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  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the subject of the invention is means, tools and methods for identifying the locus of a major gene for resistance to the yellow rice variegation virus (abbreviated to RYMV for Rice Yellow Mottle Virus). More specifically, it targets, as tools, PCR markers and primers.
• RYMV yellow rice variegation virus
• RYMV is an endemic virus in Africa. In some rare varieties of the African cultivated rice species Or ⁇ za glaberrima, very high resistance to RYMV has been identified. However, since the interspecific hybrids between the two cultivated rice species are extremely sterile, previous research has failed to describe any genetic basis or mechanism for this resistance.
• the work of the inventors in this field has shown that a variety called Gigante, originating in Mozambique and identified by WARDA, of the Asian cultivated rice species Or ⁇ za sativa, exhibits the same characteristics as those observed in O. glaberrima.
• the inventors characterized resistance to RYMV by demonstrating that it is linked to a major recessive resistance gene and identical in the two sources of resistance considered (O. Sativa and O. glaberrima).
• This resistance occurs at the level of cell-to-cell movement and results in blockage of the virus in infected cells while replication of the virus is normal.
• RYMV migration takes place in the form of a nucleoprotein complex associating viral nucleic acid, capsid protein and virus movement protein.
• a host factor possibly a protein, also contributes to the movement of the virus.
• resistant varieties on the contrary, a mutation of this protein no longer allows association with the virus and therefore its diffusion in the plant.
• the invention therefore aims to provide a method for the identification of molecular markers of the locus of resistance to RYMV.
• the invention also relates, as such, to the DNA fragments as revealed by this method, and which can be used as markers.
• the invention further relates to the applications of such markers, in particular for defining other markers of high specificity with respect to the resistance locus and for predicting a resistant phenotype.
• the invention also relates, as new products, to the sequences of the primers used in the PCR techniques used.
• the identification of markers of the locus of a major gene for resistance to RYMV includes the use of AFLP markers (Amplified Fragments Length Polymorphism) and uses the PCR technique.
• This identification process is characterized in that it comprises: - the selective amplification of rice DNA fragments on the one hand from resistant individuals, on the other hand from sensitive individuals, descended from parental varieties, these fragments having been previously subjected to a digestion step, then of ligation to fix complementary adapters of primers having, at their end, one or more specific nucleotides, one of the primers of the pair being marked for the purpose of disclosure, - the separation of the amplification products, by gel electrophoresis under denaturing conditions, and
• the DNA fragments are obtained by digestion of the genomic DNAs of resistant plants on the one hand, and of sensitive plants on the other hand, and of their parents, using restriction enzymes. Restriction enzymes which have been found to be suitable include EcoRI and Msel.
• Short nucleotide sequences are attached to the digestion fragments (adapters) to generate blunt ends to which adapters are then attached.
• the primers used in the amplification step are complementary to these adapters with, at their 3 ′ end, from 1 to 3 nucleotides which may be variable.
• the amplification step is advantageously carried out according to the PCR technique.
• Specific amplification profiles are obtained with pairs of primers having at their end, respectively, AAC and CAG, ACC and CAG, or AGC and CAG motifs.
• sequences corresponding to the EcoRI and Msel adapters are respectively GAC TGC GTA CCA ATT C (SEQ ID No.1) and GAT GAG TCC TGA GTA A (SEQ ID No.2).
• the pairs of primers used for the amplification are chosen from E-AAC / M-CAG; E-ACC / M-CAG; and E-AGC / M-CAG; in which E and M correspond respectively to SEQ ID N ° 1 and SEQ ID N ° 2.
• Other couples are given in table 6 in the examples.
• the polymorphic bands identified as specific markers of the locus of resistance to RYMV are isolated from the gels.
• the electrophoresis gels are excised. This isolation step is followed by purification by proceeding according to conventional techniques. DNA fragments are thus available.
• said purified fragments are cloned in an appropriate vector, such as a plasmid, introduced into host cells, in particular bacterial cells such as those of E. coll.
• the purified and cloned DNA fragments are sequenced.
• the invention also provides a method for obtaining markers of high specificity with respect to the locus of a major gene for resistance to RYMV.
