BRASSICA PLANT WITH POD SHATTERING TOLERANCE
20210169029 · 2021-06-10
Assignee
Inventors
- Stefan ABEL (Peine, DE)
- Laurent HANNETON (Verneuil L'Etang, FR)
- Vasilis GEGAS (Rothwell, GB)
- Jordi COMADRAN (Riom, FR)
- Jean Pierre MARTINANT (Vertaizon, FR)
Cpc classification
C12Q1/6876
CHEMISTRY; METALLURGY
A01H1/06
HUMAN NECESSITIES
International classification
A01H1/04
HUMAN NECESSITIES
A01H1/06
HUMAN NECESSITIES
Abstract
A Brassica plant including a Raphanus genomic fragment within its genome, wherein the fragment confers pod shattering tolerance phenotype POSH+ and the fragment is characterized by the absence of at least one SNP within one or more of the following Raphanus markers: SEQ ID NOs: 4-18.
Claims
1. A Brassica plant comprising a Raphanus genomic fragment within its genome, wherein the fragment confers pod shattering tolerance phenotype POSH.sup.+ to the Brassica plant, and: contains at least one Raphanus SNP that is linked to POSH.sup.+ phenotype as identified within at least one marker selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and does not contain at least one Raphanus SNP identified within any of the markers of SEQ ID NOs: 4-18, each Raphanus SNP being a nucleotide that is present in the Raphanus genome at a polymorphic position and that is not present in the Oleracea genome.
2. The Brassica plant according to claim 1, wherein the fragment does not contain the Raphanus SNPs identified within the markers of SEQ ID NO:9 and SEQ ID NOs: 12-18.
3. The Brassica plant according to claim 1, wherein the fragment does not contain the Raphanus SNPs identified within the markers of SEQ ID NOs: 4-18.
4. The Brassica plant according to claim 1, wherein the plant further comprises a Raphanus FRUITFULL allele.
5. The Brassica plant according to claim 4, wherein the Raphanus FRUITFULL allele comprises the Raphanus SNP within the marker of SEQ ID NO:22.
6. The Brassica plant according to claim 1, wherein the plant comprises the male fertility restoration locus Rf0 within the Raphanus fragment.
7. The Brassica plant according to claim 1, wherein the Brassica plant comprises a CMS Ogura cytoplasm.
8. A hybrid Brassica plant obtained by crossing the Brassica plant according to claim 1 with another Brassica plant which does not have the Raphanus fragment conferring POSH.sup.+ phenotype, wherein the hybrid plant comprises the Raphanus genomic fragment which confers pod shattering tolerance phenotype POSH.sup.+.
9. A seed, a plant part, or a progeny of the Brassica plant according to claim 1.
10. A method of identifying the Brassica plant according to claim 1, the method comprising: detecting the presence of at least one Raphanus SNP linked to POSH.sup.+ phenotype as identified within at least one of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and detecting the absence of at least one Raphanus SNP as identified within any of SEQ ID NOs: 4-18.
11. The method of claim 10, further comprising identifying the POSH.sup.+ locus using a Raphanus FRUITFULL allele.
12. The method according to claim 10, further comprising identifying the Rf0 locus.
13. Means for detecting one or more Raphanus SNPs within one or more of the markers of SEQ ID NOs: 4-22, the means comprising a nucleic acid probe, a primer or a set of primers, or combinations thereof.
14. The means according to claim 13 comprising one or more primers selected from the group consisting of SEQ ID NOs: 64-99, 106-108, 112-114, and 52-54.
15. A method of producing oil for food applications, the method comprising pressing seeds of the Brassica plant according to claim 1.
16. A method of producing a POSH.sup.+ Brassica plant, the method comprising: a) crossing (i) a first Brassica plant that is a POSH.sup.+ Brassica plant according to claim 1 with (ii) a second Brassica plant that is either a POSH.sup.−or POSH.sup.+Brassica plant, thereby obtaining a F1 hybrid plant; b) selfing or backcrossing the F1 hybrid plant with the second Brassica plant; and c) selecting the POSH.sup.+ Brassica plant from among the plants obtained in step b) by: detecting the presence of at least one Raphanus SNP linked to POSH.sup.+ phenotype as identified within at least one of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21; and optionally, detecting the absence of at least one Raphanus SNP as identified within any of SEQ ID NOs: 4-18.
17. The method of claim 16, wherein the first plant is a plant obtained from a representative sample of the seeds deposited at the NCIMB collection under the number 42444.
18. The method of claim 16, wherein the first Brassica plant comprises the Rf0 Ogura fertility restoration gene.
19. A Brassica plant obtainable or obtained by the method of claim 16.
20. A method of producing the Brassica plant according to claim 1, the method comprising: a) providing a first Brassica plant comprising a Raphanus introgression conferring the POSH.sup.+ trait to the first Brassica plant, the Raphanus introgression including at least one of the Raphanus SNPs as identified within the markers of SEQ ID NOs: 4-18; b) crossing the first Brassica plant with a second Brassica plant that is a POSH.sup.− or POSH.sup.+ Brassica plant, thereby obtaining a F1 hybrid plant; c) selfing or backcrossing the F1 hybrid plant with the second Brassica plant; and d) selecting the POSH.sup.+ Brassica plant from among the plants obtained in step c) by: detecting the presence of at least one Raphanus SNP linked to POSH.sup.+ phenotype as identified within at least one of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, and detecting the absence of at least one Raphanus SNP as identified within any of the markers of SEQ ID NOs: 4-18.
21. A method of improving the agronomical value of a Brassica plant, the method comprising performing pedigree breeding using the Brassica plant according to claim 1 as a parent plant.
22. A method of producing cake for feed applications, the method comprising grinding seeds of the Brassica plant according to claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
DETAILED DESCRIPTION
The Brassica Plant
[0052] As used herein, the term “Brassica plant” includes a plant of Brassica species, including B. napus, B. juncea and B. rapa; preferably B. napus.
[0053] As used herein, “pod shattering” also referred as “fruit or pod dehiscence” refers to a process that takes place in a fruit after seed maturation, whereby the valves detach from the central septum freeing the seeds. The region that breaks (i.e. the “dehiscence zone”) runs the entire length of the fruit between the valves and the replum (external septum). At maturity, the “dehiscence zone” is essentially a non-lignified layer of cells between a region of lignified cells in the valve and the replum. Shattering occurs due to the combination of cell wall loosening in the dehiscence zone and the tensions established by the differential mechanical properties of the drying cells in the silique.
[0054] The pod shattering trait is usually measured through laboratory tests that simulate the forces acting on the pods in the natural conditions. Different methods are available as described in Kadkol et al 1984—Evaluation of brassica accessions for resistance to shatter—Euphytica, 33, 61-71, Liu et al, 1994—Pendulum test for evaluation of rupture strength of seed pods—Journal of texture studies, 25, 179-189 or the method as described in example 3 of the present disclosure. Using this last method, the pods are harvested at complete maturity stage (BBCH97). The pod shattering tolerance is corresponding to the tension necessary to tear the two halves of the pod apart.
[0055] As used herein, the Brassica plant harboring pod with tension values more or equal to 2.3 Newton (N) are defined as pod shattering tolerant. In the present disclosure, they are also defined as POSH+ referring to the presence of the Raphanus POSH region in the plant genome. In specific embodiments, the POSH+ Brassica plant will harbor pods with a tolerance comprised between 2.3 and 7N (unit). Preferably, the POSH+ Brassica plant will harbor pods with a tolerance comprised between 2.3 and 5 N. More generally a POSH+ Brassica plant can be a Brassica plant harboring within its genome a long introgression of the Raphanus genome or it also can be one of the Brassica plant of the present disclosure.
