Abstract
BackgroundSequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. To combine next generation sequencing technology with SRAP, Illumina's Solexa sequencing was used to sequence tagged SRAP PCR products.ResultsThree sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR reactions and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB of sequence with over 13 million paired-end sequences was obtained and used to match Solexa sequences with their corresponding SRAP markers and to integrate Solexa sequences on an ultradense genetic map. The ultradense genetic bin map with 465 bins was constructed using a recombinant inbred (RI) line mapping population in B. rapa. For this ultradense genetic bin map, 9,177 SRAP markers, 1,737 integrated unique Solexa paired-end sequences and 46 SSR markers representing 10,960 independent genetic loci were assembled and 141 unique Solexa paired-end sequences were matched with their corresponding SRAP markers. The genetic map in B. rapa was aligned with the previous ultradense genetic map in B. napus through common SRAP markers in these two species. Additionally, SSR markers were used to perform alignment of the current genetic map with other five genetic maps in B. rapa and B. napus.ConclusionWe used SRAP to construct an ultradense genetic map with 10,960 independent genetic loci in B. rapa that is the most saturated genetic map ever constructed in this species. Using next generation sequencing, we integrated 1,878 Solexa sequences on the genetic map. These integrated sequences will be used to assemble the scaffolds in the B. rapa genome. Additionally, this genetic map may be used for gene cloning and marker development in B. rapa and B. napus.
Highlights
Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information
There were 388 bins with ten or more SRAP markers, three bins with over 100 SRAP markers and only three bins with one SRAP marker, suggesting that the SRAP markers were evenly distributed in the B. rapa genome (Additional file 5)
Since the A genome in B. napus comes from B. rapa [20], the alignment of these two genetic maps would allow the identification of the corresponding linkages groups in these two related genomes
Summary
Sequence related amplified polymorphism (SRAP) is commonly used to construct high density genetic maps, map genes and QTL of important agronomic traits in crops and perform genetic diversity analysis without knowing sequence information. There are several PCR-based molecular marker detection methods such as amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) and sequence related amplified polymorphism (SRAP) that are commonly used in molecular marker development, genetic mapping, genetic diversity analysis and high density genetic map construction [1]. Compared with other PCR-based molecular marker detection methods, SRAP is more flexible and easier to use. SRAP is an effective method for map-based gene cloning and molecular marker assisted selection (MAS). SRAP was adequate to perform QTL analysis, which was demonstrated in B. napus [13]
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