• This process is characterized in that pairs of PCR primers are defined which are complementary to a part of the sequence of the DNA fragment which has been sequenced, and a specific amplification of this fragment is carried out using these pairs of primers, then the amplification products are subjected to migration on an electrophoresis gel.
• DNA sequences can be used to identify a polymorphism linked to the resistance locus in a variety to be studied according to different methods as described in the examples:
• the invention relates, as new products, to the polymorphic AFLP bands as identified by the method defined above, from DNA of rice plants, and where appropriate isolated, purified and sequenced.
• AFLP bands are characterized in that they are specifically demonstrated in a variety sensitive to RYMV (IR64) and in the fraction of sensitive plants resulting from the crossing of this variety with the Gigante resistance variety as described in the examples.
• the invention particularly relates to the DNA sequences corresponding to these polymorphic bands, and which make it possible to define a segment of chromosome 4 of 10-15 cM carrying the locus of resistance to RYMV.
• the AFLP bands correspond to restriction fragments and in particular, in accordance with an embodiment of the method of the invention, to EcoRI - Msel fragments.
• markers M1 and M2 are characterized by a size, respectively, of 510 bp and 140 bp in electrophoresis gel under denaturing conditions. These fragments are characterized in that they correspond to DNA sequences flanking the resistance locus and located on either side of the latter at 5-10 cM.
• the invention also relates to fragments cloned into vectors such as plasmids, these cloning vectors as such, characterized by the fact that they contain such fragments, and the host cells transformed using these vectors, such than bacterial cells like E. coli.
• the invention relates in particular to the DNA sequence corresponding to the fragment identified as a M1 marker, and corresponding to the following sequence SEQ ID No. 3: CGTGCTTGCTTATAGCACTACAGGAGAAGGAAGGGGAACACAACAGCCATGGCGAG CGAAGGTTCAACGTCGAGAGGGGGGGGGGGGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGAGGAGGAGAGAGAGGAGGAGGAGAGAGAGGAGGAGGAGGAGGAGGAGAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGA
• the DNA sequence of the marker M1 has a size of 471 bp.
• the invention also relates, as new products, to the nucleotide sequences used as PCR amplification primers.
• Such primers include the E-AAC / M-CAG pairs; E-ACC / M-CAG; E-ACC / M-CAG; in which E and M correspond respectively to SEQ ID N ° 1 and SEQ ID N ° 2.
• sequences identified in the sequence of the fragment called the M1 marker are in particular sequences (5 ′, 3 ′) chosen from:
• the invention also relates to the DNA sequence corresponding to the fragment identified as marker M2 and corresponding to the sequence SEQ ID No. 9 AATTCACCCC ATGCCCTAAG TTAGGACGTT CTCAGCTTAG TGGTGTGGTA GCTTTTTCTA TTTTCCTAAG CACCCATTGA AGTATTTTGC ATTGGAGGTG
• the size of M2 is 120 bp.
• the invention relates to the use of the DNA sequences obtained with the above primers to define polymorphisms allowing the identification of resistant phenotypes.
• the invention also relates to a method for identifying the DNA sequence carrying the major gene for resistance to RYMV. This process is characterized by the screening of a library made up of DNA fragments of 100 to 150 kb of the IR64 variety or other, such as the BAC (Bacterial Artificial Chromosomes) library, clones in bacteria, for selecting the clone (s) of the library containing the markers defined above above and the RYMV resistance gene.
• BAC bank is available from IRRI.
• mapping of the sequence corresponding to the marker M1 makes it possible to identify a chromosomal area on chromosome 4 of rice carrying the locus of resistance to RYMV.
• the mapping of the resistance gene to RYMV on chromosome 4 of the genetic map of rice makes it possible to identify markers closer to the resistance locus.
• markers closer to the resistance locus are in particular microsatellite markers RM252 and RM273 or any other marker within the interval (4-5cM) defined by these markers making it possible to identify a polymorphism between the parents IR64 and Gigante such as the RFLP markers from genomic or cDNA libraries, microsatellites, AFLP markers or markers derived from the physical mapping of the region such as the BAC, YAC clones or their cosmids.
