PlantCeli
Plant Cell Reports (1995) 15:268-270
Reports ~ Springer-Vcrlag 1995
Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions, substitutions and microsatellites C. E Quiros, M.J. Truco *, and J. Hu Department of Vegetable Crops, University of California, Davis, CA 95616, USA * Present address." IRTA, Centre de Cabrils, 08348 Cabrils (Barcelona), Spain Received 27 February 199J/Revised version received 18 May 1995 - Communicated by W. Parrott
Summary. Two RAPD fragments segregating codominantly were investigated in a F2 population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an intemal 41 nucleotide deletion in one o f the fragments. Minor deletions o f one to three bases, including a microsatellite of CTI" motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A05 ~ whereas the shorter one, denominated A05 ~, had 1116 bp.
Introduction In general, RAPD markers are dominant in most plant species studied (Kesseli et al. 1992). Scarcity of codominant RAPD markers seems to be due to the lack of sequence homology between the primers and the target DNA being amplified within the range of polymerase amplification (Rafalski and Tingey 1993). Therefore, it is expected that potential codominant forms resulting fi'om insertions increasing the length of a sequence beyond the range for polymerase synthesis, will not be produced. However, in a few instances putative codominant markers have been disclosed which fit the expected 1:2:1 Mendelian segregation (Ayaliffe et al. 1994; Kesseli et al. 1992). Unless further analysis is performed, it is not possible solely by following segregation ratios, to determine whether these markers originate fi'om the same sequence or represent instead tightly linked unrelated adjacent sequences. During construction of a B. nigra DNA-based marker linkage map (Truco and Quiros 1994), of a total o f 85 markers Correspondence to. C. E Quiros
analyzed, we observed a pair of putative codominant RAPD markers mapping on chromosome B3. We report in this paper the molecular analysis o f these markers by comparing their sequences and describing their differences.
Material and Methods Plant Material. 13. nigra (2n=2x=16, B genome) UC Davis accessions B1148 (Pakistan) and B1157 (India) and derived 83 F2 progeny used by Truco and Quiros (1994) to construct a B genome linkage map was
used for this study. DNA extraction. Single plant extraction for each of the F2 plants and parentals was accomplished by CsCI gradient as described by Truco and Quiros(1994). DNA amplification. Approximately 10 ng of DNA were amplified by 10-merprimerA05 (5' A ~ T C I " I G 3~)fromOperon Technologies (Alameda, CA) following the RAPD protocol described by Hu and Quiros (1991). Isolation of specific RAPD products. Major DNA fragments were isolated from the gel and purified with the GeneClean kit (Bio 101 Inc., La Jolla, CA). Cloning of RAPD products. Amplified DNA products of B. nigra "Junius' by primer A05 were cloned using the TA cloning kit (InvilrogenCorp. San Diego,CA). DNA hybridizations. AmplifiedDNA ofB. nigra and genomic DNA of cultivars of this species "Junius' and B1148 digested with EcoRI, HindllI or BamHl was blotted to HyBond-N(Amersham, IL) or Zeta Probe GT (BioRad, CA) nylon membranes using the manufacturers protocols. Afterhybridization,high stringencywashes were performed followingthe manufacturer'sprotocols. DNA sequencing. The MI3 universal (-40) and MI3 reverse sequencing primers were used to sequence the inserts fiom both ends. l:or sequencing, tile deoxy termination method on double strand DNA templates (Sanger et al. 1977) was followedusing the Sequenase 2.0 kit of IJSB (Cleveland, Oil). The sequences were analyzed by the programs GCG (U. of Wisconsin, Madison) and DNA Strider 1.2 (Marck 1988). A second clone of each allele was partially sequenced as controls for amplificationartifacts causingsequence modifications
Results and Discussion Genetic segregation
In a segregating F 2 mapping population of B. nigra, one
269
or two fragments of approximately 1200 and 1100 bp resulted from genomic DNA amplification by primerA05. These fragments segregated in the 1:2:1 ratio (x2=1.58, P=0.45, n=83, see Truco and Quiros 1994), as expected for codominant markers (Fig 1). Since no additional fragments were observed, the A05 products did not seem to have the ability to form heteroduplex molecules (Ayliffe et al 1994).
Hybridization experiments After isolation, the 1100 bp fragment used as a probe hybridized to both segregating bands in the amplification profile suggesting homology. Homology was further conf'mned by hybridizing the two RAPD products to B. nigra genomic DNA digested by three different restriction enzymes. The resulting hybridizing profiles had 5 to 7 bands, ranging from approximately 8Kbp to 300 bp depending on the accession and enzyme tested (Fig 2). The profiles disclosed by both probes were virtually identical to each other. Therefore, this experiment indicated that the two clones were closely related, which were then designated A05 t for the 1200 bp one and A052 for the 1100 band. The number of bands observed suggest that A05 corresponded to moderately repeated sequences in the genome.
