Patent Publication Number: US-2021169029-A1

Title: Brassica plant with pod shattering tolerance

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This is a Continuation of application Ser. No. 15/747,122 filed Jan. 23, 2018, which in turn is a 371 of PCT/EP2016/068612 filed Aug. 4, 2016, which claims priority to EP 15306287.2 filed Aug. 11, 2015. The disclosure of the prior applications is hereby incorporated by reference herein in its entirety. 
    
    
     The field of the invention is related to plant breeding, particularly the development of new  Brassica  plants with pod shattering tolerance. 
     BACKGROUND OF THE INVENTION 
     Rape culture, also called canola across the Atlantic, is widespread on all continents due to the fact of its multiple strengths in both the food and industrial sectors. Indeed, rapeseed produces a large oil widely used as a food product but also as a biofuel especially in the automotive industry or the like. Rape also allows the production of cakes that are a good source of protein in animal feed (cattle, pigs and poultry). 
     Despite these strengths, the food use of oilseed rape oil has long been restricted because of his excessive erucic acid content. Indeed, based on rapeseed varieties, the level of erucic acid could be up to 50% of total fatty acids of the plant and have a detrimental impact on human health. 
     Similarly, the use of rapeseed for the manufacture of meals has also long been hampered due to the high content of glucosinolates in the seed. When forming cake, grinding seeds frees the myrosinase enzyme that converts glucosinolates seeds in various by-products such as glucose, thiocyanates, isothiocyanates and nitriles that may lead to metabolic disorders in mammals. The extensive development of rape crop is mainly due to two major technical advances: the decrease of the level of erucic acid in the oil and the reduction of the level of glucosinolates in the seed. Indeed, today, a network of plant breeding has produced commercial varieties whose level of erucic acid is less than 2% of total fatty acids of the rapeseed plant. Moreover, in Europe, the Decree 2294/92 has set the maximum acceptable rate of glucosinolates in seed 25 micromol per gram of seed at 9% humidity. Glucosinolates rate and the level of erucic acid being two interesting parameters in the production of products derived from rapeseed, breeders have therefore sought to develop varieties called “double zero” that is to say the varieties rapeseed which present very low level of erucic acid in the oil and low levels of glucosinolates in the seeds. 
     Although the oilseed traits are of major importance for  Brassica  breeding activities, other agronomic traits are also selected in order to improve the creation of competitive new varieties. For example, disease resistance, yield, morphological trait like silique length or physiological trait like male sterility, fertility restoration or shattering tolerance. Pod shattering is agronomically important because it may result in the premature shedding of seed before the crop can be harvested. Adverse weather conditions can exacerbate the process resulting in a greater than 50% loss of seed. This loss of seed not only has a dramatic effect on yield but also results in the emergence of the crop as a weed in the subsequent growing season. 
       Arabidopsis  mutants have been used to better understand the genetic determinism of pod shattering. Different genes encoding for transcription factors have been identified to be involved in the regulation of pod shattering tolerance. For example, SHATERPROOF 1 (SHP1) and 2 (SPH2), NAC, INDEHISCENT (IND), ALCATRAZ (ALC) are involved in valve-margin development. REPLUMLESS (RPL) and FRUITFULL (FUL) are involved in repressing the expression of valve-margin identity genes. FUL and IND function have been validated through ectopic expression. 
     Natural genetic variation for shatter tolerance has been identified in oilseed crop species like  Brassica napus . One major locus was identified from a F2 population derived from Chinese parental lines of  B. napus  and has been mapped on chromosome A09 (Hu Z et al, 2012—Discovery of pod shatter-resistant associated SNPs by deep sequencing of a representative library followed by bulk segregant analysis in rapeseed—PLoS ONE 7:e34253). Recently, experiments carried on a biparental population and a diverse germplasm panel from  Brassica napus  allowed the identification of few QTLs for pod shattering tolerance. They have been mapped on different chromosomes of  Brassica napus  genome (Raman et al, 2014, Genome-wide delineation of natural variation for pod shatter resistance in  Brassica napus —PLoS ONE 9, 7, e101673). Interestingly, the authors conclude on the difficulties to demonstrate the function of the allelic variation in conferring pod shattering resistance notably due to the expected high level of number of copy of the different genes involved in pod shattering and the complexity of their organization in the genome. 