• Gigante or O. glaberrima with varieties of rice sensitive to RYMV can be used for the transfer of resistance to RYMV in sensitive varieties by successive crossover followed by selection assisted by markers.
• - Figure 1 the cloning of the M1 marker in the plasmid PGEMTeasy. Digestion of the plasmid shows a DNA fragment of 510 bp corresponding to the Ml band; - Figure 2: amplification of the Ml marker in the four varieties of rice (Azucena, Gigante, IR64 and Tog5681) using the pairs of primers
• IR64 and Tog5681 releases an 86 bp fragment which reduces the size of the amplified fragment accordingly;
• FIG. 8 hybridization of the marker Ml used as a probe on membranes carrying the DNA of the 4 varieties (IR64, Azucena, Gigante and Tog5681) digested with 6 restriction enzymes Apal, Kpnl, PstI, Seal, HaelII.
• the variety Tog5681 has a different restriction profile from the other varieties for the Seal enzyme which can be used to mark the resistance locus of this variety;
• the varieties used in the study of resistance and in particular the two resistant varieties Gigante and Tog5681 were characterized using microsatellite markers on a representative sampling of loci.
• Polymorphism is manifested by the number of repetitions of a short nucleotide motif, most often binucleotide which is characteristic of a given variety.
• Table 1 gives the results from a reference system established by Chen et al, above according to which the alleles are identified by the number of repeats of the motif compared to the IR36 variety which serves as a control.
• the two varieties Gigante and Tog5681 are thus described specifically on 15 loci with respect to all other varieties (the microsatellite markers are given in the 1st column).
• Resistance was characterized from the artificial inoculation of young seedlings with virus compared to an extremely sensitive control variety IR64.
• the virus content was monitored for 60 days after inoculation using ELISA tests on the last leaves issued.
• the FI hybrids obtained between the susceptible and resistant varieties were tested for resistance to the RYMV virus by ELIS A test and symptom monitoring.
• This table gives the distribution of ELISA responses (at 405 nm) in the leaves infected by the systemic route of the FI hybrids, backcrosses and F2 descendants obtained from the backcrosses between the sensitive IR64 variety and the 2 resistant cultivars Gigante and Tog5681.
• Gigante the inheritance of the resistance was confirmed by a resistance test on 55 F3 families of the cross (IR64 x Gigante). The results are given in Table 3.
• the DNAs were then mixed stoichiometrically to constitute two pools of DNA corresponding respectively to 10 sensitive or resistant F2 plants with a final concentration of the mixture of 50 ng / ⁇ l. These mixtures served as a basis for the identification of resistance markers by the AFLP method (Amplified Fragments Length Polymorphism) for amplified fragment length polymorphism which was developed by Zabeau et al (4), and Vos et al. (5 ).
• the products used are in the form of a commercial kit (Gibco BRL) issued by Keygene & Life Technologies.
• Digestion reaction 25 ⁇ l: 5 ⁇ l of DNA (50 ng / ml) 0.2 ⁇ l (2 U) of EcoRI (10 U / ⁇ l) 0.2 ⁇ l (2 U) of Msel (5 U / ⁇ l ) 5 ⁇ l of T4 ligase 5X buffer 14.5 ⁇ l of H 2 O.
• the digestion reaction is carried out for two hours at 37 ° C., then 15 min. at 70 ° C to inactivate restriction enzymes. After digestion, a ligation reaction is carried out.
• the ligation reaction is carried out at 37 ° C for 3 hours, followed by inactivation of the enzyme at 60 ° C for 10 min. c - Amplification
• the actual amplification was carried out in two stages: preamplification and specific amplification.