[0056] As used herein, the Brassica plants with pod having tension values less than 2.3 N are defined as not pod shattering tolerant. Particularly, the pod tension will be above 0.6 N. In the present disclosure, they are also referred as POSH−, referring to the absence of the Raphanus POSH+ region in the plant genome. More generally, a POSH− Brassica plant could be fertile or not and for example could comprise or not the Rf0 fertility restorer gene, it could also be sterile or not and for example could comprise or not the Ogura male sterile cytoplasm, and it could also be or not a maintainer plant. Moreover, said POSH− Brassica plant may comprise a Raphanus introgression or no Raphanus introgression.
[0057] As used herein, the term “allele(s)” means any of one or more alternative forms of a gene at a particular locus. In a diploid (or amphidiploidic cell of an organism), alleles of a given gene are located at a specific location or locus on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes.
[0058] Whenever reference to a “plant” or “plants” is made, it is understood that also plant parts (cells, tissues or organs, seed pods, seeds, severed parts such as roots, leaves, flowers, pollent, etc.), progeny of the plants which retain the distinguishing characteristics of the parents (especially, pod shattering tolerance associated to the Raphanus fragment), such as seed obtained by selfing or crossing, e.g. hybrid seeds (obtained by crossing two inbred parent plants), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated.
[0059] As used herein, a “Raphanus genomic fragment” refers to an introgression, and preferably the original introgression and any of their recombinant fragment of the Raphanus sativa genome within the Brassica napus genome, which introgression is found in many commercialized Brassica varieties, including without limitation Albatros or Artoga varieties. For ease of reading, the original introgression will be defined hereafter as the “long introgression of the Raphanus genome”.
[0060] Such long introgression of the Raphanus genome within the Brassica napus genome further comprises the Rf0 gene for the fertility restoration of the Ogura CMS system. This Raphanus long introgression may not comprise any Brassica napus genome fragments. Examples of commercialized Brassica varieties comprising a long Raphanus introgression fragment are depicted in
[0061] A shorter introgression of the Raphanus genome within the Brassica napus genome have also been described in the art and said introgression comprises the Rf0 gene for the fertility restoration of the Ogura CMS system but the inventors now identified that such shorter introgression did not comprise a genome region conferring the pod shattering tolerance named POSH+. Examples of commercialized Brassica varieties comprising such shorter Raphanus introgression fragment is listed in
[0062] As used herein, the term “introgression” refers to a DNA fragment of a particular species, in the present case, from Raphanus sativus species, and transferred into another plant species, in the present case, Brassica, more preferably Brassica napus.
[0063] As used herein a “marker” refers to a specific DNA sequence identified within the genome of a plant and which can be used to determine whether a plant has inherited a particular phenotype or allele of interest from a parent plant. Said marker may include coding or non-coding sequences. In particular, said marker may include one or more Single Nucleotide Polymorphism or SNP identified between the Raphanus and the napus genome. It is also possible to identify sequence deletion/insertion (indel) polymorphism. In the present invention, the rapa genome is not considered, therefore the napus genome will also be identified as oleracea genome.
[0064] As used herein, a “Raphanus SNP” corresponds to the nucleotide present in the Raphanus genome at a polymorphic position compared to the oleracea genome.
[0065] It is herein disclosed Raphanus SNPs within markers (identified by their nucleotide sequence) for determining, in a Brassica plant, whether any recombinant fragment of the long Raphanus introgression further retains the POSH+ allele conferring pod shattering tolerance. Accordingly, the Brassica plant of the present disclosure includes a recombinant fragment of said long introgression, which is advantageously shorter than the long introgression while retaining at least the POSH+ allele.
[0066] More specifically, certain Raphanus SNPs found in said Raphanus long introgression have been characterized as not being linked to the POSH+ allele. Such SNPs are included in any of the following fifteen markers: SEQ ID NOs: 4-18.
[0067] Accordingly, a Brassica plant according to the present disclosure comprises a Raphanus genomic fragment within its genome, wherein said fragment confers pod shattering tolerance phenotype (POSH+) and said fragment is characterized by the absence of at least one Raphanus SNP within at least one of the following markers: SEQ ID NOs: 4-18.
[0068] For each of these markers, a Raphanus SNP has been identified (see Table 2). Preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of these SNPs are absent in said Brassica plant.
[0069] Other markers, and in particular Raphanus SNPs have been found in said Raphanus long introgression and characterized as being linked to the POSH+ allele. Such Raphanus SNPs are identified in any of the following three sequence markers: SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.
[0070] In a specific embodiment, said Brassica plant comprises a Raphanus genome fragment within its genome, but not the region including the Raphanus SNPs within the markers: SEQ ID NOs: 12-18 and SEQ ID NO: 9.
[0071] In a specific embodiment, said Brassica plant comprises a Raphanus genome fragment within its genome, but not the region including the Raphanus SNPs within the markers: SEQ ID NOs: 4-18.
[0072] In another specific embodiment that may be combined with the previous embodiments, said Brassica plant comprises at least the region of the Raphanus genome fragment including 1, 2 or 3 of the Raphanus SNPs within the following markers: SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, said SNP being identified in Table 1.
[0073] Alternatively, in another specific embodiment, said Brassica plant comprises at least the region of the Raphanus genome fragment including the Raphanus FRUITFULL allele, as identified within the following marker: SEQ ID NO: 22 or SEQ ID NO: 31.
[0074] In another specific embodiment, the Brassica plant is non-transgenic plant. Transgenic or “genetically modified organisms” (GMO) as used herein are organisms whose genetic material has been altered using techniques generally known as “recombinant DNA technology”. Recombinant DNA technology is the ability to combine DNA molecules from different sources into one molecule ex vivo (e.g. in a test tube). This terminology generally does not cover organisms whose genetic composition has been altered by conventional cross-breeding, or by “mutagenesis” breeding. “Non-transgenic” thus refers to plants and food products derived from plants that are not “transgenic” or “genetically modified organisms”.
[0075] The invention also relates to hybrid Brassica plants which can be produced by crossing a Brassica plant obtained above with a second plant. For example, a hybrid Brassica plant may be obtained by crossing a Brassica plant as disclosed herein which have a Raphanus fragment conferring POSH+ phenotype and another Brassica plant which does not have said Raphanus fragment conferring POSH+ phenotype, wherein said hybrid plant comprises the Raphanus genomic fragment which confers pod shattering tolerance phenotype POSH+.
[0076] Methods to produce hybrid plants are well-known in the art. Typically, hybrid plants are produced by preventing self-pollination of female parent plants, permitting pollen from male parent plant to fertilize such female parent plant and allowing F1 hybrid seeds to form on the female plants. Self-pollination can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. Hybrid plants can be obtained by different genetic systems well known from the person skilled in the art like for example, the CMS systems like Ogura system or the Kosena system (See Yamagashi and Bhat, 2014, Breeding Science, 64: 38-47), or the MSL (Male Sterility Lembke) system (Pinochet et al., 2000 OCL-Leagineux Corps Gras Lipides 7:11-16). Preferably, the hybrid plants of the invention are obtained with the Ogura system.
[0077] Therefore, it is also disclosed herein the Brassica plants or lines according to the present disclosure developed to obtain such hybrid plants. Such plants or lines typically comprise the genetic and/or cytoplasmic elements necessary for the implementation of the corresponding hybrid system. Preferably, the plants or lines comprise the fertility restoration gene Rf0 and/or the cytoplasm of the Ogura system.
Method of Producing a Brassica Plant with Pod Shattering Tolerance Phenotype (POSH+)
[0078] The present disclosure also relates to new methods to produce Brassica plants with pod shattering tolerance phenotype (POSH+) as described in the previous section.
