCCCACGCGGAGAAACYGCCTGRGGAGAAG CCAAACGATGAGGAATTGGTGCWCGTTCATCRAGG CCGYGCTTCTCTGGGCTACGACTCCT 35 18 Chr9_24213487 AAGAATTCTTTGTGTTAAGGTGGTTGTGTGATGTAT GTTTGATAGTAGCAAAGATTCCTTTGATATATGTAG ATTAATATAGTCAATTTTTATCTATCAT[C]TGTAGT AGCACTCTCAAGTTCATCTGATATTCGTGTAATAAT TCTGAACGCTTGAATGCATTGGTTGTTTTTGTTTAG ACATTAAACTCTTTTGTTCAGG 36 18 Chr9_24213487 AAGAATTCTTTGTGTTAAGGTGGTTGTGTGATGTAT GTTTGATAGTAGCAAAGATTCCTTTGATATATGTAG ATTAATATAGTCAATTTTTATCTATCAT[T]TGTAGT AGCACTCTCAAGTTCATCTGATATTCGTGTAATAAT TCTGAACGCTTGAATGCATTGGTTGTTTTTGTTTAG ACATTAAACTCTTTTGTTCAGG 37 19 Chr9_24369620 AACTTATATTACATGAGTGACATTCATATAGGTCAC TTACGCGAACATCTGGTAGATTATCAATGCCTAAAT TTGAAGTATCCTTCTCATATTCAACAGG[T]CCACAT GAATTACTTATAGTTGCAAGGGAAAGGTCCAGCGT GGAGTTGCCATCCTCAGAATTCACGCACATATGCT GTTAGATAGCAGCATACAAATGTT 38 19 Chr9_24369620 AACTTATATTACATGAGTGACATTCATATAGGTCAC TTACGCGAACATCTGGTAGATTATCAATGCCTAAAT TTGAAGTATCCTTCTCATATTCAACAGG[C]CCACAT GAATTACTTATAGTTGCAAGGGAAAGGTCCAGCGT GGAGTTGCCATCCTCAGAATTCACGCACATATGCT GTTAGATAGCAGCATACAAATGTT