• the characteristics of the preamplification by PCR are as follows: 0 cycles with denaturation: 30 sec at 94 ° C hybridization: 30 sec at 56 ° C elongation: 1 min at 72 ° C Selective amplification is done from an aliquot of the first amplification diluted 1/30 by using primers having 3 selective nucleotides at the 3 'end, and by labeling one of the primers to reveal the bands on a autoradiographic film. The following pairs of primers are used: E-AAC / M-CAG
• E-ACC / M-CAG E-AGC / M-CAG in which E corresponds to the sequence GAC TGC GTA CCA ATT C (SEQ ID N ° 1), and
• the hybridization temperature is reduced by 0.7 ° C per cycle, during the following 1 1 cycles: last 20 denaturation cycles: 30 sec at 90 ° C hybridization: 30 sec at 56 ° C elongation: 1 min at 72 ° C
• the EcoRI primer is labeled (reduced to a tube of 0.5 ⁇ l): 0.18 ⁇ l of the EcoRI primer (5 ng) 0.1 ⁇ l of ⁇ 33 P ATP (10 mCu / ⁇ l)
• the labeling reaction is carried out at 37 ° C for 1 hour and is stopped for 10 minutes at 70 ° C.
• the amplification products are separated by electrophoresis on a denaturing polyacrylamide gel.
• the gel is transferred to 3M Wattman paper and dried for 45 minutes at 80 ° C with a gel dryer.
• the gel is placed in a cassette with an ultra-sensitive film.
• the segregation data between the AFLP markers Ml to M6 and the resistance locus for the F2 pools (IR64 x Gigante) and the interspecific backcross (IR64 x Tog5681) x Tog5681 are summarized in Tables 4 and 5.
• Data analysis of segregation and of the rare recombinants observed in the two crossings makes it possible to evaluate the rates of recombination between these different markers and the locus of resistance.
• the markers M1 on the one hand and the markers M2 to M6 on the other hand determine a segment lower than 10-15 cM carrying the resistance locus. Ml and M2 are thus less than 5-10 cM and placed on either side of this locus.
• Resistant F2 pool (IR64 x gigantic) 10 Sensitive F2 pool (IR64 x gigantic) 10 Interspecific backcross Tog5681 1 1
• Resistant F2 pool (IR64 x gigantic) 1 1 0 - Sensitive F2 pool (IR64 x gigantic) 0 10 Interspecific backcross Tog5681 10 2 - 0 8
• Resistant F2 pool (IR64 x gigantic) 11 0 - Sensitive F2 pool (IR64 x gigantic) - - 0 10 Interspecific backcross Tog5681 9 3 - 0 8 TABLE 5
• Resistant F2 pool (IR64 x gigantic) 0 1 0 10 Sensitive F2 pool (IR64 x gigantic) 10 0 0 0 Interspecific backcross Tog5681 11 2 2 1 1
• the amplification product was separated again on 6% denaturing acrylamide gel, and compared to the parents and to the sensitive and resistant pools.
• the track corresponding to this amplification product shows a single band of 510 bp migrating at exactly the same level as the original band which had been excised.
• Another 5 ⁇ l aliquot was also amplified with the same primers and was separated on 1.8% agarose gel.
• the band corresponding to the expected size (510bp) was again excised and purified with a clean gene kit (Promega).
• plasmid DNA was carried out with the Wizard plus kit (Promega).
• the plasmid DNA containing the insert was digested with the EcoRI enzyme to verify the presence of the M1 marker.
• a 1.8% agarose gel made it possible to verify the presence of the 3 kb band corresponding to the plasmid and the 510 bp band corresponding to the Ml marker (photo 1).
• sequence of the insert (SEQ ID N ° 3) is as follows (5 ', 3'): SEQ ID N ° 3
• the real size of the cloned rice DNA fragment is 471 bp.
• the marker was tested on the F2 individuals from the sensitive pool and the cross-resistant pool (IR64 x Gigante). All resistant individuals have the profile of the Gigante variety (absence of the AFLP M1 marker associated with the absence of the Hpall / Mspl restriction site) with the exception of the individual (5.11). Sensitive individuals present the Hpall / Mspl restriction site in the homozygous state like the IR64 variety with the exception of two heterozygous individuals who are recombined (fig. 5).
• the sequence of the M1 marker which can be amplified by specific primers corresponds well to the AFLP M1 marker. Digestion with the enzyme Hpall / Mspl makes it possible to distinguish the allele coming from the sensitive parent (IR64) from the resistant parent (Gigante).