     Rape is a self-pollinating species, these varieties have long been only population varieties, not hybrids. However, the development of hybrid plants has multiple interests rape for both the farmer and the breeder, since it allows to obtain improved plants, exhibiting qualities of heterosis (or hybrid vigor), homeostasis (stability of the plant in different environments), the possibility of introducing and combining resistance genes to insects, fungi, bacteria or viruses, or adaptation to abiotic stress. But this development of rapeseed hybrids requires effective means of pollination control. To do this, cytoplasmic male sterility (or CMS Cytoplasmic Male Sterility) systems have been developed such as Polima, and especially Kosena and Ogura systems. 
     The Ogura cytoplasmic male sterility system is based on the use of a determinant of male sterility derived from the cytoplasm of radish ( Raphanus sativus ), which was transferred from the radishes in  Brassica napus  by inter-specific crosses, bailouts of embryos and backcrosses (Bannerot et al, 1974). Protoplast fusion was needed to produce cytoplasmic male sterile hybrids (Pelletier et al., 1983). But CMS Ogura cytoplasmic male sterility is dominant, hybrid rapeseed plant does not produce pollen, and without pollen, the plant does not produce seeds. To remedy this situation and get a harvest, it is necessary that the male parent of the hybrid contains a gene restoring the male fertility. Such a male fertility restorer gene of Ogura system was identified in radish  Raphanus sativus  and  Brassica  plants transferred to the carrier of cytoplasmic male sterility by the National Institute of Agronomic Research in 1987 (Pelletier et al, 1987, Proc 7th Int. Rapeseed Conf. Poznan, Poland, 113/119). Rf restorer gene has been described in the WO92/05251 patent application and in Delourme et al, 1991, Proc 8th Int. Rapeseed Conf. Saskatoon, Canada, 1506/1510. However, the resulting plants carrying this Rf gene restoring the male fertility have two major disadvantages: a significant increase in glucosinolates in the seed and a significant decrease of the agronomic characteristics of the plant such as a decrease in the amount of seeds produced, decreased disease resistance and increased susceptibility to lodging. These disadvantages appear to be directly linked to the wearer introgression fragment including the gene Rf restoration of the cytoplasmic male fertility transferred from  Raphanus sativus . This chromosomal region not having the Rf-restoring gene, it also comprises one or more genes that result in the abovementioned disadvantages. To remedy this situation, various research programs have sought recombination events in this chromosomal region, recombination to break the existing linkage between the DNA segments encoding the various characters. Although research has been hampered by the fact that the chromosomal region surrounding the restorer gene Rf is very difficult to subject to recombination, different patent applications describe the generation of recombination event in the  Raphanus  fragment leading to new recombinant lines harboring the Rf gene and a reduced level of glucosinolates and better pod size (see WO97/02737, WO98/27806, WO 2005/002324, WO 2005/074671, and WO2011020698). Each document describes the generation of specific recombination event between the Rf gene restoring the male fertility and the genes linked to high levels of glucosinolates in the seeds or genes linked to small pod size and each event is characterized by using specific markers. 
     Low glucosinolate levels in seeds and a good pod size are necessities for any commercialized plant. One effective way to reduce the glucosinolate levels and to improve the pod size in Ogura restorers is to shorten the  Raphanus  introgression. On the other hand the reduction of the size of the  Raphanus  fragment may lead to the elimination of agronomic traits of interest. One of these agronomic traits lost after  Raphanus  fragment size reduction is pod shattering tolerance which is of big importance to reduce the seed losses before harvest and a main advantage of Ogura hybrids. New development in this region is therefore needed but are strongly hampered by the very low recombination rate in the  Raphanus  fragment, by the lack of any  Raphanus  genome mapping or sequence and therefore by the lack of any marker specific to this fragment and finally by the complexity of the  Brassica  genome. 
