SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley

A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene btwd1 in Huaai 11 was mapped onto the high density linkage maps. The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley.

Huaai 11 is a new source of dwarfism, and consisted of desirable agronomic traits such as shortened stature and early maturity 8 . This dwarf phenotype was controlled by a new dwarfing gene btwd1 mapped on chromosome 7H. The dwarfing gene btwd1 is non-allelic with the uzu and sdw1, which have widely been used in China. The chromosome location of btwd1 is different from those of the uzu, sdw1/denso, br1, br2 genes and QTL PH-7 8 . It is a new source for broadening the genetic base of dwarfism and provides barley breeders with a new gene in China 8 . At present, the btwd1 gene has been successfully applied in breeding program by Huazhong Agricultural University. In 2011, new barley cultivar Huadamai 9 was registered in Hubei Province with Huaai 11 as male parent. In 2014, new barley cultivar Huadamai 10 was registered in Anhui, Henan and Hubei Provinces with Huaai 11 as male parent. Both cultivars have high yield and good quality.
In order to efficiently use this new germplasm for barley breeding program, we have constructed a high-density genetic map based on SNP genotyping and further mapping of btwd1 dwarfing gene using a double haploid population derived from a cross of Huadamai 6 and Huaai 11. This information could be useful for position cloning of this gene and developing new varieties for plant breeders using MAS.

Methods
Plant materials and field trial. A DH population including 122 lines was developed from a cross between a common feed barley cultivar Huadamai 6 and Huaai 11 using anther culture in this study. The DH population and parents were planted on the Experimental Farm of Huazhong Agricultural University, Wuhan, China. The field trials were conducted following a randomized complete block design with three replications in 2006-2008. Each of the DH and parental lines were grown in three rows in a plot of 0.6 × 1.5 m 2 . Height of plants before ripening was measured in the field from soil surface to top of the main culm (with the spike). The height was calculated as the mean of twelve plants 8 . Extraction of genomic DNA. The young leaves from each doubled-haploid (DH) lines and parents were collected and frozen for DNA extraction. The CTAB method was used to extract genomic DNA from about 0.6-1.0 g tissues of each accession 8,15 . DNA samples were electrophoresed on agarose gels for quality assurance purposes. Liquid DNA samples were normalized in water or 1 × TE to a standard concentration of 100-200 ng/μ l.
Genotyping. Frozen DNA samples were sent to the Personal Biotechnology Co., Ltd. (Shanghai, China). The libraries were quantified using Qubit fluorometer (Invitrogen), Agilent 2100 (Agilent Technologies) and real-time quantitative PCR, and then submitted for sequencing on the Illumina HiSeq2000 platform. Each marker was required to have an allele present in at least 85% of DH individuals. Marker genotypes not meeting the minimum thresholds were scored as missing data. The information of SSR markers was as previously described 16 . The segregation data for SSR markers in the same population were detected, and 68 of them were used for constructing an integrated map. The phenotyping data of dwarfing gene btwd1 were from Ren et al. 8 and listed in Table 1.
Genetic linkage map construction. Before constructing genetic maps, SNPs were filtered by excluding those had poor quality data. Low quality SNP included those with NormR results < 0.2 and SNP with large numbers of missing values (15% or more). Markers showing identical segregation patterns were also excluded, with one marker per co-segregating group retained. The DH population, consisting of 122 individuals, was utilized to construct a genetic map. The input datasets were constructed from 1,894 genotyped SNP markers and 68 SSR markers. The program Joinmap 4.0 was used to calculate the marker order and genetic distance 17 . The marker grouping process used a maximum LOD of 3.0 and 7 linkage groups were created. The Kosambi mapping function was employed for map length estimations. Markers were tested for segregation distortion using the chi-square test. Gene mapping was performed with the computer software MAPMAKER and the genetic distance (centimorgan, cM) was derived using Kosambi function 18,19 .