[0079] In one embodiment, said method comprises the following steps: [0080] a. providing a first POSH+ Brassica plant comprising a Raphanus introgression conferring the pod shattering tolerance POSH+; said Raphanus introgression including at least one of the Raphanus SNP within one or more of the following markers: SEQ ID NOs: 4-18; [0081] b. crossing said first POSH+ Brassica plant with said second POSH.sup.− or POSH+Brassica plant, thereby obtaining a F1 hybrid plant; [0082] c. selfing or backcrossing the F1 hybrid plant with the second plant POSH.sup.− or POSH+; [0083] d. selecting the POSH+ plant among the plant obtained in step c), optionally by selecting for the presence of at least one Raphanus SNP within at least one of the markers SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and/or optionally further selecting for the absence of at least one Raphanus SNP within at least one of the markers SEQ ID NOs: 4-18.
[0084] Advantageously, one of the known varieties comprising the long or short introgression can be used as the first Brassica plant. In specific embodiment, the second (recurrent) Brassica that is used in the above method is a Brassica plant characterized by the absence of any Raphanus genome fragment within its genome, i.e; a plant wherein the Raphanus genome fragment have not been introgressed. Alternatively, said second Brassica plant does not contain at least the Rf gene for fertility restoration of the Ogura CMS system and does not contain the pod shattering tolerant region (POSH.sup.−). This second Brassica plant can be for example any wild type non restorer and POSH.sup.− Brassica napus plant. Alternatively, said second plant comprises the short introgression, including the Rf0 Ogura fertility restoration gene. In another specific embodiment, the first Brassica plant further includes the Rf0 Ogura fertility restoration gene.
[0085] At step c, backcrossing of F1 hybrid plant with the recurrent second plant aims at reducing the percentage of the genome of the recurrent plant and decrease the percentage of genome of the parent plant (containing the Raphanus introgression). Thanks to the selection markers as disclosed in the above methods, it is possible to select/retain the POSH+ phenotype during the selection process.
[0086] The applicant has deposited a sample of seeds of the disclosed Brassica plant with said Raphanus introgression conferring the POSH+ trait, under the Budapest treaty, at NCIMB collection under the number 42444.
[0087] The present disclosure further includes and provides for methods of identifying a POSH+ Brassica plant as disclosed in the previous section, and more generally methods of selecting or breeding Brassica plants for the presence or absence of POSH+ allele as comprised in the Raphanus introgression, for example, as molecular guided programs. Such methods of identifying, selecting or breeding Brassica plants comprise obtaining one or more Brassica plants and assessing their DNA to determine the presence or absence of the POSH+ allele contained in the Raphanus introgression and/or the presence or absence of other alleles or markers, for example other markers of the Raphanus introgression not associated with the POSH+ allele. Such methods may be used, for example, to determine which progeny resulting from a cross have the POSH+ allele and accordingly to guide the preparation of plants having POSH+ allele in combination with the presence or absence of other desirable traits.
[0088] In specific embodiments, determining the presence of the POSH+ allele or other markers, comprises determining the presence of markers of the Raphanus introgression associated to the POSH+ allele and/or the absence of markers of the Raphanus introgression not associated to the POSH+ allele. Accordingly, plants can be identified or selected by assessing them for the presence of one or more individual SNPs appearing in Table 1 for POSH+, and/or the absence of one or more individual SNPs appearing in Table 2 for Raphanus fragment not related to POSH+ allele.
[0089] In a specific embodiment, Rf0 locus may further be identified.
TABLE-US-00001 Table 1 Raphanus SNP associated to POSH+ allele (one identified SNP is highlighted in bold font) SEQ ID Sequence NO 19 TAGAGCTGAAGCTAGGTATAGGAGGCACATCATAYAAAGATT TCATTCAAAGCCTTCATCTACCTATGCAATTGAGTCAAGTAG ACCCAATAGTAGCGTCCTTCTCYGGAGGAGCTGTTGGTGTGA TCTCRGCKYTGATGGTWGTWGAAGTCAACAACGTGAAGCAGC AAGAGCACAAGAGATGCAAATACTGTCTAGGAA NO 20 TTAAGAACTGTGTCACTGACATTGACCCTGAGAGGGAGAAGG AGAAGAGAGAAAGGATGGAAAGCCAAAACCTCAAGGCTAGTA CAAAGCTGAGTCAAGCGAGGGAGAAAATCAAGCGCAAGTATC CACTTCCTGTTGCAAGGAGRCAACTYTCCACTGGRTACNTGG AAGATGCTCTCGAAGAGGATGAAGAGACAGACC NO 21 GCTCAGGTAGATCTCCCACGGGTTGGGGAAGAGGATCCGGAT ATGGGTATGGGTCTGGATCTGGATCAGGTAGCGGATATGGGT ACGGTTCCGGAGGTGGAGGAGSACGTGGTGGTGGGTATGGTT ATGGAAGCGGAAATGGTCGGTCTGGAGGWGGTGGTGGTGGCT CTAATGGTGAAGTTGCCGCTTTGGGCCACGGTG NO 22 GGGAGAGAGAGGAAACCTGGAGGATGTTACGCAGTACTGGGG CTGAAGAACTGAAGAATTGTTGGAGCATTGGATTAATTGTCC TTCKTGCTGACCCGTGTTCTTCT
TABLE-US-00002 TABLE 2 Raphanus SNP associated to Raphanus fragment but not POSH+ allele SEQ ID Sequence NO 18 AGAAGATGGAGTTCTTGATGTTTGATCTYGATCGGGTTTTGAARCCCGGTGGGTTGTTC TGGTTGGATAACTTCTACTGCGCTAGTGACGTGAAGAAGAAAGAGCTGACGCGTTTGAT YGAGAGGTTTGGGTATAAGAAGCTGAAATGGGTTATTGGAGAGAAGGCTGATGGGCAAG TGWATCTCTCTGCTGTTCTKCAAA NO 17 CACAACATGCCGGTGATTGGTATCCAGCTGACCTTGGATCCAACGATTTCAAAGGTCTC TATGGATATAAGGTCTTTATTGCCATTGCCATTATCCTTGGGGACGGTCTCTACAATCT TGTCAAGATCATTGCTGTCACTGTGAAGGAATTATGCAGCAATAGCTCTAGACACCTCA ATCTACCCGTTGTTRCCAACGTTG NO 16 ACTTTGTTGAYAGYCTTACMGGAGTAGGACTTGTTGATCAAATGGGAAACTTCTTCTGC AAAACGCTCTTGTTTGTGGCTGTAGCTGGAGTTCTTTTCATTCGCAAGAACGAAGATTT AGATAAGCTCAAGGGTCTRWTYGAAGAGACGACGYTRTATGACAAGCARTGGCAAGCGG CTTGGAAAGAGCCGGAAATAATCA NO 15 GTCCATGTTTGATGCAATTGTATCAGCAGACGCATTTGAGAACTTGAAACCAGCTCCAG ATATTTTCTTGGCTGCTTCCAAKATCTTGGGTGTGCCCACATGCGAGTGTATTGTTATT GAAGATGCACTTGCTGGAGTCCAGGCTGCTCAAGCTGCAAACATGAGATGCATAGCTGT