     In this context, one of the essential objectives of the invention is to obtain a  Brassica  plant overcoming all the disadvantages mentioned above. In particular, one objective is to obtain a  Brassica  plant comprising a shortened  Raphanus  fragment including the pod shattering tolerance alleles. Said  Brassica  plant may advantageously be used for breeding to readily transfer such pod shattering tolerance alleles to other  Brassica  plant with other genetic background. In particular, one objective of the present invention is to provide a  Brassica  plant comprising a shortened  Raphanus  fragment including the pod shattering tolerance alleles and the male fertility restoration Rf0 gene. 
     Another objective of the invention is to identify a new  Raphanus  pod shattering tolerance that can be used in  Brassica  breeding activities. 
     Yet another objective of the invention is to obtain a  Brassica  plant comprising the  Raphanus  pod shattering tolerance and the use of this plant to drive the introgression pod shattering tolerance in  Brassica  plants harboring a pod shattering tolerance phenotype. 
     Another object of the invention is to obtain seeds, hybrid plants and progeny of said  Brassica  plants. 
     The invention also relates to methods for identifying the presence of said pod shattering tolerance allele in  Brassica  plants and in particular, suitable markers associated (or not associated) to said new pod shattering tolerance. 
     SUMMARY 
     It is therefore disclosed herein a  Brassica  plant comprising 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 the at least one of the following markers: SEQ ID NOs: 4-18. 
     In specific embodiment of said  Brassica  plant, the  Raphanus  genomic fragment is further defined by the presence of at least one of the  Raphanus  SNP within SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 marker. For example, said  Raphanus  SNPs within SEQ ID NO:9 and SEQ ID NOs:12-18 markers are absent. For example, said  Raphanus  SNPs within all markers of SEQ ID NOs: 4-18 are absent. 
     In another specific embodiment, the  Brassica  plant as disclosed herein further comprises a  Raphanus  FRUITFULL allele. Typically, said  Raphanus  FRUITFULL allele comprises the  Raphanus  SNP within marker SEQ ID NO: 22. 
     In specific embodiments, said  Brassica  plant as disclosed above further comprises the male fertility restoration locus Rf0 within the  Raphanus  fragment. 
     In other specific embodiments, the  Brassica  plant as defined above comprises a CMS Ogura cytoplasm. 
     It is further disclosed herein a hybrid  Brassica  plant obtained by crossing a  Brassica  plant having a  Raphanus  fragment conferring POSH+ phenotype as disclosed above, with 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 + . 
     The seed, or part of plants or their progenies of said  Brassica  plant are also disclosed herein. 
     Another aspect disclosed herein relates to methods for identifying a POSH+  Brassica  plant as described above, wherein said  Brassica  plant is identified by detecting the presence of one or more of the  Raphanus  SNPs within at least one of the following markers SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and/or by the absence of the  Raphanus  SNPs within at least one of the following markers: SEQ ID NOs: 4-18. 
     In specific embodiments of such method, the POSH+  Brassica  is identified by using a  Raphanus  fruitfull allele, and more specifically the  Raphanus  marker SEQ ID NO: 22. 
     Another aspect disclosed herein relates to new means for detecting one or more  Raphanus  SNP within one or more of the following markers: SEQ ID NOs: 4-22. 
     Such means may typically be nucleic acid probes or primers or a set of primers or their combinations, for example one or more primers including any of SEQ ID NOs: 64-99, 106-108, 112-114 and 52-54. 
     Such  Brassica  plants or seeds as disclosed herein are useful for food applications, preferably for oil production and for feed applications, preferably for cake production, or breeding applications for example for use as a parent plant in breeding for improving agronomical value of a  Brassica  plant, line, hybrid or variety. 
     It is also disclosed a method of production of a POSH+  Brassica  plant, wherein the method comprises the following steps:
         a. crossing a first  Brassica  plant of the present invention as disclosed above with the POSH+ phenotype, with a second POSH −  or POSH+  Brassica  plant; thereby obtaining a F1 hybrid plant;   b. selfing or backcrossing the F1 hybrid plant with said second POSH −  or POSH +    Brassica  plant;   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   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 markers SEQ ID NOs: 4-18.       

