DNA‐based molecular markers play a significant role in gene mapping, genetic diversity analysis, germplasm evaluation and molecular marker‐assisted selection. A combination of desirable marker characteristics such as abundant polymorphism, good stability, ease of production and high efficiency is difficult to achieve when utilizing traditional molecular marker systems. MicroRNAs are a type of endogenous non‐coding RNAs prevalent in the genomes of many organisms. The high conservation of miRNA and pre‐miRNA sequences provides an opportunity to develop a novel molecular marker type. We downloaded 82 miRNA sequences from the Brassica miRBase database and designed 46 single miRNA‐based primers which could be randomly combined to generate primer pairs. A proportion of these primer pairs were validated for transferability and polymorphism using DNA from 16 varieties of six Brassica species. The percentage of polymorphic primer pairs were 28.1%, and the average polymorphism information content (PIC) value for the 34 primer pairs was 0.43. Good transferability was verified across species. These results indicate that miRNA‐based markers provide a novel genotyping technique with low costs, high efficiency, stability and good transferability.
Long terminal repeat (LTR) retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferentially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well understood. In this study, a total of 1.52Gb of Brassica sequence containing 2020 bacterial artificial chromosomes (BACs) was scanned, and six bacterial artificial chromosome (BAC) clones with extremely nested LTR-TEs (LTR-TEs density: 7.24/kb) were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23Mya. LTR-TEs also preferably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes (3/207) could be matched to the Brassica expressed sequence tag (EST) database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.
Simple sequence repeats (SSRs) are important components of eukaryotic genomes and may play an important role in regulating gene expression. However, the characteristics of genic SSRs and the effect of interspecific hybridization and polyploidization on genic SSRs seem not to have received desired attention in terms of scientific investigations. To determine the features of genic SSRs and elucidate their role in polyploidization process of the Brassica family, we identified SSRs in Plant Genome Database-assembled unique transcripts (PUTs) of Brassica species. A higher density of SSRs and a greater number of compound motif SSRs and mononucleotide motif types with large average number of repeats were detected in allotetraploid Brassica napus than in the diploid parental species (Brassica rapa and Brassica oleracea). In addition, a greater proportion of SSR-PUTs were found to be associated with the stress response and developmental processes in B. napus than in the parents. A negative correlation between the repeat number and the motif type and the total length, and a positive correlation between the repeat number and the total length of SSRs were observed. PUT-SSR might be generated from A/T-rich regions. The successful development of 123 pairs of SSR primers for Brassica PUTs showed that SSR-PUTs could be exploited as gene-based SSR functional markers for application in Brassica breeding. These results indicate that interspecific hybridization and polyploidization could trigger the amplification of SSRs, and long SSRs might become shorter to enable the plant to adapt to environmental and artificial selection.
Numerous quantitative trait loci (QTL) have been detected in Brassica species, but fine-mapping of major QTL has advanced slowly. The development of functional markers can overcome this barrier. We used publicly available PlantGDB-assembled unique transcripts (PUTs) from Brassica species to design 7836 functional simple sequence repeat (SSR) primer pairs. Functional annotation of the PUTs containing SSRs was done by Blast2GO. The PUTs harbouring SSRs were mainly involved with nucleotide or protein binding and enzyme activity, and preferentially functioned in membranes and cytoplasm. Totally, 210 PUT primer pairs were selected to test their polymorphism, stability, and PCR quality. Approximately 70% (147) of the primer pairs resulted in successful amplification with an average polymorphic information content (PIC) value of 0.49. The highest level of polymorphism was dinucleotide repeat SSRs, followed by tri- and mononucleotide repeats. Approximately 60% of the primer pairs showed good transferability among Brassica species. These results show that the development of markers from PUTs is a feasible and simple approach to develop functional SSR markers on a large scale across Brassica species. In addition, these markers can provide a novel alternative that is a putative approach for rapid determination of candidate genes, genetic mapping, genetic diversity analysis, and comparative mapping in Brassica species.
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