GAAAACTACTTTATCTGAAGCAAT NO 14 CTTTTGCTGGTTTTGGTGAAATAGTATCTGTCAAGATACCAGTTGGGAAAGGATGTGGA TTCATTCAGTTTGTCAACAGAGAAAACGCAGAGGAGGCTTTAGAGAAACTAAATGGTTC TGTAATTGGAAAACAAACCGTTCGCCTTTCMTGGGGTCGTAAYCAAGGCAAYAAACAGC CTCGAGGTGGGTATGG NO 13 CTAAGGCAATGAAGTACCTGTCAATAGGTGAAGAAGACGATATATCATGGTCACTTATC AAAGCTGCCTTCTCTTCAGTAGCTCAAACCGCAATCATACCAATGCAAGACATTCTCGG WCTYGGAAGTTCTGCCAGGATGAACACTCCAGCCACTGAGGTGGGGAACTGGGGTTGGA GGATTCCGAGTTCAACGAACTTTG NO 12 TTGGCCCTGAAGGTTCTACAGTGCTTCATTATAGACAATCTTCAACTTCTGCTTCTATT GGGAAAATCAGTTGCAAGGTGTACTATTGCAAAGAAGACGAAGTTTGCTTGTACCAGTC TGTTCAGTTTGAGGTACCTTTCAAGRTGGAATCAGAAKCRTCTYCTTCYCAGGTGATCG CATTCACCGTTAAACCTAGAGCAT NO 11 TCAAGGACTTTGGTGATAGTATTCCAGGACATGGTGGAATCACTGATAGAATGGACTGC CAGATGGTAATGGCAGTATTTGCTTACATATATCTCCAGTCCTTTATCGTCTCCCAAAG CGTTTCGGTTGACAAAATCCTGGACCAGATATTGACGAACCTTAGCTTCGAGGAACAAC AAGCTCTCTTCACTAGATTAGGGC NO 10 CTCCTCCKCCGAATCCGTTTGGGGAYGCGTTCAAGGGGCCMGAGATGTGGGCSAAGCTG ACGGCGGATCCGTCGACGAGGGGGTTCTTGAAGCAGCCTGACTTCGTCAACATGATGCA GGAGATCCAGAGGAACCCTAGCAGTCTCAATCTCTACTTGAAGGACCAGAGGGTGATGC AGTCTCTYGGGGTTTTGTTGAATG NO 9 AGTATGAAGAAGAGGGYGAGTATGAGAGAGGTGGGTCGAAGCAGAGGAGAGGAGAGTCA GAGGAAGGKCATGGRTACTACGAAGGGCGTAGTAGACGTTCAAGCCATTATGAGCGTGA GGAGGAACAAGGAGGTGASCAAGACCGKTACGAYGACCGTTATGGGAGAGTGGAGGAAG AAGAATACCGTTATGATGATCGTG NO 8 TCAAGAAGACTTACCCAACAGTCCAGCTTACAGCATGGACATTTTTCCCCATTGTGGGA TGGGTAAAYTACAAGTATGTGCCACTGCACTTCCGGGTCATCTTGCACAGCCTCGTYGC ATTCTTCTGGGGAATCTTCCTGACCCTGCGAGCAAGGTCAATGACACTAGCTTTGGCAA AGGCTAAGTGATCAGGGAAACACA NO 7 CTAGTTTCAGGGAATGGTTTRCAGAAGGTTGAATTGATGAAGACGAGAGCTTCTTCATC AGACGAGACCTCAACGTCCATTGACACCAACGAACTCTTTACWGACTTGAAGGAAAAGT GGGATGGTCTTGAGAACAARACRACYGTGGTTATCTAYGGAGGAGGAGCCATTGTWGCT GTTTGGTTATCTTCCATTCTTGTT NO 6 GAAGTGTTCTGGACACAGCTGAGAAAGCCCACGAAGGGGATATCACATGCATTTCGTGG GCACCCAAGGCAATGACAGTTGGGGAGAGAAAGGCGCAGGTATTAGCGACAGCAGGGGT TGACAARAAAGTGAAGCTGTGGGAAGCTCCAAMGTTGCAGTCTGTGTAGACTTGCTACT GCTGCTGCAATACAAAGAAAGTCT NO 5 TAAAGTATACTCGAAATGGCCCAAATCTCACTCTTTCAAGATCGGCGACTCCCTCTTGT TCTTGTACCCACCAAGCGAAGATTCAATGATTCAAGTGACACCTTCCAACTTCAAGAGC TGCAACACCAAAGATCCGATCTTGTACATGAACGACGGCAACTCTCTCTTCAACCTCAC CCAAAACGGAACCTTTTACTTCAC NO 4 TCAGACTCATCCAGATAAAGAAGAACAAAATCTCATCTTCTGTGCACTCTATGGTACAA ACTCCTTCAGGTACAGCWCGAACGCACAGGTTTGCCACTGAAACAGCCGAGCTCCCTGC GCAAGAAGGAGAAAGAGTGACAATTGCATCTGCTGCTCCATCAGATGTTTACAGACAAG TGGGACCTTTCAAGTTTACCCCCA
[0090] A specific Raphanus SNP within each of the above marker sequences in Table 1 and Table 2 have been shown under bold font. Of course, the skilled person may use other Raphanus SNPs identified within the above markers as depicted in Table 1 and Table 2. Some of these SNPs are indicated by the IUPAC code in the above sequence.
[0091] More generally, it is disclosed herein the specific means for detecting the POSH+ allele of the Raphanus introgression in a plant, more specifically a Brassica plant.
[0092] Said means thus include any means suitable for detecting the following Raphanus SNP markers within one or more of the following markers: SEQ ID NOs: 4-22.
[0093] Any method known in the art may be used in the art to assess the presence or absence of a SNP. Some suitable methods include, but are not limited to, sequencing, hybridization assays, polymerase chain reaction (PCR), ligase chain reaction (LCR), and genotyping-by-sequence (GBS), or combinations thereof.
[0094] Different PCR based methods are available to the person skilled of the art. One can use the RT-PCR method or the Kaspar method from KBioscience (LGC Group, Teddington, Middlesex, UK).
[0095] The KASP™ genotyping system uses three target specific primers: two primers, each of them being specific of each allelic form of the SNP (Single Nucleotide Polymorpshism) and one other primer to achieve reverse amplification, which is shared by both allelic form. Each target specific primer also presents a tail sequence that corresponds with one of two FRET probes: one label with FAM® dye and the other with HEX® dye.
[0096] Successive PCR reactions are performed, the last one presence of the probes amplification. The nature of the emitted fluorescence is used to identify the allelic form or forms present in the mix from the studied DNA.
[0097] The primers identified in Table 3 are particularly suitable for use with the KASP™ genotyping system. Of course, the skilled person may use variant primers or nucleic acid probes of the primers as identified in Table 3, said variant primers or nucleic acid probes having at least 90%, and preferably 95% sequence identity with any one of the primers as identified in Table 3, or with the DNA genomic fragment amplified by the corresponding set of primers as identified in Table 3.
[0098] Percentage of sequence identity as used herein is determined by calculating the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. For example, nucleic acid sequences may be aligned using the BLAST 2 sequences (Bl2seq) using BLASTN algorithms (www.ncbi.nlm.nih.gov).
[0099] As used herein, a primer encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form though single-stranded form is preferred. Alternatively, nucleic acid probe can be used. Nucleic acid probe encompass any nucleic acid of at least 30 nucleotides and which can specifically hybridizes under standard stringent conditions with a defined nucleic acid. Standard stringent conditions as used herein refers to conditions for hybridization described for example in Sambrook et al 1989 which can comprise 1) immobilizing plant genomic DNA fragments or library DNA on a filter 2) prehybridizing the filter for 1 to 2 hours at 65° C. in 6×SSC 5×Denhardt's reagent, 0.5% SDS and 20 mg/ml denatured carrier DNA 3) adding the probe (labeled) 4) incubating for 16 to 24 hours 5) washing the filter once for 30 min at 68° C. in 6×SSC, 0.1% SDS 6) washing the filter three times (two times for 30 min in 30 ml and once for 10 min in 500 ml) at 68° C. in 2×SSC 0.1% SDS.