• Example 9 Marking of the resistance locus of the variety Tog5681
• the presence of the Hpall / Mspl restriction site in the Tog5681 variety does not allow the strategy of Example 8 to be used to verify that the marker M1 is also a marker of the resistance originating from Tog5681.
• the 4 varieties Azucena, Gigante, IR64 and Tog5681 were digested with 12 restriction enzymes (BamHI, Bg / II, Dral, EcoRI, EcoRV, HindIII, Apal, Kpnl, PstI, Seal, Xbal, HaelII) restriction polymorphism using the DNA sequence of the M1 marker as a probe.
• the Seal enzyme identifies a polymorphism between IR64 and Tog5681 (fig. 8). This polymorphism was used to validate the Ml marker on a crossover (IR64 x Tog5681) x IR64 in segregation for resistance. 5 individuals sensitive to this crossover have been tested and all show the characteristic band of IR64. The 9 resistant individuals show only the Tog5681 band with the exception of one which is recombinant (fig. 9). The restriction polymorphism demonstrated by the Seal enzyme using the Ml marker as a probe is therefore well linked to the resistance locus of Tog5681. The genetic analysis and the identification of resistance markers are consistent in considering that the marker Ml maps the same resistance locus in the two varieties Gigante and Tog5681.
• the AFLP band obtained with the pair of primers E-ACC / M-CAG and corresponding to the M2 band visible in the sensitive parent (IR64) and present in all the individuals making up the sensitive pool was cloned according to the same protocol as for the marker Ml. The sequence corresponding to this band was determined and 3 primers were defined (1 forward - 2 reverse) to allow the transformation of this marker into a PCR-specific marker.
• the M2 marker can be amplified alone with a hybridization temperature of 60.5 ° C, the other parameters remaining unchanged. Under these amplification conditions, the M2 marker appears as a dominant marker characterized by the presence of a band in the sensitive parent (IR64) and the absence of a band in the parent (Gigante).
• Example 11 Creation of a population of recombinant resistant plants between the markers M1 and M2 to order within this interval the markers AFLP candidates for the marking of the resistance 750 individuals F2 (IR64 x Gigante) were artificially inoculated with the RYMV virus (strain BF1). The symptomless plants were transplanted in the greenhouse either
• Example 13 Anchoring the RYMV Resistance Locus Using Microsatellite Markers
• the markers RM241, RM252, RM273 were mapped on an F2 population (IR64 x Gigante) evaluated in parallel for resistance to RYMV.
• the results on 183 F2 individuals make it possible to characterize an interval of approximately 3.6 cM bounded by the two microsatellite loci RM 252 and RM273 flanking the gene for resistance to RYMV.
• Example 14 Fine mapping of the interval carrying the resistance locus and ordering of the resistance markers in the interval M1-M2
• the 45 resistant and recombinant F2 individuals (IR64 x Gigante) for the markers M1 and M2 were characterized for the microsatellite markers identified in example 13.
• the mapping of the markers in segregation on all the available F2 individuals (IR64 x Gigante) (321) confirms the order and the distance between the markers of the interval M1 - M2 and in particular the interval RM252 - RM273 which is evaluated at 3.6 cM (FIG. 10 (b)).
• the 45 resistant and recombinant F2 individuals (IR64 x Gigante) for the markers M1 and M2 make it possible to confirm the order of the AFLP markers identified in example 12.
• AFLP EACG / MACA remains in the range RM252 - RM273 and represents the marker closest to the locus of resistance to RYMV (Table 9).
• Table 9 the marker closest to the locus of resistance to RYMV.
• Example 15 Transfer of resistance assisted by markers The markers close to the resistance locus were tested on irrigated varieties very sensitive to the RYMV virus (var. BG90-2, Bouakél89, Jaya).
• 3 markers (Ml, RM241, RM252) show a polymorphism between these 3 varieties and the Gigante variety, which makes it possible to envisage the use of these markers to transfer resistance into sensitive genotypes.
• the experimental transfer of resistance in these varieties was carried out up to the level of the 2nd crossover. At each crossing, the plants are checked for the presence of markers from the Gigante variety and segregation for resistance is checked by progeny tests on F2. The results are given in Table 10.

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