     In specific embodiments of such method, the second POSH −    Brassica  plant is characterized by the absence of any  Raphanus  genome fragment within its genome. 
     In another specific embodiment of such method, the first plant is a plant obtained from a representative sample of the seeds as deposited at NCIMB collection under the number 42444. 
     It is also disclosed a method of production of a POSH+  Brassica  plant of the present invention as described above, wherein the method comprises the following steps:
         a. providing a first POSH+  Brassica  plant, comprising a  Raphanus  introgression conferring the POSH+ trait, said  Raphanus  introgression including at least one of the  Raphanus  SNP within one or more of the following markers: SEQ ID NO:4-18;   b. crossing said first POSH+  Brassica  plant with a second POSH− or POSH+  Brassica  plant, thereby obtaining a F1 hybrid plant;   c. selfing or backcrossing the F1 hybrid plant with said second plant POSH −  or POSH+;   d. selecting the POSH+ plant among the plant obtained in step c), optionally 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.       

     In specific embodiments of the above method, said first POSH+  Brassica  plant comprises the Rf0 Ogura fertility restoration gene. 
     The disclosure further pertains to the  Brassica  plant obtainable or obtained by the above methods of production. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  (SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122) shows the non-genome specific design strategy used for markers FRUITFULL_H1_04. 
         FIG. 2  (SEQ ID NO: 123, SEQ ID NO: 124, and SEQ ID NO: 125) shows the genome specific design strategy used for markers FRUITFULL_spe_01 on the alignment of  Raphanus  and  Brassica  sequences. 
         FIGS. 3A and 3B  show the measurement of the pod size of different lines or hybrids carrying the POSH trait. 
         FIG. 4A  to  FIG. 4H  show the segregation of POSH in a Double Hybrid population. 
         FIG. 5  shows examples of commercialized  Brassica  varieties comprising a long  Raphanus  introgression, examples of commercialized  Brassica  varieties comprising shorter  Raphanus  introgression fragments, and the results of the measurements of different Ogura hybrids. 
         FIG. 6  shows the results of the phenotyping and genotyping of the F5 progeny compared to other lines and hybrids and the results for pod stability and genotype profile on the pod shattering tolerant recombinant line R51542141F (also called R42141F) and a panel of reference genotypes. 
         FIG. 7  shows genotypic profiles are given for the molecular characterization of the F3 recombinant lines on the  Raphanus  introgression. 
         FIG. 8  is a table showing that the increased pod shattering tolerance is limited to some Ogura restorers and hybrids with a long introgression from  Raphanus sativus.    
         FIG. 9  is a table showing that the increased pod shattering tolerance is limited to some Ogura restorers and hybrids with a long introgression from  Raphanus sativus.    
     
    
    
     DETAILED DESCRIPTION 
     The  Brassica  Plant 
     As used herein, the term “ Brassica  plant” includes a plant of  Brassica  species, including  B. napus, B. juncea  and  B. rapa ; preferably  B. napus.    
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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”. 
     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  FIG. 5  like Albatros or Artoga. 
     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  FIG. 5  like Anterra or are also described in patent application WO2011/020698, WO97/02737, WO98/27806, WO 2005/002324 or WO 2005/074671. 
     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.    
     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. 
     As used herein, a “ Raphanus  SNP” corresponds to the nucleotide present in the  Raphanus  genome at a polymorphic position compared to the  oleracea  genome. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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”. 
     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+. 
     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. 
     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+) 
     The present disclosure also relates to new methods to produce  Brassica  plants with pod shattering tolerance phenotype (POSH+) as described in the previous section. 
     In one embodiment, said method comprises the following steps:
         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;   b. crossing said first POSH+  Brassica  plant with said second POSH −  or POSH+ Brassica  plant, thereby obtaining a F1 hybrid plant;   c. selfing or backcrossing the F1 hybrid plant with the second plant POSH −  or POSH+;   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.       

     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 − ). This second  Brassica  plant can be for example any wild type non restorer and POSH −    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. 
     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. 
     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. 
     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. 
     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. 
     In a specific embodiment, Rf0 locus may further be identified. 
     
