CASE7——RAD-seq for Grape genetic map construction Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing. BMC Plant Biology .2012, 12:148 Results 1,81 4 high-quality SNP markers were developed. The density for linkage maps developed for the F1 population of Z180 × Beihong was very high. The average intervals between two adjacent mapped markers were 1.68 cM, 2.29 cM and 1.16 cM for the Z180, Beihong and integrated maps ,respectively A comparison of the genetic maps to the published Vitis vinifera gen ome revealed both conservation and variations Research Objective Develop SNP markers and construct high density genetic map for grape using next generation restriction-site associated DNA sequencing Material and methods Two parents Z180 and Beihong and F1 mapping population consisted of 100 progeny. RAD sequencing PE76 Material：Z180 ( V. monticola × V. riparia)and Beihong ( V. vinifera× V. amurensis) in 2003. Background： High-density genetic map, one of the most valuable genomic resources, can largely reveal genome compositions and meet the requirement of high throughput superior traits selection among a lot of germplasms in most species, including plant and animal. Although some genetic maps for grapes already exist, the total marker number on the linkage groups (LGs) of these existing maps is generally <1,000 and some of these mapped markers have no sequence information. Result： Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations.

Figure 2 Valid read number and coverage for each plant in the F1 population and their parents. The X axis in a and b indicates the plant accession, including the two parents and their average one; the Y axis in a indicates read number, and in b, cluster (locus) coverage The polymorphic loci for each F1 plant and its parents were calculated. According to Fig. 2, we obtained an average of ~12,840 reads involved in the polymorphic loci and thus a 17.0-fold coverage per cluster per each individual. An average of ~12,840 reads involved in the polymorphic loci and a 17.0-fold coverage per cluster per each individual.

the 1,814 SNP markers were imported into JoinMap4 the 1,814 SNP markers were imported into JoinMap4.0 for map construction. In total, 1,121 markers fell into 19 LGs for Z180 (female), 759 markers for the Beihong (male), and 1,646 markers for the integrated map. The difference in the number of markers between Z180 and Beihong might indicate the heterozygosity of Z180 is larger than Beihong 1,121 markers fell into 19 LGs for Z180 (female), 759 markers for the Beihong (male), and 1,646 markers f or the integrated map.

Figure 7 Collinear analysis of the consensus between genetic and physical maps.The X axis indicates the physical position of each SNP marker; the Y axis indicates the genetic position of each SNP marker. Red diamonds indicate the female genetic position against the reference physical position; black diamonds indicate the male genetic position To compare the order of the common markers, a dot-plot diagram (Figure 7) was generated using the physical position of each common marker on the reference genome against its genetic position on the LGs.Among the 19 LGs, Chr01, 03, 04, 05, 06, 08 (two LGs for male), 09, 10, 12, 13, 14, 17, 18, 19 showed high collinear results for both female and male maps. The remaining LGs only showed high collinear results for one map。the same order for the two types of map most probably indicates conservation of genomes among the different grape species; the non-collinearity for some chromosome regions might indicate some variations among different grape species during evolution. A comparison of the genetic maps to the published Vitis vinifera genome revealed most of the markers showed good linear agreement indicating conservation among the different grape species and the non-collinearity for some chromosome regions might indicate some variations among different grape species during evolution.

CASE8——RAD-seq assist anthracnose disease resistance study in Lupinus angustifolius L. Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L. BMC Genomics 2012, 13:318 Results Material and methods Research Objective discovered 8207 SNP markers. 38 markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective,simple PCR-based markers. Linkage analysis confirmed that randomly selected five markers were linked to the R gene. Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. Strategy： PE91 RAD The objectives of this research were to examine the utility of RAD sequencing, applied as DNA fingerprinting, for rapid marker development for MAS in plant breeding, and to develop molecular markers more closely linked to the disease. Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program.

Paired-end reads were aligned to the Arabidopsis thaliana ref-erence genome version TAIR9 (www.arabidopsis.org) using Bowtie (Langmead et al. 2009), included in the TopHat software v1.0.14 (Trapnell et al. 2009). We mapped nearly 74% of the starting reads to the reference genome, of which almost 97% aligned uniquely (Fig. 1A). Additionally, the number of aligned reads per chromo-some correlated with the chromosome size (Fig. 1B), implying extensive chromosome coverage. The read distribution along each chromosome in windows of 1 kb is shown in Figure 1C A flow diagram illustrating the marker development procedures in this study

Two of the newly developed markers, AnSeq3 and AnSeq4, were flanking the R gene at a genetic distance of 0.9 cM

CASE9—— Double enzyme digestion Genotyping-by-sequencing Results Material and methods map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map; 20,000 SNPs and 367,000 tags on the Synthetic SynOpDH wheat reference map; D-genome markers was lower than either the A or B-genomes in Wheat; assembled 1,485 of the SNP markers into a genetic linkage map of 21 linkage groups. Material: Barley：OWB Wheat：SynOpDH Enzyme：PstI (CTGCAG)；MspI (CCGG) Sequencing： Illumina GAII/ Illumina HiSeq 2000 PE 64bp Background GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost.

Figure 1. Adapter Design, PCR amplification of fragments. Figure1.The ligation product of a genomic DNA fragment (black) containing a PstI restriction site and a MspI restriction site. The forward adapter (blue) binds to a PstI generated overhang. The 4–9 bp barcode for this adapter is in bold with ‘‘X’’. The MspI generated overhang corresponds to the reverse Y-adapter (green). The unpaired tail of the Y-adapter is underlined. 2) During the first round of PCR only the forward primer (red) can anneal. PCR synthesis of the complementary strand proceeds to the end of the fragment synthesizing the compliment of the Y-adapter tail. 3) During the second round of PCR the reverse primer (orange) can anneal to the newly synthesized compliment of the Y-adapter tail. This PCR reaction then proceeds to fill in the compliment of the forward adapter/primer on the other end of the same fragment. Fig.2 Histogram of number of markers in the three wheat genomes for DArT and GBS SNP genetic maps.A) The number of markers assigned to each genome from the DArT genetic map [25] and B) the number of markers in each genome from thede novogenetic map constructed using GBS SNP markers and the AntMap Algorithm. C) The total number of SNPs assigned to each genome using the bin mapping approach in SynOpDH. Figure 2. Histogram of number of markers in the three wheat genomes for DArT and GBS SNP genetic maps

Figure 3. Distribution of GBS SNP markers in the Oregon Wolfe Barley (OWB) bin map. Histogram showing the number of markers from the set of GBS SNPs mapping to each bin in the OWB bin map. The number of SNPs mapping to a single bin is shown by the height of the blue bars. Additional markers that could not be placed in a single bin are show in grey. If a marker mapped to more than one bin (due to missing data), that marker was attributed to its middle bin. Bins that did not have definitive placement of any GBS SNP marker are noted with a red triangle below the plot.