[0100] In specific embodiments, said primers for detecting the SNP markers of the present disclosure are as listed in the following table:
TABLE-US-00003 TABLE 3 Primers for use in detecting Raphanus SNP markers of the invention (as indicated in the primer name) SEQ ID Nucleotide Sequence Primer name 64 GAAGGTGACCAAGTTCATGCTCGAAGGGCGTAGTAGACGTTCA SEQ ID NO: 9_A1 65 GAAGGTCGGAGTCAACGGATTGAAGGGCGTAGTAGACGTTCG SEQ ID NO: 9_A2 66 CCTTGTTCCTCCTCACGCTCATAAT SEQ ID NO: 9_C 67 GAAGGTGACCAAGTTCATGCTCCACTGCACTTCCGGGTCATA SEQ ID NO: 8_A1 68 GAAGGTCGGAGTCAACGGATTCCACTGCACTTCCGGGTCATC SEQ ID NO: 8_A2 69 GAAGAATGCGACGAGGCTGTGCAA SEQ ID NO: 8_C 70 GAAGGTGACCAAGTTCATGCTAGAGAAAACGCAGAGGAGGCTTTA SEQ ID NO: 14_A1 71 GAAGGTCGGAGTCAACGGATTGAGAAAACGCAGAGGAGGCTTTG SEQ ID NO: 14_A2 72 GCGAACGGTTTGTTTTCCAATTACAGAA SEQ ID NO: 14_C 73 GAAGGTGACCAAGTTCATGCTCAAGTAGACCCAATAGTAGCGTCA SEQ ID NO: 19_A1 74 GAAGGTCGGAGTCAACGGATTAAGTAGACCCAATAGTAGCGTCC SEQ ID NO: 19_A2 75 ACCATCAACGCTGAGATCACACCAA SEQ ID NO: 19_C 76 GAAGGTGACCAAGTTCATGCTGGTACGGTTCCGGAGGTGGA SEQ ID NO: 21_A1 77 GAAGGTCGGAGTCAACGGATTGTACGGTTCCGGAGGTGGC SEQ ID NO: 21_A2 78 CGACCATTTCCGCTTCCATAACCAT SEQ ID NO: 21_C 79 GAAGGTGACCAAGTTCATGCTAGTACAAAGCTGAGTCAAGCA SEQ ID NO: 20_A1 80 GAAGGTCGGAGTCAACGGATTCTAGTACAAAGCTGAGTCAAGCG SEQ ID NO: 20_A2 81 CAGGAAGTGGATACTTGCGCTTGAT SEQ ID NO: 20_C 82 GAAGGTGACCAAGTTCATGCTTTATTGCCATTGCCATTATCCTTGGA SEQ ID NO: 17_A1 83 GAAGGTCGGAGTCAACGGATTATTGCCATTGCCATTATCCTTGGG SEQ ID NO: 17_A2 84 GTGACAGCAATGATCTTGACAAGATTGTA SEQ ID NO: 17_C 85 GAAGGTGACCAAGTTCATGCTAAGGTGTACTATTGCAAAGAAGACGAA SEQ ID NO: 12_A1 86 GAAGGTCGGAGTCAACGGATTGGTGTACTATTGCAAAGAAGACGAG SEQ ID NO: 12_A2 87 TCAAACTGAACAGACTGGTACAAGCAAA SEQ ID NO: 12_C 88 GAAGGTGACCAAGTTCATGCTGGGAGAGAAAGGCGCAGGTA SEQ ID NO: 6_A1 89 GAAGGTCGGAGTCAACGGATTGGGAGAGAAAGGCGCAGGTT SEQ ID NO: 6_A2 90 TTTTGTCAACCCCTGCTGTCGCTAA SEQ ID NO: 6_C 91 GAAGGTGACCAAGTTCATGCTCAAGATCTTGGGTGTGCCCACAA SEQ ID NO: 15_A1 92 GAAGGTCGGAGTCAACGGATTCAAGATCTTGGGTGTGCCCACAT SEQ ID NO: 15_A2 93 CTCCAGCAAGTGCATCTTCAATAACAATA SEQ ID NO: 15_C 94 GAAGGTGACCAAGTTCATGCTCGAAGATTCAATGATTCAAGTGACAC SEQ ID NO: 5_A1 95 GAAGGTCGGAGTCAACGGATTCGAAGATTCAATGATTCAAGTGACAG SEQ ID NO: 5_A2 96 GGTGTTGCAGCTCTTGAAGTTGGAA SEQ ID NO: 5_C 97 GAAGGTGACCAAGTTCATGCTGTAGCTGGAGTTCTTTTCATC SEQ ID NO: 16_A1 98 GAAGGTCGGAGTCAACGGATTGGCTGTAGCTGGAGTTCTTTTCATT SEQ ID NO: 16_A2 99 CCCTTGAGCTTATCTAAATCTTCGTTCTT SEQ ID NO: 16_C 100 GAAGGTGACCAAGTTCATGCTAGTAGCTCAAACCGCAATCATACCA SEQ ID NO: 13_A1 101 GAAGGTCGGAGTCAACGGATTAGCTCAAACCGCAATCATACCG SEQ ID NO: 13_A2 102 TTCCGAGACCGAGAATGTCTTGCAT SEQ ID NO: 13_C 103 GAAGGTGACCAAGTTCATGCTCAGTATTTGCTTACATATATCTCCAGTCA SEQ ID NO: 11_A1 104 GAAGGTCGGAGTCAACGGATTGTATTTGCTTACATATATCTCCAGTCC SEQ ID NO: 11_A2 105 CCGAAACGCTTTGGGAGACGATAAA SEQ ID NO: 11_C 106 GAAGGTGACCAAGTTCATGCTGGGTTCTTGAAGCAGCCTGAC SEQ ID NO: 10_A1 107 GAAGGTCGGAGTCAACGGATTGGGGTTCTTGAAGCAGCCTGAT SEQ ID NO: 10_A2 108 GGATCTCCTGCATCATGTTGACGAA SEQ ID NO: 10_C 109 GAAGGTGACCAAGTTCATGCTGAACGCACAGGTTTGCCACTGAA SEQ ID NO: 4_A1 110 GAAGGTCGGAGTCAACGGATTAACGCACAGGTTTGCCACTGAG SEQ ID NO: 4_A2 111 AGATGCAATTGTCACTCTTTCTCCTTCTT SEQ ID NO: 4_C 112 GAAGGTGACCAAGTTCATGCTCCATTGACACCAACGAACTCTTTAA SEQ ID NO: 7_A1 113 GAAGGTCGGAGTCAACGGATTCCATTGACACCAACGAACTCTTTAC SEQ ID NO: 7_A2 114 GTCTTGTTCTCAAGACCATCCCACTT SEQ ID NO: 7_C 115 GAAGGTGACCAAGTTCATGCTGCGCTAGTGACGTGAAGAAGAAA SEQ ID NO: 18_A1 116 GAAGGTCGGAGTCAACGGATTGCGCTAGTGACGTGAAGAAGAAG SEQ ID NO: 18_A2 117 GCTTCTTATACCCAAACCTCTCAATCAAA SEQ ID NO: 18_C
Use of Brassica Plants of the Disclosure
[0101] Brassica plants of the present disclosure may be used for breeding applications. As used herein, breeding applications encompass pedigree breeding to improve the agronomical value of a plant, line, hybrid, or variety. In specific embodiment, it relates to backcrossing activities in order to create new recombinant lines in a genomic region of interest or to introgress a region of interest in another plant not comprising such region. Typically, in the present disclosure, the Brassica plants are used to introgress the Raphanus region conferring POSH+ phenotype in another plant.
[0102] Accordingly, it is a further disclosed a method of production of a POSH+ Brassica plant, wherein the method comprises the following steps: [0103] a. crossing a first Brassica plant as described in the previous section with a second POSH.sup.− or POSH+ Brassica plant; thereby obtaining a F1 hybrid plant; [0104] b. selfing or backcrossing said F1 hybrid plant with said second POSH.sup.−or POSH+Brassica plant; [0105] c. selecting the POSH+ Brassica plant among the plant obtained in step b), optionally using at least one Raphanus SNP within at least one of the markers SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and [0106] d. optionally, further selecting said POSH+ Brassica plant for the absence of at least one of the Raphanus SNPs within at least one of the marker SEQ ID NOs: 4-18.
[0107] In a specific embodiment, a first plant as used in the above method is the plant obtained from a representative sample of the seeds as deposited on Jul. 27, 2015 at NCIMB collection under the accession number 42444, obtained from Brassica napus R42141F as described in Example 5 below.