Fig. 1. B. n!~a F2 population segregating in a 1:2:1 Mendelian fashionfor A05 and A052RAPDfragments.
Sequence comparisons The program DNA Strider (Marck et al. 1988) estimated a sequence similarity of 93% between the two RAPD products. Sequence comparison revealed a few differences. The most important one was a deletion of 41-nucleotides in the smaller fragment, A052, which had an actual size of 1116 nucleotides (Fig 3). The larger fragment, A05 ~, had an actual size of 1154 nucleotides. Besides this deletion, there were a few other small deletions ranging from one to three bases in both sequences (Table 1, Fig 4). These included a microsatellite sequence of CTT motif. Sequence A05 ~ had three perfect repeats whereas A05 z had five (Fig 4). This trinucleotide does not seem to be abundant in plants, since it was not reported in the surveys of short tandem repeats by the sequence searches of Morgante and Olivieri (1993) and Wang et al. (1994).
Table 1. Deletionsize and frequencyin two codominantsequences for RAPDmarkersamplifiedby OperonprimerA05. Allele
Size
A051
1154
A052
1116
Numberof bases
Frequency
1
3
2 3
1 1
2 3 41
1 1 1
Other differences observed included several base substitutions, mostly transitions and a few transversions (Table 2, Fig 3). The deletions and base substitutions were evenly distributed along the length of the two fragments. No changes in sequence were observed between different clones of the same fragment. The two sequences have been filed in GenBank under the accession numbers L35235 and L35243 for alleles A051 and A05 ~, respectively.
Fig. 2. Hybridizationprofilesfor B. nigra "Junius' (1) and Bl148 (2) genomicDNA digestedby BamHI disclosedby fragmentA05~usedas a probe. The presenceof five fragmentsranging from 6.3 to 0.8 Kb indicatesthatthis sequencein not single copy. A search in GenBank with the FASTA program did not reveal homology of A051 or A052 to any previously reported sequence. Six potential open reading frames were detected by DNA Strider. However, since the fragments were amplified fi'om genomic DNA, it is not known whether they correspond to coding sequences. The relatively small differences in both sequences demonstrated that indeed A051 and A052 were different versions of the same sequence. Although they display codominant segregation, the possibility that these sequences represent two duplicated loci tightly linked in trans, cannot be discarded. This is specially true in view that they are not single copy sequences.
270 Table 2. Type and frequency of base substitutions in two codominant RAPD markers amplified by Operon primer A05.
Type
Base change
Frequency
Transitions
TC AG
8 8
Transversions
TG AC AT CG
2 1 3 3
Acknowledgements. We are indebted to Jan Sadowski for critical reading of the manuscript, and to Ariadna Benet and Xiao Feng Yang for technical assistance. Research supported in part by USDA Competitive Grant 9201568 to CFQ.
Referenees Ayliffe MA, Lawrence GJ, Ellis JG, Pryor AJ (1994) Heteroduplex molecules formed between allelie sequences cause nonparental RAPD bands. Nucleic Acids Res. 22:1632-1636 Hu J, Quires CF (1991) Identification of broccoli and cauliflower cultivars with RAPD markers. Plant Cell Rpts 10:505-511 Kesseli RV, Paran I, Michelmore RW (1992) Efficient mapping of specifically targeted genomic regions and the tagging of these regions with reliable PCR-based genetic markers. In: Applications of RAPD to Plant Breeding pp. 31-36. Crop Science Society of America, Minneapolis Marck C (1988) "DNA Strider", a "C" program for fast analysis of DNA and protein sequences in the Apple Mclntosh family of computers. Nucleic Acid Res. 16:1829-1836 Fig. 3. Partial sequences corresponding to codominant RAPDs. Merging lines show the location of a 41 base deletion in A05 2. Single asterisk indicate transitions, double asterisks to transpositions.
Morgante M, Olivieri AM (1993) PCR-amplified microsatellites as markers in plant genetics. The Plant Journal 3:175-182 Rafalski JA, Tingey SV (1993) Genetic diagnosis in plant breeding: RAPDs, microsatellites and machines. Trends in Genetics 9:275280 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating iahibitors. Proc Natl Acad Sci 74:5463-5467 Truce MJ, Quires CF (1994) Structure and organization of the B genome based on a linkage map of Brassica nigra. Theor. Appl. Genet. 89:590-598 Wang Z, Weber JL, Zhong G Tanksley SD (1994) Survey of plant short tandem DNA repeats. Theor Appl Genet 88:1-6.
Fig 4. Illustration of 1 to 3 base deletions in codominant RAPDs. Microsatellite of CTT motif indicated by bar.