       
         
           
               
             
               
                 Table 1 
               
             
            
               
                   
               
               
                 Raphanus SNP associated to POSH+ 
               
               
                 allele (one identified SNP is 
               
               
                 highlighted in bold font)  
               
            
           
           
               
               
            
               
                 SEQ 
                   
               
               
                 ID 
                 Sequence 
               
               
                   
               
               
                 NO 19 
                 TAGAGCTGAAGCTAGGTATAGGAGGCACATCATAYAAAGATT 
               
               
                   
                 TCATTCAAAGCCTTCATCTACCTATGCAATTGAGTCAAGTAG 
               
               
                   
                 ACCCAATAGTAGCGTC   C   TTCTCYGGAGGAGCTGTTGGTGTGA 
               
               
                   
                 TCTCRGCKYTGATGGTWGTWGAAGTCAACAACGTGAAGCAGC 
               
               
                   
                 AAGAGCACAAGAGATGCAAATACTGTCTAGGAA 
               
               
                   
               
               
                 NO 20 
                 TTAAGAACTGTGTCACTGACATTGACCCTGAGAGGGAGAAGG 
               
               
                   
                 AGAAGAGAGAAAGGATGGAAAGCCAAAACCTCAAGGCTAGTA 
               
               
                   
                 CAAAGCTGAGTCAAGC   G   AGGGAGAAAATCAAGCGCAAGTATC 
               
               
                   
                 CACTTCCTGTTGCAAGGAGRCAACTYTCCACTGGRTACNTGG 
               
               
                   
                 AAGATGCTCTCGAAGAGGATGAAGAGACAGACC 
               
               
                   
               
               
                 NO 21 
                 GCTCAGGTAGATCTCCCACGGGTTGGGGAAGAGGATCCGGAT 
               
               
                   
                 ATGGGTATGGGTCTGGATCTGGATCAGGTAGCGGATATGGGT 
               
               
                   
                 ACGGTTCCGGAGGTGG   A   GGAGSACGTGGTGGTGGGTATGGTT 
               
               
                   
                 ATGGAAGCGGAAATGGTCGGTCTGGAGGWGGTGGTGGTGGCT 
               
               
                   
                 CTAATGGTGAAGTTGCCGCTTTGGGCCACGGTG 
               
               
                   
               
               
                 NO 22 
                 GGGAGAGAGAGGAAACCTGGAGGA   T   GTTACGCAGTACTGGGG 
               
               
                   
                 CTGAAGAACTGAAGAATTGTTGGAGCATTGGATTAATTGTCC 
               
               
                   
                 TTCKTGCTGACCCGTGTTCTTCT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Raphanus SNP associated to Raphanus 
               
               
                 fragment but not POSH+ allele 
               
            
           
           
               
               
            
               
                 SEQ ID 
                 Sequence 
               
               
                   
               
               
                 NO 18 
                 AGAAGATGGAGTTCTTGATGTTTGATCTYGATCGGGTTTTGAARCCCGGTGGGTTGTTC 
               
               
                   
                 TGGTTGGATAACTTCTACTGCGCTAGTGACGTGAAGAAGAA   A   GAGCTGACGCGTTTGAT 
               
               
                   
                 YGAGAGGTTTGGGTATAAGAAGCTGAAATGGGTTATTGGAGAGAAGGCTGATGGGCAAG 
               
               
                   
                 TGWATCTCTCTGCTGTTCTKCAAA 
               
               
                   
               
               
                 NO 17 
                 CACAACATGCCGGTGATTGGTATCCAGCTGACCTTGGATCCAACGATTTCAAAGGTCTC 
               
               
                   
                 TATGGATATAAGGTCTTTATTGCCATTGCCATTATCCTTGG   G   GACGGTCTCTACAATCT 
               
               
                   
                 TGTCAAGATCATTGCTGTCACTGTGAAGGAATTATGCAGCAATAGCTCTAGACACCTCA 
               
               
                   
                 ATCTACCCGTTGTTRCCAACGTTG 
               
               
                   
               
               
                 NO 16 
                 ACTTTGTTGAYAGYCTTACMGGAGTAGGACTTGTTGATCAAATGGGAAACTTCTTCTGC 
               
               
                   
                 AAAACGCTCTTGTTTGTGGCTGTAGCTGGAGTTCTTTTCAT   T   CGCAAGAACGAAGATTT 
               
               
                   