Results
Construction of linkage map. 38,268 polymorphic SNPs were detected between the parental lines Huadamai 6 and Huaai 11, of which 10,367 polymorphic SNPs were detected in the mapping population. All 10,367 polymorphic SNP markers showed segregation within the DH population. Low quality SNP markers excluded as a result of filtering processes comprised of 5,375 markers with inconsistent parental scores, monomorphic and more than 15% missing data. The remaining 4992 polymorphic SNP markers and 153 of the obtained before polymorphic SSR marker met the requirements for use in the construction of a genetic map. After removing some of the co-segregated markers and non-linked markers, the remaining 1894 SNP markers and 68 SSR markers were used to create 7 high-density genetic linkage groups. This high-density genetic linkage map was generated from 1,962 markers covering seven linkage groups with a total map distance of 1,375.8 cM. The averaged distance between two positions across the whole map was 0.7 cM. The number of markers on different chromosomes ranged from 209 on 1H to 396 on 7H. The genetic distance on different chromosome ranged from 145.0 cM on 1H to 230.9 cM on 3H (Fig. 1, Table 2, Table S1).
Molecular mapping of the dwarf gene btwd1. The DH population was constructed from the crosses between Huaai 11 and Huadamai 6 varieties 8 . Plant height of the DH lines showed a bimodal distribution, the segregation ratio between tall and dwarf is 1:1 8 . Huaai 11 was a six-row new source of dwarf that was controlled by a recessive dwarfing gene btwd1, and Huadamai 6 was a two-row common feed barley cultivar 16 . Previous linkage analysis between SSR markers and plant height found that the dwarfing gene was located on the long arm of chromosome 7H, associated with the marker Bmac167 and Bmac031 at a genetic distance of 2.2 cM 8 . A set of 40 SNP markers near the SSR marker Bmac031 and Bmac167 was used to analyze the 122 individuals of DH population. The results showed that the gene btwd1 controlling plant height in Huaai 11 was positioned between SNP marks 7HL_6335336 and 7_249275418 with genetic distance of 0.9 cM and 0.7 cM on chromosome 7HL, respectively (Fig. 2).

Discussion
Constructing a high-density linkage map is a vital prerequisite for genetic analysis and efficient molecular breeding. For the past ten years there has been a surge in marker density and convergence toward consensus maps for barley 2,4-6,20-29 (Table 3). In the previous published barley genetic maps, limited number of markers were included using traditional marker technologies such as RFLP, RAPD, DArT, SSR and EST based markers 4,5,20,21,30 . SNP markers have been recognized as important candidate markers due to their high abundance and relatively even distribution in barley genome. Our results demonstrated the value of RAD sequencing for the development of SNP markers and for the production of the most saturated linkage maps. RAD is one such NGS-based method which utilizes the restriction enzyme digestion of genome to reduce the genome complexity 1 . In RAD, only a small fraction of the genome is sequenced to identify many genetic markers along the genome. It is a simple and more cost-effective than other NGS methods not only for large-scale polymorphism discovery but also for genotyping-by-sequencing approach 2 . RAD sequencing data are easier to handle because less overall data are detected in comparison with whole-genome sequencing data, and therefore different individuals of populations can be easily genotyped. Using RAD sequencing approach, we identified thousands of SNPs in the Huaai 11 and Huadamai 6 DH population. In this paper, we reported the construction of a new SNP based genetic map of barley from Huaai 11 and Huadamai 6 DH population. The genetic linkage map is composed of 1894 SNP markers and 68 SSR markers. The total length of the map is 1375.8 cM with an average distance of 0.7 cM between loci ( Table 2). These markers can be used for genetic diversity analysis, marker-trait association and marker-assisted selection for barley improvement.
We have used these SNP markers to map the dwarfing gene btwd1 of barley in Huaai 11 and Huadamai 6 DH population, which previous mapped using SSR markers 8 . The results showed that the gene btwd1 controlling plant height of Huaai 11 was positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7HL, respectively. Our results are comparable to those in a few previous studies on dwarf and semi-dwarf genes in barley, and corresponded well with our previous result reported by Ren et al. 8 . The chromosome location of btwd1 is different from the hcm1 gene (2HL) 29 , sdw3 gene (2HS) 31 , uzu gene (3HL) 8,12,29,32,33 , sdw1/denso geng (3HL) 8,14,29,34,35 , br2 (4H) 8,36,37 , ari-e gene (5HL) 29 , br1 gene (7HS) 8  germplasm Huaai 11 is a new source of dwarfs for broadening the genetic base of dwarfism. Two barley cultivars with high yield and good quality were bred with Huaai 11 as the male parent. The dwarfing gene btwd1 in Huaai 11 has widely been used in China, and provides barley breeders with a new source for barley genetic improvement. In order to efficiently use this new germplasm Huaai 11 for barley breeding program, we will verify the identified SNP markers associated with btwd1 in the lines developed from Huaai 11. We have also constructed  Table 2. Genetic distance and number of markers from the high-density map. a large F 2 population including 16000 lines derived from a cross of Huadamai 6 and Huaai 11. We will fine map dwarfing gene btwd1 using this large F 2 population and the SNP markers. In near future, we will clone the dwarf gene btwd1 and verify its function.
In conclusion, we have constructed a high-density barley linkage map using SNP markers derived from the DH population RAD sequencing. Our study provides a valuable genetic resource for molecular markers, map-based gene cloning, MAS and the sequence assembly of the barley reference genome. At the same time, the linked SNP markers identified in the present study can provide a useful marker-assisted selection tool to transfer the dwarfing gene btwd1 in barley breeding.