Microsatellite or SSR marker is an efficient tool for plant genotype identification, molecular mapping and marker-assisted selection. Objective of this study is to analyze the mutagenized microsatellite variations in ...Microsatellite or SSR marker is an efficient tool for plant genotype identification, molecular mapping and marker-assisted selection. Objective of this study is to analyze the mutagenized microsatellite variations in soybean genome and reveal nature of these mutations. In the present study, mutations at fifteen microsatellite loci were detected in genomic DNAs of soybean mutant E182 induced by EMS （ethyne metyl sulfate） using PCR amplification of 485 pairs of SSR primers. These fifteen mutagenized microsatellite loci with repeat number variation were Satt005, Satt117, Satt185, Satt282, Satt290, Satt420, Satt452, Satt483, Satt569, Satt579, Satt600, Satt602, Sat086, Sat107 and Sat135, respectively. Sequencing results of these fifteen loci indicated that microsatellite sequences at Satt282, Satt483, Satt579, Satt600 and Satt602 loci were respectively deleted 1-, 3-, 8-, 20- and 1-trinucleotide （all [ATT] 1-20 except for [CAA] 8[TAA] 12 at Satt600 locus） repeats, which made allele sizes at these five loci decrease 3, 9, 24, 60 and 3 bp, respectively. And while microsatellite sequences at the other ten mutated loci, Satt005, Satt117, Satt185, Satt290, Satt420, Satt452, Satt569 and Sat086, Sat107, Sat135, were respectively inserted 1-, 6-, 6-, 3-, 4-, 3-, 8-trinucleotide repeats （ATT） 1-8 and 12-, 6-, 16-dinucleotide repeats （AT） 6-16 , making allele sizes at these ten loci increase 3, 18, 18, 9, 12, 9, 24, 24, 12, 32 bp, respectively. On the other hand, eleven events of base mutations were detected in flanking regions at seven （Sat-107, Satt185, Satt282, Satt420, Satt569, Satt579 and Satt600） of fifteen mutated microsatellite loci. These base mutations consisted of 6 transitions （4T→C and 2 A→G）, 2 transvertions （A→T and T→A）, 1 insertion （T） and 2 deletions （A and T）. The experimental results proved that EMS mutagenesis could cause different types of mutations at microsatellite multilocus in soybean genome, including repeat number variations in microsatellite regions and random base mutations in flanking regions. We found three mutational biases, which were frequent insertion mutations of repeat units, initiating positions of microsatellite sequences of repeat unit insertions/deletions and both flanking-base T↓A of these insertion/deletion positions. In addition, the resolution capacity of high-quality agarose gels was sufficient to distinguish differences of only three base pairs in this experiment.展开更多
基金supported in part by the National“863”Projects(No.2002AA211051,2002AA207007,2001AA241061-4)“973”Projects(2002CB111301)Nowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-1-01).
文摘Microsatellite or SSR marker is an efficient tool for plant genotype identification, molecular mapping and marker-assisted selection. Objective of this study is to analyze the mutagenized microsatellite variations in soybean genome and reveal nature of these mutations. In the present study, mutations at fifteen microsatellite loci were detected in genomic DNAs of soybean mutant E182 induced by EMS （ethyne metyl sulfate） using PCR amplification of 485 pairs of SSR primers. These fifteen mutagenized microsatellite loci with repeat number variation were Satt005, Satt117, Satt185, Satt282, Satt290, Satt420, Satt452, Satt483, Satt569, Satt579, Satt600, Satt602, Sat086, Sat107 and Sat135, respectively. Sequencing results of these fifteen loci indicated that microsatellite sequences at Satt282, Satt483, Satt579, Satt600 and Satt602 loci were respectively deleted 1-, 3-, 8-, 20- and 1-trinucleotide （all [ATT] 1-20 except for [CAA] 8[TAA] 12 at Satt600 locus） repeats, which made allele sizes at these five loci decrease 3, 9, 24, 60 and 3 bp, respectively. And while microsatellite sequences at the other ten mutated loci, Satt005, Satt117, Satt185, Satt290, Satt420, Satt452, Satt569 and Sat086, Sat107, Sat135, were respectively inserted 1-, 6-, 6-, 3-, 4-, 3-, 8-trinucleotide repeats （ATT） 1-8 and 12-, 6-, 16-dinucleotide repeats （AT） 6-16 , making allele sizes at these ten loci increase 3, 18, 18, 9, 12, 9, 24, 24, 12, 32 bp, respectively. On the other hand, eleven events of base mutations were detected in flanking regions at seven （Sat-107, Satt185, Satt282, Satt420, Satt569, Satt579 and Satt600） of fifteen mutated microsatellite loci. These base mutations consisted of 6 transitions （4T→C and 2 A→G）, 2 transvertions （A→T and T→A）, 1 insertion （T） and 2 deletions （A and T）. The experimental results proved that EMS mutagenesis could cause different types of mutations at microsatellite multilocus in soybean genome, including repeat number variations in microsatellite regions and random base mutations in flanking regions. We found three mutational biases, which were frequent insertion mutations of repeat units, initiating positions of microsatellite sequences of repeat unit insertions/deletions and both flanking-base T↓A of these insertion/deletion positions. In addition, the resolution capacity of high-quality agarose gels was sufficient to distinguish differences of only three base pairs in this experiment.