                 AGATAAGCTCAAGGGTCTRWTYGAAGAGACGACGYTRTATGACAAGCARTGGCAAGCGG 
               
               
                   
                 CTTGGAAAGAGCCGGAAATAATCA 
               
               
                   
               
               
                 NO 15 
                 GTCCATGTTTGATGCAATTGTATCAGCAGACGCATTTGAGAACTTGAAACCAGCTCCAG 
               
               
                   
                 ATATTTTCTTGGCTGCTTCCAAKATCTTGGGTGTGCCCACA   T   GCGAGTGTATTGTTATT 
               
               
                   
                 GAAGATGCACTTGCTGGAGTCCAGGCTGCTCAAGCTGCAAACATGAGATGCATAGCTGT 
               
               
                   
                 GAAAACTACTTTATCTGAAGCAAT 
               
               
                   
               
               
                 NO 14 
                 CTTTTGCTGGTTTTGGTGAAATAGTATCTGTCAAGATACCAGTTGGGAAAGGATGTGGA 
               
               
                   
                 TTCATTCAGTTTGTCAACAGAGAAAACGCAGAGGAGGCTTT   A   GAGAAACTAAATGGTTC 
               
               
                   
                 TGTAATTGGAAAACAAACCGTTCGCCTTTCMTGGGGTCGTAAYCAAGGCAAYAAACAGC 
               
               
                   
                 CTCGAGGTGGGTATGG 
               
               
                   
               
               
                 NO 13 
                 CTAAGGCAATGAAGTACCTGTCAATAGGTGAAGAAGACGATATATCATGGTCACTTATC 
               
               
                   
                 AAAGCTGCCTTCTCTTCAGTAGCTCAAACCGCAATCATACC   A   ATGCAAGACATTCTCGG 
               
               
                   
                 WCTYGGAAGTTCTGCCAGGATGAACACTCCAGCCACTGAGGTGGGGAACTGGGGTTGGA 
               
               
                   
                 GGATTCCGAGTTCAACGAACTTTG 
               
               
                   
               
               
                 NO 12 
                 TTGGCCCTGAAGGTTCTACAGTGCTTCATTATAGACAATCTTCAACTTCTGCTTCTATT 
               
               
                   
                 GGGAAAATCAGTTGCAAGGTGTACTATTGCAAAGAAGACGA   A   GTTTGCTTGTACCAGTC 
               
               
                   
                 TGTTCAGTTTGAGGTACCTTTCAAGRTGGAATCAGAAKCRTCTYCTTCYCAGGTGATCG 
               
               
                   
                 CATTCACCGTTAAACCTAGAGCAT 
               
               
                   
               
               
                 NO 11 
                 TCAAGGACTTTGGTGATAGTATTCCAGGACATGGTGGAATCACTGATAGAATGGACTGC 
               
               
                   
                 CAGATGGTAATGGCAGTATTTGCTTACATATATCTCCAGTC   C   TTTATCGTCTCCCAAAG 
               
               
                   
                 CGTTTCGGTTGACAAAATCCTGGACCAGATATTGACGAACCTTAGCTTCGAGGAACAAC 
               
               
                   
                 AAGCTCTCTTCACTAGATTAGGGC 
               
               
                   
               
               
                 NO 10 
                 CTCCTCCKCCGAATCCGTTTGGGGAYGCGTTCAAGGGGCCMGAGATGTGGGCSAAGCTG 
               
               
                   
                 ACGGCGGATCCGTCGACGAGGGGGTTCTTGAAGCAGCCTGA   C   TTCGTCAACATGATGCA 
               
               
                   
                 GGAGATCCAGAGGAACCCTAGCAGTCTCAATCTCTACTTGAAGGACCAGAGGGTGATGC 
               
               
                   
                 AGTCTCTYGGGGTTTTGTTGAATG 
               
               
                   
               
               
                 NO 9 
                 AGTATGAAGAAGAGGGYGAGTATGAGAGAGGTGGGTCGAAGCAGAGGAGAGGAGAGTCA 
               
               
                   
                 GAGGAAGGKCATGGRTACTACGAAGGGCGTAGTAGACGTTC   A   AGCCATTATGAGCGTGA 
               
               
                   