Plant Cell Reports (1995) 15:268-270
Reports ~ Springer-Vcrlag 1995
Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions, substitutions and microsatellites C. E Quiros, M.J. Truco *, and J. Hu Department of Vegetable Crops, University of California, Davis, CA 95616, USA * Present address." IRTA, Centre de Cabrils, 08348 Cabrils (Barcelona), Spain Received 27 February 199J/Revised version received 18 May 1995 - Communicated by W. Parrott
Summary. Two RAPD fragments segregating codominantly were investigated in a F2 population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an intemal 41 nucleotide deletion in one o f the fragments. Minor deletions o f one to three bases, including a microsatellite of CTI" motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A05 ~ whereas the shorter one, denominated A05 ~, had 1116 bp.
Introduction In general, RAPD markers are dominant in most plant species studied (Kesseli et al. 1992). Scarcity of codominant RAPD markers seems to be due to the lack of sequence homology between the primers and the target DNA being amplified within the range of polymerase amplification (Rafalski and Tingey 1993). Therefore, it is expected that potential codominant forms resulting fi'om insertions increasing the length of a sequence beyond the range for polymerase synthesis, will not be produced. However, in a few instances putative codominant markers have been disclosed which fit the expected 1:2:1 Mendelian segregation (Ayaliffe et al. 1994; Kesseli et al. 1992). Unless further analysis is performed, it is not possible solely by following segregation ratios, to determine whether these markers originate fi'om the same sequence or represent instead tightly linked unrelated adjacent sequences. During construction of a B. nigra DNA-based marker linkage map (Truco and Quiros 1994), of a total o f 85 markers Correspondence to. C. E Quiros
analyzed, we observed a pair of putative codominant RAPD markers mapping on chromosome B3. We report in this paper the molecular analysis o f these markers by comparing their sequences and describing their differences.
Material and Methods Plant Material. 13. nigra (2n=2x=16, B genome) UC Davis accessions B1148 (Pakistan) and B1157 (India) and derived 83 F2 progeny used by Truco and Quiros (1994) to construct a B genome linkage map was
used for this study. DNA extraction. Single plant extraction for each of the F2 plants and parentals was accomplished by CsCI gradient as described by Truco and Quiros(1994). DNA amplification. Approximately 10 ng of DNA were amplified by 10-merprimerA05 (5' A ~ T C I " I G 3~)fromOperon Technologies (Alameda, CA) following the RAPD protocol described by Hu and Quiros (1991). Isolation of specific RAPD products. Major DNA fragments were isolated from the gel and purified with the GeneClean kit (Bio 101 Inc., La Jolla, CA). Cloning of RAPD products. Amplified DNA products of B. nigra "Junius' by primer A05 were cloned using the TA cloning kit (InvilrogenCorp. San Diego,CA). DNA hybridizations. AmplifiedDNA ofB. nigra and genomic DNA of cultivars of this species "Junius' and B1148 digested with EcoRI, HindllI or BamHl was blotted to HyBond-N(Amersham, IL) or Zeta Probe GT (BioRad, CA) nylon membranes using the manufacturers protocols. Afterhybridization,high stringencywashes were performed followingthe manufacturer'sprotocols. DNA sequencing. The MI3 universal (-40) and MI3 reverse sequencing primers were used to sequence the inserts fiom both ends. l:or sequencing, tile deoxy termination method on double strand DNA templates (Sanger et al. 1977) was followedusing the Sequenase 2.0 kit of IJSB (Cleveland, Oil). The sequences were analyzed by the programs GCG (U. of Wisconsin, Madison) and DNA Strider 1.2 (Marck 1988). A second clone of each allele was partially sequenced as controls for amplificationartifacts causingsequence modifications
Results and Discussion Genetic segregation
In a segregating F 2 mapping population of B. nigra, one
269
or two fragments of approximately 1200 and 1100 bp resulted from genomic DNA amplification by primerA05. These fragments segregated in the 1:2:1 ratio (x2=1.58, P=0.45, n=83, see Truco and Quiros 1994), as expected for codominant markers (Fig 1). Since no additional fragments were observed, the A05 products did not seem to have the ability to form heteroduplex molecules (Ayliffe et al 1994).
Hybridization experiments After isolation, the 1100 bp fragment used as a probe hybridized to both segregating bands in the amplification profile suggesting homology. Homology was further conf'mned by hybridizing the two RAPD products to B. nigra genomic DNA digested by three different restriction enzymes. The resulting hybridizing profiles had 5 to 7 bands, ranging from approximately 8Kbp to 300 bp depending on the accession and enzyme tested (Fig 2). The profiles disclosed by both probes were virtually identical to each other. Therefore, this experiment indicated that the two clones were closely related, which were then designated A05 t for the 1200 bp one and A052 for the 1100 band. The number of bands observed suggest that A05 corresponded to moderately repeated sequences in the genome.