[0108] Any Brassica plants obtained or obtainable by the disclosed methods for producing Brassica plant with POSH+ phenotype are also part of the present invention.
[0109] Brassica plants disclosed herein are further useful for example for producing canola oils. Seeds harvested from plants described herein can be used to make a crude canola oil or a refined, bleached, deodorized (RBD) canola oil. Harvested canola seed can be crushed by techniques known in the art. The seed can be tempered by spraying the seed with water to raise the moisture to, for example about 8.5%. The tempered seed can be flaked using a smooth roller with, for example a gap setting of 0.23 to 0.27 mm. Heat may be applied to the flakes to deactivate enzymes, facilitate further cell rupturing, coalesce the oil droplets, or agglomerate protein particles in order to ease the extraction process. Typically, oil is removed from the heated canola flakes by a screw press to press out a major fraction of the oil from the flakes. The resulting press cake contains some residual oil.
[0110] Crude oil produced from the pressing operation typically is passed through a settling tank with a slotted wire drainage top to remove the solids expressed out with the oil in the screw operation. The clarified oil can be passed through a plate and frame filter to remove the fine solid particles. Canola press cake produced from the screw pressing operation can be extracted with commercial n-Hexane. The canola oil recovered from the extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil.
[0111] The Brassica plants or their oil are also useful as food compositions, for human or animal. The oil may also be used in biofuel.
EXAMPLES
Example 1: Creation of New Recombinant Brassica Plant
[0112] Brassica napus is a relatively young crop and does still show some characteristics of wild species. One of these characteristics is a tendency for pod shattering at harvest time. It has been shown that some B. napus Ogura-hybrids show a much better pod shatter tolerance. In order to characterize this trait and obtain new recombinant lines, 204 crosses were done in January 2011. Here Ogura males and hybrids with the original Raphanus introgression and carrying the pod shatter tolerance have been crossed with Ogura males with shortened Raphanus introgressions or with inbred lines not carrying a Raphanus introgression. Moreover Ogura males with shortened Raphanus introgressions have been crossed with inbred lines not carrying a Raphanus introgression. In November 2011 the resulting F2 plants were genotyped using SNP markers located on C09 and flanking the Raphanus introgression. One SNP Marker is flanking the Raphanus introgression on telomeric region, four other SNP markers are flanking the Raphanus introgression on centromeric region.
[0113] Hence all marker profiles combinations of telomeric and centromeric SNP markers which were not present in the parents of the respective cross indicate a recombination between these markers and consequently probably within the Raphanus introgression located between these markers. By this approach 62 potential recombinant plants have been identified from 11770 F2 plants. Such screening was repeated in 2013, 2014 and 2015 selfed seed of all 62 potential recombinants from 2012 were sown in F3 to validate the results from F2 plants. F3 plants were analysed again with the telomeric and centromeric SNP markers flanking the introgression and SNP markers BnRfo5 (depicted as SEQ ID NO: 1) and SSR markers C08 and Boljon (depicted as SEQ ID NO:2) located on the Raphanus introgression. F3 lines where the recombination was validated were continued to F4.
Example 2: New Markers Development
[0114] Characterization with molecular markers of the new recombinant plants is very difficult. Indeed, on one hand the introgression has replaced a part of the Brassica napus genome and it is difficult to find markers that work in both Raphanus and napus species. Moreover due to the low level of recombination rate in this region, it is not possible to map the position of markers on the introgression based on linkage. Therefore the possibilities to describe the introgression were very limited. To address the problem of SNP discovery, we employed a Next Generation Sequencing (NGS)-based approach on the transcriptome of vegetative tissue.
[0115] Specifically 118 fixed restorer lines and 27 fixed female lines were sampled at 4-weeks post emergence stage and flush frozen in preparation for RNA extraction. RNA concentration of each combined sample was measured using 1 μl of each RNA sample on the Qubit fluorometer (Invitrogen). The current version IIlumina mRNA-Seq kit was used according to the manufacturer's protocol to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing for subsequent cluster generation. Libraries were prepared using 5 μg of total RNA, with quantification and quality assessment being carried out by running 1 μl of library on an Agilent DNA 1000 LabChip (Agilent Technology 2100 Bioanalyzer). The libraries were multiplexed two per lane, loaded onto the Illumina HiSeq2000 instrument following the manufacturer's instructions and run for 100 cycles (single end reads) to produce at least 2.0 Gb of sequence per sample.
[0116] In order to develop the markers of the disclosure useful to identify new recombinant lines, initial sequence alignment and SNP discovery across the panel of lines was performed using MAQ (Li et al Genome Research 18:1851-1858, 2008) and Perl scripts (Trick et al Plant Biotech J. 7:334, 2009; Bancroft et al, 2011).
[0117] Across the 143 OSR lines for which sequence data were obtain, on average, 1.59×107 sequence reads of 100 bases (1.59 Gb sequence data) were aligned to 50.4 Mb of reference sequences, resulting in 31.5-fold coverage. As a result we developed a marker dataset comprising 84,022 simple SNPs (28,402 after removing those with minor allele frequency below 5%) and 119,523 hemi-SNPs (80,100 after removing those with minor allele frequency below 5%). The advantage of simple SNPs is that these markers can be assigned with more confidence to one of the two genomes of oilseed rape than can hemi-SNP markers. An analysis was conducted with the aim of identifying SNP markers associated with line designation of male parent (MP) or female parent (FP). 169 markers were identified that fully differentiated between the types. The 169 markers all shared the characteristic of the allele comprising an ambiguity code (i.e. indicating the presence of 2 bases) for MP lines and a resolved base for FP lines, consistent with the addition of an additional genomic segment (i.e. that associated with the CMS restorer locus from radish). The markers were clustered predominantly in two pairs of homoeologous regions on linkage groups A9 and C9, with a few in regions paralogous to these.
[0118] The inventors developed around 550 new SNP markers which are specific for the Raphanus introgression. By BLASTing these SNP to the oleracea genome it was concluded that the specific markers for the Raphanus introgression covers around 24 Mbp (length of the raphanus introgression) which is about 50% of chromosome 009.
[0119] Moreover we have identified a high number of markers which are functional in original B. napus but that are not present in Ogura-restorer lines. By BLASTing these to the oleracea genome it was found that these markers cover again around 24 Mbp (lost B. napus chromosome segment). This was also validated by results from the IIlumina 50k Chip array, where also Markers were not present in restorer material covering a fragment of about 22 Mbp.
[0120] This result clearly shows that one arm of the chromosome C09 was replaced by one arm of a Raphanus chromosome when the Ogura-introgression was created.
Example 3: Phenotype Characterization of the New Recombinant Lines
[0121] The stability of the pod can be measured with a test developed by Dr. Schulz at the Institute LFA-Mecklenburg-Vorpommern as described in March 2013 in Abschlussbericht 2013, Forschungsnummer 1/29, im Forschungskomplex, Verfahrensoptimierung zur Verbesserung der Wirtschaftlichkeit. Pods are sampled at complete maturity (BBCH 97) from the middle part of the main stem. After sampling the pods are kept under dry conditions at room temperature for at least 21 days in order to ensure complete maturity of all pods. In the test the measured parameter for pod shatter tolerance is the tension measured to tear the two halves of the pod apart. For the measurement a Sauter Digital Force Gauge FK 50 was used. 20 individual pods of each genotype have been measured and the average of the 20 measurements was calculated.
[0122] Example of the results of these measurements made with different lines or hybrids are given in
[0123] The table in
[0124] The table in
Example 3: Pod Shattering Tolerance Provided by Raphanus is Partial Dominant
[0125] Ogura restorers are used to produce hybrids with a sterile CMS line. In the resulting hybrid the Raphanus introgression is in the heterozygote state and therefore these hybrids are suitable to test if the pod shattering tolerance is inherited dominant, recessive or intermediate.