                 GGAGGAACAAGGAGGTGASCAAGACCGKTACGAYGACCGTTATGGGAGAGTGGAGGAAG 
               
               
                   
                 AAGAATACCGTTATGATGATCGTG 
               
               
                   
               
               
                 NO 8 
                 TCAAGAAGACTTACCCAACAGTCCAGCTTACAGCATGGACATTTTTCCCCATTGTGGGA 
               
               
                   
                 TGGGTAAAYTACAAGTATGTGCCACTGCACTTCCGGGTCAT   C   TTGCACAGCCTCGTYGC 
               
               
                   
                 ATTCTTCTGGGGAATCTTCCTGACCCTGCGAGCAAGGTCAATGACACTAGCTTTGGCAA 
               
               
                   
                 AGGCTAAGTGATCAGGGAAACACA 
               
               
                   
               
               
                 NO 7 
                 CTAGTTTCAGGGAATGGTTTRCAGAAGGTTGAATTGATGAAGACGAGAGCTTCTTCATC 
               
               
                   
                 AGACGAGACCTCAACGTCCATTGACACCAACGAACTCTTTA   C   WGACTTGAAGGAAAAGT 
               
               
                   
                 GGGATGGTCTTGAGAACAARACRACYGTGGTTATCTAYGGAGGAGGAGCCATTGTWGCT 
               
               
                   
                 GTTTGGTTATCTTCCATTCTTGTT 
               
               
                   
               
               
                 NO 6 
                 GAAGTGTTCTGGACACAGCTGAGAAAGCCCACGAAGGGGATATCACATGCATTTCGTGG 
               
               
                   
                 GCACCCAAGGCAATGACAGTTGGGGAGAGAAAGGCGCAGGT   A   TTAGCGACAGCAGGGGT 
               
               
                   
                 TGACAARAAAGTGAAGCTGTGGGAAGCTCCAAMGTTGCAGTCTGTGTAGACTTGCTACT 
               
               
                   
                 GCTGCTGCAATACAAAGAAAGTCT 
               
               
                   
               
               
                 NO 5 
                 TAAAGTATACTCGAAATGGCCCAAATCTCACTCTTTCAAGATCGGCGACTCCCTCTTGT 
               
               
                   
                 TCTTGTACCCACCAAGCGAAGATTCAATGATTCAAGTGACA   C   CTTCCAACTTCAAGAGC 
               
               
                   
                 TGCAACACCAAAGATCCGATCTTGTACATGAACGACGGCAACTCTCTCTTCAACCTCAC 
               
               
                   
                 CCAAAACGGAACCTTTTACTTCAC 
               
               
                   
               
               
                 NO 4 
                 TCAGACTCATCCAGATAAAGAAGAACAAAATCTCATCTTCTGTGCACTCTATGGTACAA 
               
               
                   
                 ACTCCTTCAGGTACAGCWCGAACGCACAGGTTTGCCACTGA   A   ACAGCCGAGCTCCCTGC 
               
               
                   
                 GCAAGAAGGAGAAAGAGTGACAATTGCATCTGCTGCTCCATCAGATGTTTACAGACAAG 
               
               
                   
                 TGGGACCTTTCAAGTTTACCCCCA 
               
               
                   
               
            
           
         
       
     
     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. 
     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. 
     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. 
     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. 
     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). 
     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. 
     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. 
     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. 
     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). 
     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&#39;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. 
     In specific embodiments, said primers for detecting the SNP markers of the present disclosure are as listed in the following table: 
     
       
         
           
               
             
               
                 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 
       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. 
     Accordingly, it is a further disclosed a method of production of a POSH+  Brassica  plant, wherein the method comprises the following steps:
         a. crossing a first  Brassica  plant as described in the previous section with a second POSH −  or POSH+  Brassica  plant; thereby obtaining a F1 hybrid plant;   b. selfing or backcrossing said F1 hybrid plant with said second POSH − or POSH+ Brassica  plant;   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   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.       

