Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L.

Background: 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. To combine next generation sequencing technology with SRAP, Illumina’s Solexa sequencing was used to sequence tagged SRAP PCR products. Results: Three sets of SRAP primers and three sets of tagging primers were used in 77,568 SRAP PCR and the same number of tagging PCR reactions respectively to produce a pooled sample for Illumina's Solexa sequencing. After sequencing, 1.28 GB 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. Consequently, on 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 make alignment of the current genetic map with other five genetic maps in B. rapa and B. napus. Conclusion: We 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 B. rapa genome. Additionally, this genetic map might be applied in gene cloning and marker development in B. rapa and B. napus.

背景：序列相关扩增多态性（Sequence Related Amplified Polymorphism, SRAP）无需知晓序列信息，即可用于构建高密度遗传图谱、定位作物重要农艺性状的基因与数量性状位点（Quantitative Trait Locus, QTL），并开展遗传多样性分析。为将下一代测序技术与SRAP相结合，本研究采用Illumina Solexa测序技术对带有标签的SRAP聚合酶链式反应（Polymerase Chain Reaction, PCR）产物进行测序。
结果：本研究分别使用3套SRAP引物与3套标签引物，完成77,568次SRAP PCR反应与等量的标签PCR反应，最终获得混合样本用于Illumina Solexa测序。测序完成后，共获得1.28GB的测序数据，包含超过1300万条双端序列（Paired-end Sequence），并利用这些数据将Solexa序列与其对应的SRAP标记进行匹配，同时将Solexa序列整合至超高密度遗传图谱中。本研究利用白菜型油菜（Brassica rapa, B. rapa）的重组自交系（Recombinant Inbred Line, RI）作图群体，构建了包含465个bin的超高密度遗传bin图谱。据此，在该超高密度遗传bin图谱上，共组装得到9,177个SRAP标记、1,737个整合的唯一Solexa双端序列，以及代表10,960个独立遗传位点的46个简单序列重复（Simple Sequence Repeat, SSR）标记；同时有141条唯一Solexa双端序列与其对应的SRAP标记完成匹配。通过两个物种共有的SRAP标记，将白菜型油菜的该遗传图谱与此前发布的甘蓝型油菜（Brassica napus, B. napus）超高密度遗传图谱进行共线性比对。此外，本研究还利用SSR标记，将当前构建的遗传图谱与白菜型油菜和甘蓝型油菜中的另外5张遗传图谱进行了比对。
结论：本研究利用SRAP技术在白菜型油菜中构建了包含10,960个独立遗传位点的超高密度遗传图谱，为该物种目前已构建的饱和度最高的遗传图谱。借助下一代测序技术，本研究将1,878条Solexa序列整合至该遗传图谱中。这些整合后的序列将用于白菜型油菜基因组的支架（Scaffold）组装。此外，该遗传图谱可应用于白菜型油菜与甘蓝型油菜的基因克隆及标记开发工作。