[0126] Examples of the results of the measurements of different Ogura hybrids are given in
Example 4: Identification of Other Markers Strongly Associated to POSH Locus
[0127] The inventors have shown that surprisingly a FRUITFULL locus is localized on the Raphanus introgression as all the markers developed from the FRUITFULL gene sequence as identified on the Raphanus genome are strongly associated with the POSH locus markers described above (
[0128] In particular the inventors have also identified the predicted Open Reading Frame (SEQ ID NO:31) of the Raphanus FRUITFULL gene and the corresponding protein as predicted (SEQ ID NO:32) or corresponding predicted cDNA (SEQ ID NO:33). Such sequences may further advantageously be used to identify Raphanus SNP associated to POSH+ locus in Brassica plants.
[0129] Two different types of markers were identified. A first type is not genome specific. It is derived from a classic design with a SNP between napus and Raphanus, and a common marker shared with oleracea, rapa and radish. Thus, the one allele will amplify B. rapa and B. oleracea, and the other allele is specific of the radish genome. In this type of design, the A genome is always amplified and therefore giving a background signal that decreases the resolution of the observations. This kind of marker does not permit us to distinguish AA/CC and AA/Ø.
[0130] The second type of markers is genome specific. Therefore, there is no amplification of ‘A’ rapa genome. The design was realized between a SNP between napus and Raphanus and a HSV (Homeologous sequence variation) shared with oleracea and raphanus.
[0131] Examples of primers sequences to identify the non genome specific marker FRUITFULL_H1_04 are FRUITFULL_H1_04_F_A1 (SEQID NO 40), FRUITFULL_H1_04_F_A2 (SEQID NO 41) and (FRUITFULL_H1_04_F_C) SEQIDNO 42 and primers to identify the genome specific marker FRUITFULL_spe_01 are FRUITFULL_spe_01_R_A1 (SEQIDNO 52), FRUITFULL_spe_01_R_A2 (SEQIDNO 53) and FRUITFULL_spe_01_R_C (SEQIDNO 54).
[0132] These markers have been used to identify and follow the POSH region in breeding programs as shown in table 5.
Example 5: Development of New Pod Shattering Tolerant Brassica napus Lines with Shortened Raphanus Introgression
[0133] The F4 progeny of the lines obtained in example 1 was systematically phenotyped for pod shattering tolerance and screened with codominant SNP markers developed in example 3.
[0134] The following Table 6 show the SNP codominant markers which were used to analyze all the new recombinant plants generated:
TABLE-US-00004 SEQ ID NO Nucleotide sequence 4 TCAGACTCATCCAGATAAAGAAGAACAAAATCTCATCTTCTGTGCACTCTATGGTACAAA CTCCTTCAGGTACAGCWCGAACGCACAGGTTTGCCACTGA[A/G]ACAGCCGAGCTCCCT GCGCAAGAAGGAGAAAGAGTGACAATTGCATCTGCTGCTCCATCAGATGTTTACAGACAA GTGGGACCTTTCAAGTTTACCCCCA 5 TAAAGTATACTCGAAATGGCCCAAATCTCACTCTTTCAAGATCGGCGACTCCCTCTTGTT CTTGTACCCACCAAGCGAAGATTCAATGATTCAAGTGACA[C/G]CTTCCAACTTCAAGA GCTGCAACACCAAAGATCCGATCTTGTACATGAACGACGGCAACTCTCTCTTCAACCTCA CCCAAAACGGAACCTTTTACTTCAC 6 GAAGTGTTCTGGACACAGCTGAGAAAGCCCACGAAGGGGATATCACATGCATTTCGTGGG CACCCAAGGCAATGACAGTTGGGGAGAGAAAGGCGCAGGT[A/T]TTAGCGACAGCAGGG GTTGACAARAAAGTGAAGCTGTGGGAAGCTCCAAMGTTGCAGTCTGTGTAGACTTGCTAC TGCTGCTGCAATACAAAGAAAGTCT 7 CTAGTTTCAGGGAATGGTTTRCAGAAGGTTGAATTGATGAAGACGAGAGCTTCTTCATCA GACGAGACCTCAACGTCCATTGACACCAACGAACTCTTTA[C/A]WGACTTGAAGGAAAA GTGGGATGGTCTTGAGAACAARACRACYGTGGTTATCTAYGGAGGAGGAGCCATTGTWGC TGTTTGGTTATCTTCCATTCTTGTT 8 TCAAGAAGACTTACCCAACAGTCCAGCTTACAGCATGGACATTTTTCCCCATTGTGGGAT GGGTAAAYTACAAGTATGTGCCACTGCACTTCCGGGTCAT[C/A]TTGCACAGCCTCGTY GCATTCTTCTGGGGAATCTTCCTGACCCTGCGAGCAAGGTCAATGACACTAGCTTTGGCA AAGGCTAAGTGATCAGGGAAACACA 9 AGTATGAAGAAGAGGGYGAGTATGAGAGAGGTGGGTCGAAGCAGAGGAGAGGAGAGTCAG AGGAAGGKCATGGRTACTACGAAGGGCGTAGTAGACGTTC[A/G]AGCCATTATGAGCGT GAGGAGGAACAAGGAGGTGASCAAGACCGKTACGAYGACCGTTATGGGAGAGTGGAGGAA GAAGAATACCGTTATGATGATCGTG 10 CTCCTCCKCCGAATCCGTTTGGGGAYGCGTTCAAGGGGCCMGAGATGTGGGCSAAGCTGA CGGCGGATCCGTCGACGAGGGGGTTCTTGAAGCAGCCTGA[C/T]TTCGTCAACATGATG CAGGAGATCCAGAGGAACCCTAGCAGTCTCAATCTCTACTTGAAGGACCAGAGGGTGATG CAGTCTCTYGGGGTTTTGTTGAATG 11 TCAAGGACTTTGGTGATAGTATTCCAGGACATGGTGGAATCACTGATAGAATGGACTGCC AGATGGTAATGGCAGTATTTGCTTACATATATCTCCAGTC[C/A]TTTATCGTCTCCCAA AGCGTTTCGGTTGACAAAATCCTGGACCAGATATTGACGAACCTTAGCTTCGAGGAACAA CAAGCTCTCTTCACTAGATTAGGGC 12 TTGGCCCTGAAGGTTCTACAGTGCTTCATTATAGACAATCTTCAACTTCTGCTTCTATTG GGAAAATCAGTTGCAAGGTGTACTATTGCAAAGAAGACGA[A/G]GTTTGCTTGTACCAG TCTGTTCAGTTTGAGGTACCTTTCAAGRTGGAATCAGAAKCRTCTYCTTCYCAGGTGATC GCATTCACCGTTAAACCTAGAGCAT 13 CTAAGGCAATGAAGTACCTGTCAATAGGTGAAGAAGACGATATATCATGGTCACTTATCA AAGCTGCCTTCTCTTCAGTAGCTCAAACCGCAATCATACC[A/G]ATGCAAGACATTCTC GGWCTYGGAAGTTCTGCCAGGATGAACACTCCAGCCACTGAGGTGGGGAACTGGGGTTGG AGGATTCCGAGTTCAACGAACTTTG 14 CTTTTGCTGGTTTTGGTGAAATAGTATCTGTCAAGATACCAGTTGGGAAAGGATGTGGAT TCATTCAGTTTGTCAACAGAGAAAACGCAGAGGAGGCTTT[A/G]GAGAAACTAAATGGT TCTGTAATTGGAAAACAAACCGTTCGCCTTTCMTGGGGTCGTAAYCAAGGCAAYAAACAG CCTCGAGGTGGGTATGG 15 GTCCATGTTTGATGCAATTGTATCAGCAGACGCATTTGAGAACTTGAAACCAGCTCCAGA TATTTTCTTGGCTGCTTCCAAKATCTTGGGTGTGCCCACA[T/A]GCGAGTGTATTGTTA TTGAAGATGCACTTGCTGGAGTCCAGGCTGCTCAAGCTGCAAACATGAGATGCATAGCTG TGAAAACTACTTTATCTGAAGCAAT 16 ACTTTGTTGAYAGYCTTACMGGAGTAGGACTTGTTGATCAAATGGGAAACTTCTTCTGCA AAACGCTCTTGTTTGTGGCTGTAGCTGGAGTTCTTTTCAT[T/C]CGCAAGAACGAAGAT TTAGATAAGCTCAAGGGTCTRWTYGAAGAGACGACGYTRTATGACAAGCARTGGCAAGCG GCTTGGAAAGAGCCGGAAATAATCA 17 CACAACATGCCGGTGATTGGTATCCAGCTGACCTTGGATCCAACGATTTCAAAGGTCTCT ATGGATATAAGGTCTTTATTGCCATTGCCATTATCCTTGG[G/A]GACGGTCTCTACAAT CTTGTCAAGATCATTGCTGTCACTGTGAAGGAATTATGCAGCAATAGCTCTAGACACCTC AATCTACCCGTTGTTRCCAACGTTG 18 AGAAGATGGAGTTCTTGATGTTTGATCTYGATCGGGTTTTGAARCCCGGTGGGTTGTTCT GGTTGGATAACTTCTACTGCGCTAGTGACGTGAAGAAGAA[A/G]GAGCTGACGCGTTTG ATYGAGAGGTTTGGGTATAAGAAGCTGAAATGGGTTATTGGAGAGAAGGCTGATGGGCAA GTGWATCTCTCTGCTGTTCTKCAAA 19 TAGAGCTGAAGCTAGGTATAGGAGGCACATCATAYAAAGATTTCATTCAAAGCCTTCATC TACCTATGCAATTGAGTCAAGTAGACCCAATAGTAGCGTC[C/A]TTCTCYGGAGGAGCT GTTGGTGTGATCTCRGCKYTGATGGTWGTWGAAGTCAACAACGTGAAGCAGCAAGAGCAC AAGAGATGCAAATACTGTCTAGGAA 20 TTAAGAACTGTGTCACTGACATTGACCCTGAGAGGGAGAAGGAGAAGAGAGAAAGGATGG AAAGCCAAAACCTCAAGGCTAGTACAAAGCTGAGTCAAGC[G/A]AGGGAGAAAATCAAG CGCAAGTATCCACTTCCTGTTGCAAGGAGRCAACTYTCCACTGGRTACNTGGAAGATGCT CTCGAAGAGGATGAAGAGACAGACC 21 GCTCAGGTAGATCTCCCACGGGTTGGGGAAGAGGATCCGGATATGGGTATGGGTCTGGAT CTGGATCAGGTAGCGGATATGGGTACGGTTCCGGAGGTGG[A/C]GGAGSACGTGGTGGT GGGTATGGTTATGGAAGCGGAAATGGTCGGTCTGGAGGWGGTGGTGGTGGCTCTAATGGT GAAGTTGCCGCTTTGGGCCACGGTG 22 TCAGACTCATCCAGATAAAGAAGAACAAAATCTCATCTTCTGTGCACTCTATGGTACAAA CTCCTTCAGGTACAGCWCGAACGCACAGGTTTGCCACTGA[A/G]ACAGCCGAGCTCCCT GCGCAAGAAGGAGAAAGAGTGACAATTGCATCTGCTGCTCCATCAGATGTTTACAGACAA GTGGGACCTTTCAAGTTTACCCCCA