     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. 
     Any  Brassica  plants obtained or obtainable by the disclosed methods for producing  Brassica  plant with POSH+ phenotype are also part of the present invention. 
       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. 
     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. 
     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 
       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. 
     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 
     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. 
     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&#39;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&#39;s instructions and run for 100 cycles (single end reads) to produce at least 2.0 Gb of sequence per sample. 
     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). 
     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. 
     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. 
     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. 
     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 
     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. 
     Example of the results of these measurements made with different lines or hybrids are given in  FIGS. 8 and 9 . The tables in these figures clearly show that the increased pod shattering tolerance is limited to some Ogura restorers and hybrids with a long introgression from  Raphanus sativus  and it can be concluded that the increased pod shattering tolerance is coded on the  Raphanus  introgression. 
     The table in  FIG. 8  provides the results for pod stability and genotype profile on a panel of genotypes from harvest 2013. The given alleles represent the calling of alleles from the  Raphanus  and  oleracea  genome disregarding the alleles from the  rapa  genome. Black colour indicates the presence of  Raphanus  genome, white colour indicates the presence of  oleracea  genome and grey the presence of both genomes. 
     The table in  FIG. 9  provides results for pod stability and genotype profile on a panel of genotypes from harvest 2014. The given alleles represent the calling of alleles from the  Raphanus  and  oleracea  genome disregarding the alleles from the  rapa  genome. Black colour indicates the presence of  Raphanus  genome, white colour indicates the presence of  oleracea  genome and grey the presence of both genomes. 
     Example 3: Pod Shattering Tolerance Provided by  Raphanus  is Partial Dominant 
     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. 
     Examples of the results of the measurements of different Ogura hybrids are given in  FIG. 5 . Black colour indicates the presence of  Raphanus  genome, white colour indicates the presence of  oleracea  genome and grey the presence of both genomes. For these measurements in total 100 pods were measured and the average of 100 measurements was calculated. The table clearly shows that the pod shattering tolerance is inherited at least partial dominant from long introgression Ogura restorers to the respective hybrids (genotypes 1-7 and 11). Genotypes 7 to 10 are also Ogura hybrids but for these the respective restorers are short introgressions lacking the pod shatter region from the  Raphanus  introgression. Consequently these hybrids do not show pod shattering tolerance. 
     Example 4: Identification of Other Markers Strongly Associated to POSH Locus 
     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 ( FIG. 5 ). 
     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. 
     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. 
     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.    
     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). 
     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 
     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. 
     The following Table 6 show the SNP codominant markers which were used to analyze all the new recombinant plants generated: 
     
       
         
           
               
               
             
               
                   
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     The SNP is shown under bracket in the above marker sequences, the first nucleotide representing  Raphanus  SNP, the second nucleotide representing  Oleracea  SNP. 
     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. 
       FIG. 6  shows the results of the phenotyping and genotyping of the F5 progeny compared to other lines and hybrids. Amalie and Arabella are non restorer lines. R7011-AB is a restorer line with a long introgression comprising the Rf0 gene (BnRF0 marker as described in SEQIDNO:1) and the POSH region markers. Arsenal is a hybrid variety with a long introgression comprising the Rf0 gene (BnRF0 marker as described in SEQIDNO:1) and the POSH region markers. RD153-101 is a restorer line not pod shattering tolerant with a short introgression described in patent application WO2011020698. R101540103-AACCBA is a restorer line not pod shattering tolerant with a short introgression. 
     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. 
     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  FIG. 6 . The given alleles represent the calling of alleles from the  Raphanus  and  oleracea  genome disregarding the alleles from the  rapa  genome. Black colour indicates the presence of  Raphanus  genome, white colour indicates the presence of  oleracea  genome and grey the presence of both genomes. 
     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 
     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 +  have been crossed with POSH −  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. 
     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). 
     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  FIG. 7  which presents the molecular characterization of the F3 recombinant lines on the  Raphanus  introgression. Black colour indicates the presence of  Raphanus  genome, white colour indicates the presence of  oleracea  genome and grey the presence of both genomes.). 
     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 
     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  FIGS. 3A and 3B . 
     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. 
     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. 
     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 
     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. 
     The results in  FIG. 4A  to  FIG. 4H  show the segregation of POSH in a Double Hybrid population. 
     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.