Mutation Mechanisms And Evolutionary Patterns Of Microsatellite Sequences In Common Almond (Prunus Amygdalus L.) And Other Prunus Species

Expressed sequence tag (EST) derived simple sequence repeats ( SSRs, microsatellites) were screened and identified from 3864 almond EST sequences, and the spectra of SSRs in the non-redundant EST sequences from 1388 contigs were investigated by MISA software after sequence assembly. 965 (59.09%) SSRs were detected. The EST-SSR occurs every 1.0kb in almond ESTs, and SSRs with mono-repeat motifs are the most abundant in almond. The spectra of di- and trinucleotide repeated SSR types are average and less than mononucleotide repeat type. The content of AG/TC and AAC/GTT type with di-repeat and tri-motif are most respectively. Moreover, there are not apparently different with SSR abundance in redundant and non-redundant EST sequences of almond.To explore the process by which SSRs evolve, more SSR sequence data are required. In this study, six EST-SSR loci including ASSR63, ASSR72, ASSR60, ASSR4, ASSR61, ASSR17 and two genomic-SSR loci (BPPCT010 and BPPCT026) were randomly selected and were used to reveal the polymophisms of 38 common almond (P. amygdalus) cultivars, six Chinese wild almond and 12 other Primus species. 158 PCR fragments corresponding to 56 alleles of 8 SSR loci were cloned and sequenced from 38 common almonds. Also 88 PCR fragments of 4 loci, ASSR63, ASSR61, ASSR72 and BPPCT010 were cloned and sequenced from 18 wild and Prunus related species. The results are as follows:ASSR63 is a perfect (CAT) 5 repeat located at 3'-UTR of a almond EST putatively encoding Cp12 protein homolog and has 10 alleles, 7 present in common almond, 5 in other Prunus species. 12 fragments from common almond cultivars and 15 from other species were cloned and sequenced. Allelic variation is due to SSR repeat motif except for twice single base insertion of one allele from the individual 'Shuangren'and 'Yeshengbiantao'. The SSR evolution models at the loci basically fit the SMM for increase/decrease the number of their motif repeats.ASSR61 with a (GAA) 9imp repeat motif encoding for cold-regulated LTCOR18 homolog, which was previously found in the CDS region of the almond EST sequence(Xu et al., 2004) , two alleles were observed on the PAGE gels are actually the same one allele and SSR repeat motif is not present within all the sequenced alleles; Similarly, for two alleles of the locus ASSR60 with the (T) 9 repeat motif encoding Formate dehydrogenase homolog assumed by Xu et al (2004) , which located in the 3'-UTR of the almond EST sequence, the repeat motif is not also observed within all the sequenced alleles and their allelic variation is due to an indel of 6 bases in their flanking sequences.ASSR4 is an imperfect (AAAAAT) 3imp repeat located at 5'-UTR of a almond EST putatively encoding hexameric polyubiquitin homolog and SSR repeat motif and five bases deletions in flanking region also contributed to allelic variation.For the locus ASSR17 with the (GA) 15 repeat motif at 3'-UTR of the almond EST sequence encoding Auxin-repressed protein like protein, an indel of 10 bases in the flanking region contributed to the variation of some allelic sizes, apart from the number of the motif repeats. The substitutions of T to G in repeat region occur in the fixed location, and two new SSR types are found in flanking region. Thus, this locus mutation process is more complex than anticipation and any model is not used to explain.The motif repeats in BPPCT010 and BPPCT026 locus both with a compound (AG) repeat motif from peach genome cause allele-size variation. The interruption of GG/GGGG (BPPCT010) and G/GGG (BPPCT026) happened in the fixed location and more fit for an equilibrium model.ASSR72 is a perfect(TC )19 repeat located at 5'-UTR of a almond EST putatively encoding ADP-ribosylation factor-like protein. The twice two bases deletions in flanking and the motif repeat in repeat region both cause allele-size variation. Interestingly, the substitution of TC to GA lead to the loss of a (TC) motif repeat and increase a (GATC) repeat motif, apart from a base substitution of A to C in the flanking region. The mechanisms of two base substitution...