Fig. 1. B. n!~a F2 population segregating in a 1:2:1 Mendelian fashionfor A05 and A052RAPDfragments.
Sequence comparisons The program DNA Strider (Marck et al. 1988) estimated a sequence similarity of 93% between the two RAPD products. Sequence comparison revealed a few differences. The most important one was a deletion of 41-nucleotides in the smaller fragment, A052, which had an actual size of 1116 nucleotides (Fig 3). The larger fragment, A05 ~, had an actual size of 1154 nucleotides. Besides this deletion, there were a few other small deletions ranging from one to three bases in both sequences (Table 1, Fig 4). These included a microsatellite sequence of CTT motif. Sequence A05 ~ had three perfect repeats whereas A05 z had five (Fig 4). This trinucleotide does not seem to be abundant in plants, since it was not reported in the surveys of short tandem repeats by the sequence searches of Morgante and Olivieri (1993) and Wang et al. (1994).
Table 1. Deletionsize and frequencyin two codominantsequences for RAPDmarkersamplifiedby OperonprimerA05. Allele
Size
A051
1154
A052
1116
Numberof bases
Frequency
1
3
2 3
1 1
2 3 41
1 1 1
Other differences observed included several base substitutions, mostly transitions and a few transversions (Table 2, Fig 3). The deletions and base substitutions were evenly distributed along the length of the two fragments. No changes in sequence were observed between different clones of the same fragment. The two sequences have been filed in GenBank under the accession numbers L35235 and L35243 for alleles A051 and A05 ~, respectively.
Fig. 2. Hybridizationprofilesfor B. nigra "Junius' (1) and Bl148 (2) genomicDNA digestedby BamHI disclosedby fragmentA05~usedas a probe. The presenceof five fragmentsranging from 6.3 to 0.8 Kb indicatesthatthis sequencein not single copy. A search in GenBank with the FASTA program did not reveal homology of A051 or A052 to any previously reported sequence. Six potential open reading frames were detected by DNA Strider. However, since the fragments were amplified fi'om genomic DNA, it is not known whether they correspond to coding sequences. The relatively small differences in both sequences demonstrated that indeed A051 and A052 were different versions of the same sequence. Although they display codominant segregation, the possibility that these sequences represent two duplicated loci tightly linked in trans, cannot be discarded. This is specially true in view that they are not single copy sequences.
270 Table 2. Type and frequency of base substitutions in two codominant RAPD markers amplified by Operon primer A05.
Type
Base change
Frequency
Transitions
TC AG
8 8
Transversions
TG AC AT CG
2 1 3 3
Acknowledgements. We are indebted to Jan Sadowski for critical reading of the manuscript, and to Ariadna Benet and Xiao Feng Yang for technical assistance. Research supported in part by USDA Competitive Grant 9201568 to CFQ.
Referenees Ayliffe MA, Lawrence GJ, Ellis JG, Pryor AJ (1994) Heteroduplex molecules formed between allelie sequences cause nonparental RAPD bands. Nucleic Acids Res. 22:1632-1636 Hu J, Quires CF (1991) Identification of broccoli and cauliflower cultivars with RAPD markers. Plant Cell Rpts 10:505-511 Kesseli RV, Paran I, Michelmore RW (1992) Efficient mapping of specifically targeted genomic regions and the tagging of these regions with reliable PCR-based genetic markers. In: Applications of RAPD to Plant Breeding pp. 31-36. Crop Science Society of America, Minneapolis Marck C (1988) "DNA Strider", a "C" program for fast analysis of DNA and protein sequences in the Apple Mclntosh family of computers. Nucleic Acid Res. 16:1829-1836 Fig. 3. Partial sequences corresponding to codominant RAPDs. Merging lines show the location of a 41 base deletion in A05 2. Single asterisk indicate transitions, double asterisks to transpositions.
Morgante M, Olivieri AM (1993) PCR-amplified microsatellites as markers in plant genetics. The Plant Journal 3:175-182 Rafalski JA, Tingey SV (1993) Genetic diagnosis in plant breeding: RAPDs, microsatellites and machines. Trends in Genetics 9:275280 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating iahibitors. Proc Natl Acad Sci 74:5463-5467 Truce MJ, Quires CF (1994) Structure and organization of the B genome based on a linkage map of Brassica nigra. Theor. Appl. Genet. 89:590-598 Wang Z, Weber JL, Zhong G Tanksley SD (1994) Survey of plant short tandem DNA repeats. Theor Appl Genet 88:1-6.
Fig 4. Illustration of 1 to 3 base deletions in codominant RAPDs. Microsatellite of CTT motif indicated by bar.
