Microsatellites inBrassicaunigenes: relative abundance, marker design, and use in comparative physical mapping and genome analysis

Abstract

Microsatellites present in the transcribed regions of the genome have the potential to reveal functional diversity. Unigene sequence databases are the sources of such genic microsatellites with unique flanking sequences and genomic locations even in complex polyploids. The present study was designed to assay the unigenes of Brassica napus and B. rapa for various microsatellite repeats, and to design markers and use them in comparative genome analysis and study of evolution. The average frequency of microsatellites in Brassica unigenes was one in every 7.25 kb of sequence, as compared with one in every 8.57 kb of sequence in Arabidopsis thaliana. Trinucleotide motifs coding for serine and the dinucleotide motif GA were most abundant. We designed 2374 and 347 unigene-based microsatellite (UGMS) markers including 541 and 58 class I types in B. napus and B. rapa, respectively, and evaluated their use across diverse species and genera. Most of these markers (93.3%) gave successful amplification of target microsatellite motifs, which was confirmed by sequencing. Interspecific polymorphism between B. napus and B. rapa detected in silico for the UGMS markers was 4.16 times higher in 5' untranslated regions than in coding sequences. Physical anchoring of Brassica UGMS markers on the A. thaliana genome indicated their significance in studying the evolutionary history of A. thaliana genomic duplications in relation to speciation. Comparative physical mapping identified 85% of Brassica unigenes as single copy and gave clues for the presence of conserved primordial gene order. Complex chromosomal rearrangements such as inversions, tandem and segmental duplications, and insertions/deletions were evident between A. thaliana and B. rapa genomes. The results obtained have encouraging implications for the use of UGMS markers in comparative genome analysis and for understanding evolutionary complexities in the family Brassicaceae.

Development and linkage mapping of unigene-derived microsatellite markers in Brassica rapa L.

“…UGMS markers have been recently developed in order to use unique functional genes in the plant genomes as molecular markers (Parida et al 2006(Parida et al , 2010. This has reduced the redundancy, i.e., multiple copies of genes or repeats of the same DNA sequences, which is a general problem when SSRs are designed from random genomic DNA or EST sequences.…”

“…Of the 450 UGMS primer pairs designed, 428 (95.1%) primer pairs successfully amplified DNA fragments of expected sizes in the B. rapa genome. Several UGMS markers were designed from B. rapa and B. napus earlier (Parida et al 2010). However, due to the large genome size containing many more functional unigenes in Brassica species, representation of the developed UGMS were few.…”

“…However, due to the large genome size containing many more functional unigenes in Brassica species, representation of the developed UGMS were few. Therefore, we developed additional 450 new UGMS markers using the unigene sequence information which was not used in the earlier study (Parida et al 2010). The newly developed B. rapa UGMS markers displayed a low level of polymorphism (16.4%) between the two parental lines compared to 24.3% polymorphism detected by genomic SSRs.…”

“…Recently, Parida et al (2010) developed 347 unigene-derived microsatellite (UGMS) markers in the B. rapa genome, although there still are undoubtedly many more that remain to be identified. So, considering the advantages and utility of UGMS markers, in the present study, we attempted to develop more UGMS markers from unique and transcribed gene regions and map in the B. rapa genome along with previously developed markers.…”

Genetic Diversity Analysis for Salinity Tolerance in Indian Mustard [Brassica juncea (L.)] Using SSR Markers

“…Molecular markers particularly the microsatellite markers (SSRs) provide a valuable tool for genetic analysis and produce salt tolerant plants (Tanksley, 1989). It can help in the identification of suitable parental lines, diversity and pedigree analyses and is more suitable to fasten the selection and development of new improved genotypes, as it is independent of environmental factors and developmental stage of the plant (Parida, 2010).…”