[0135] The SNP is shown under bracket in the above marker sequences, the first nucleotide representing Raphanus SNP, the second nucleotide representing Oleracea SNP.
[0136] This systematic scoring resulted in the identification of one recombinant plant with a shortened raphanus introgression where the pod shatter coding region was still present. The F5 progeny of this plant is genotype R42141F with the pedigree (FOCTD909×NSL09/196). This recombinant line is pod shattering tolerant and has a good pod size.
[0137]
[0138] This result shows that the POSH region is localized in the region strongly associated with the POSH locus markers of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
[0139] Results for Pod stability and genotype profile on the pod shattering tolerant recombinant line R51542141F (also called R42141F) and a panel of reference genotypes are shown in
Example 6: Identification of New Pod Shattering Tolerant Brassica napus Lines without Rf0 Raphanus Region and Obtention of Pod Shatter Tolerant Females and Non Ogura Inbred Lines
[0140] In order to create new recombinant restorer lines carrying shorter Raphanus introgression, 128 crosses have been done in January 2012. Here Ogura males and hybrids with the original Raphanus long introgression and carrying the pod shatter tolerance POSH.sup.+ have been crossed with POSH.sup.− plants, Ogura males with shortened Raphanus introgressions or inbred lines not carrying a Raphanus genome fragment introgression. In November 2012, 6421 F2 plants resulting from these crossing were genotyped using 4 SNP markers located on C09 as described in “Exemple1”. Selfed seed of all 353 potential recombinants were sown in F3 to validate the results from F2 plants. F3 plants were analysed in November 2013 with the set of codominant SNP markers developed in example 3.
[0141] The same SNP codominant markers were used to analyze all the new recombinant plants generated sowed in F3 in November 2013 (see previous Table 7).
[0142] Among these F3 plants, the plants coded as FR-13C-3-03137-2 and FR-13C-3-03137-5 were identified. These plants were selected because they were carrying only the Raphanus favorable alleles of the markers SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 linked to pod shattering tolerance without any other raphanus alleles of the introgression. Since the plants were in sterile Ogura cytoplasm but did not contain the Rf0-gene, they were sterile and consequently could not be selfed. To maintain this event of recombination, 4 other recombinant fertile lines with shortened Raphanus introgression but carrying Rf0 gene were selected to be crossed with the sterile pod shatter tolerant recombinant (Genotypic profiles are given in
[0143] The resulting F1 is fertile, is carrying a shortened Raphanus fragment and the pod shatter tolerance. With this F1 it is possible on the one hand to develop inbred lines with shortened Raphanus introgression and carrying the pod shatter tolerance by inbreeding and marker assisted selection and on the other hand to cross the F1 as male parent and thereby transfer the pod shatter tolerance to any other Brassica plant. In this respect it is of special interest to cross the F1 to a Brassica napus plant to a fertile cytoplasm in order to transfer the pod shattering tolerance outside the sterile Ogura cytoplasm and to develop inbred lines with pod shattering tolerance but without the Rf0 gene. These inbred lines can subsequently be used as male/female parent in other hybrid systems (e.g. GMS) or after CMS-conversion as female parent in the Ogura hybrid system.
Example 7: Correlation Between the Reduction of Raphanus Introgression Fragment and the Increase of Pod Size
[0144] The pod size of different lines or hybrids carrying the POSH trait has been measured. Plants were grown in field and the pods were sampled at complete maturity. Pods size measurement corresponds to the measure (cm) of the upper half of the pod which does not comprise the beak and the pedicel. The results are shown in
[0145] R51542141F is the recombinant line, R4513-CA is the line with the long introgression. Adriana is a control non restorer hybrid that does not comprise the Raphanus genome introgression.
[0146] Arsenal is a hybrid variety with a long introgression comprising the Rf0 gene and the POSH region markers. RD153-101 is a restorer line with short introgression which is not pod shattering tolerant.
[0147] The results show that there is a correlation between the reduction of Raphanus introgression fragment and the increase of pod size.
Example 8: Segregation of POSH in a Double Hybrid Population
[0148] Hybrids comprising the short introgression were crossed with hybrids comprising long introgression. Segregating DH-populations were generated and plants were grown in field. The pod stability was measured according to example 3.
[0149] The results in
[0150] The results demonstrate that there is a significant correlation between the long introgression carrying the POSH markers as represented by the black boxes alleles and the high level of pod stability.