Integretion Of SOLEXA Sequences On An Ultradense Genetic Map In Brassica Rapa.

Sequence related amplified polymorphism (SRAP) was developed by Dr. Li and Dr. Quiros from University of California, Davis in 2001. It is commonly used to construct high density genetic maps, map genes and QTLs of important agronomic traits in crops and perform genetic diversity analysis. Especially for those species without prior sequence information, SRAP is one of the most effective ways to study the genome because design of SRAP primer does not need prior sequence information.Because of high polymorphic rate, and easy acquirement of SRAP primer, it is not difficult to construct an ultradense genetic map with thousands of SRAP markers in a short time. The next generation sequencing technology was extensively used for genome sequencing, gene location, and gene profiling analysis. Illumina’s Solexa sequencing technology was characterized as high throughput and low cost, it could sequence millions of SRAP PCR products in a single run and provide the sequences of both ends of SRAP markers. By using tagging primers through tagging PCR, DNA source of SRAP PCR products could be identified after sequencing. For a species whose whole genome sequence is available, Solexa sequences allow anchoring SRAP markers in the genome. For a species without genome sequence information, an ultradense genetic map constructed with evenly distributed SRAP markers with known Solexa sequences might be used to assemble whole genome sequence.A RIL mapping population was used in this study, which was developed from a cross between a yellow seeded variety“Yellow Sarson”in B. rapa and a Chinese cabbage doubled haploid (DH) line“RI16”. The material of this study included 92 RILs of the F7 generation and their parental lines. Three sets of SRAP primers were used to produce SRAP PCR products and three sets of tagging primers were used in tagging PCR to tell origin of DNA source after sequencing, respectively. The tagged PCR fragments were pooled for Illumina’s Solexa sequencing. In total, 805 SRAP primer pairs were used to amplify the mapping population. JoinMap 3.0 software was employed to construct an ultradense genetic map. There are SRAP markers that were used in both Solexa sequecing and ultradense genetic map construction. To match Solexa sequences with their corresponding SRAP markers on the ultradense genetic map, locus-specific primers were designed from Solexa sequences to test and verify it in a specific PCR. Statistical significance tests of linkage analysis between the counts distribution of each unique Solexa paired-end sequence in mapping population and 465 markers of the genetic bin map were carried out by QTL analysis software to integrate Solexa sequencs on the ultradense genetic map. Next generation sequencing technology was combined with SRAP by sequencing tagged SRAP PCR products for construction of an ultradense genetic map covering the whole genome and with sequence information. The ultradense genetic map could be used for development of markers, gene clone and QTL analysis. Besides, it is of very important reference value for the on going physical map assembling of A genome in Brassica. The main results are as follows:1. Construction of an ultradense genetic mapEight hundred and five pairs of SRAP primers were used to assemble an ultradense genetic bin map with 465 bins on 10 linkage groups and covering a genetic distance of 1,495.6 cM. The genetic map consists of 9,177 SRAP marker and 46 SSR markers. Alignment of the current map and the 5 published maps was performed based on common SRAP markers and published SSRs in B. rapa and B. napus, so 10 linkage group were assigned to R01-R10.2. Solexa sequencing of SRAP PCR productsTo sequence the SRAP PCR products, three sets of SRAP primers were selected or designed. The first set of SRAP primers included 14 forward primers and 192 reverse primers, which resulted in 2688 primer pairs in total. They were only used to amplify two parental lines. The Second and the third set included 4 forward primers and 96 reverse primers, 1 forward primer and 384 reverse primers, respectively. These two sets of 384 SRAP primer pairs were used to amplify 94 lines of the mapping population. In total, 77,568 SRAP PCR reactions were performed.Similarly, the same number of tagging PCR reactions involving three sets of tagging primers were performed to add tag sequence to the SRAP PCR products. The tagging primers were designed according to the SRAP forward primer. The tagging PCR products were pooled together for Illumina’s Solexa sequencing after size selection of 100-400bp and purification.After sequencing, a 1.28 Gb text file with over 13 million paired-end Solexa sequences was obtained. Java programs were developed to identify primer sequences in the Solexa sequences. According to tagging primers, all Solexa sequences were classified into three sequence groups corresponding to three sets of SRAP primer and three sets of tagging primer.3. Matching Solexa sequences with SRAP markers on the ultradense genetic map through locus-specific PCR Five hundred and thirty-two Solexa sequences of the first sequence group were obtained with 165 SRAP primer pairs, which were also used in the genetic map construction. To integration these Solexa sequences on the map, 532 locus-specific primers were designed from sequences located at the reverse primer end of a SRAP fragment. These locus-specific primers were combined with the original SRAP forward primers to amplify 16 SRAP PCR products of 16 individuals of RILs produced with the original SRAP forward and reverse primers in the genetic map construction procedure. When only one polymorphic fragment was generated, fragment pattern was identical to one of the SRAP markers in SRAP profiling, and fragment size was about 20bp less, then the Solexa sequences were matched with the SRAP markers. Among the 532 selected sequences, 141 were succesfully matched with their corresponding SRAP markers.4. Integration of Solexa sequence through linkage analysis between Solexa sequence counts and bin representitive markers of the ultradense genetic mapTo integrate the Solexa sequences of the second and the third sequence group on the map, 2,172 and 4,243 sequences were selected because of their better count distribution among mapping population individuals. Windows QTL cartgrapher 2.5 software was used to test linkage statistical significance between count of each Solexa sequence and 465 bin representitive SRAP markers of the genetic map. Solexa sequence with significance linkage to only one linkage group were accepted and assigned to the bin whose representitive marker had the maximum LOD score with the Solexa sequence. Among the integrated 1,737 sequences, 651 belong to the second group and 1,086 belong to the third group.Taken together, by using SRAP marker system and Illumina’Solexa sequencing technology an ultradense genetic bin map which comprise 9,177 SRAP markers, 46 SSR markers and 1,878 integrated Solexa sequences were succesfully constructed. The genetic map was succesfully aligned with the published genetic maps in B. rapa and B. napus. Totally, over 10,000 independent genetic loci were assembled in B. rapa. To our knowedge, it is the most saturated genetic map that has been constructed in B. rapa.