Kazusa Marker DataBase: a database for genomics, genetics, and molecular breeding in plants

Abstract

In order to provide useful genomic information for agronomical plants, we have established a database, the Kazusa Marker DataBase (http://marker.kazusa.or.jp). This database includes information on DNA markers, e.g., SSR and SNP markers, genetic linkage maps, and physical maps, that were developed at the Kazusa DNA Research Institute. Keyword searches for the markers, sequence data used for marker development, and experimental conditions are also available through this database. Currently, 10 plant species have been targeted: tomato (Solanum lycopersicum), pepper (Capsicum annuum), strawberry (Fragaria × ananassa), radish (Raphanus sativus), Lotus japonicus, soybean (Glycine max), peanut (Arachis hypogaea), red clover (Trifolium pratense), white clover (Trifolium repens), and eucalyptus (Eucalyptus camaldulensis). In addition, the number of plant species registered in this database will be increased as our research progresses. The Kazusa Marker DataBase will be a useful tool for both basic and applied sciences, such as genomics, genetics, and molecular breeding in crops.

Introduction

DNA polymorphisms and mutations, some of which confer phenotypic variations, can be detected by DNA marker analyses. The first DNA marker, the restriction fragment length polymorphism (RFLP), has been used for linkage analysis to determine the genomic positions responsible for Huntington’s disease in humans (Gusella et al. 1983). The RFLP marker technologies were then applied to analysis of plant genetics, particularly tomato and maize (Helentjaris et al. 1986), to construct genetic maps, which are essential tools for positional cloning and quantitative trait loci (QTL) analysis of genes of interest. Then, several types of DNA markers, e.g., random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) or microsatellite, and single nucleotide polymorphism (SNP), were made available through advances in the technologies for DNA analysis (Phillips and Vasil 2001).

DNA markers are used not only in basic sciences but also in applied studies (Kumar 1999). For example, DNA markers linking to desirable loci are used in the selection of elite lines from breeding populations, a process known as marker-assisted selection (MAS). F₁ hybrids are used in the production of commercial varieties in several cereal and vegetable crops, because the F₁ hybrids sometimes exhibit hybrid vigor and heterosis. Therefore, DNA markers can also be applied for purity testing to investigate the heterogeneity of F₁ hybrids, which is a combination of different alleles from the parental lines. In addition, in the management of genetic resources and quality control of food products, DNA markers have been employed for identification of species, cultivars, and varieties.

Whole-genome sequencing in plants was first achieved in Arabidopsis thaliana (The Arabidopsis Genome Initiative 2000), followed by rice (International Rice Genome Sequencing Project 2005). Since those initial reports, the genomes of more than 50 plants have been sequenced (Michael and Jackson 2013). In addition, massive transcriptome analysis has been performed in several plants by using the next-generation sequencers (NGSs) (Hamilton and Buell 2012). This has enabled the development of large numbers of DNA markers for several plants in a relatively short time. Numerous databases have been made available with the genome and DNA marker information for these plant species, e.g. TAIR for Arabidopsis thaliana (http://www.arabidopsis.org) and IRGSP for Oryza sativa (http://rgp.dna.affrc.go.jp/IRGSP/). In addition, there are also databases integrating the information for several plant species, such as the Gramene database for cereal plant species (http://www.gramene.org), the Sol Genomics Network (SGN) for the Solanaceae family (http://solgenomics.net), and VegMarks for seven species of vegetables analyzed in the NARO Institute of Vegetable and Tea Sciences (http://vegmarks.nivot.affrc.go.jp). Our group has also worked on DNA marker development, genetic linkage map, and QTL analysis to promote breeding programs for crops whose molecular genetic information has lagged behind those of the model species, but which are nonetheless important for food production, animal feedings, and industrial materials. In this paper, we introduce the Kazusa Marker DataBase (http://marker.kazusa.or.jp), which provides information on DNA markers, genetic linkage and physical maps developed in the Kazusa DNA Research Institute, for crops and other plant species to enrich the available molecular genetic information on agronomical plants.

Contents of the Kazusa Marker DataBase

The Kazusa Marker DataBase was constructed using Red Hat Enterprise Linux Server release 5.6 as the computer operating system. The MySQL (http://dev.mysql.com) system, which is a relational database management system, was employed for management of the database contents. Most of the contents of this database were written using html text format and Ruby on Rails (RoR), the open-source web framework (http://rubyonrails.org).

Currently, the database includes mainly SSR markers for 10 species: tomato (Solanum lycopersicum), pepper (Capsicum annuum), strawberry (Fragaria × ananassa), radish (Raphanus sativus), Lotus japonicus, soybean (Glycine max), peanut (Arachis hypogaea), red clover (Trifolium pratense), white clover (Trifolium repens), and eucalyptus (Eucalyptus camaldulensis) (Table 1). SSR markers have advantages over other marker systems because of their multi-allelic detection, co-dominant inheritance, high-transferability across species, and flexibility with various laboratory systems. The SSR markers can be classified into two categories, genome-SSR and expressed sequence tag (EST)-SSR. The genome-SSR markers are developed from random genome sequences from, for example, SSR-enrichment genomic libraries, while EST-SSR markers are from sequences of cDNAs. In the subsequent in silico analyses, SSR motifs were identified from the sequence data with the SSRIT (Temnykh et al. 2001), MISA (Thiel et al. 2003), and/or SciRoKo (Kofler et al. 2007) programs, and PCR primers were designed on the flanking sequences of the SSR motifs using the Primer3 program (Rozen and Skaletsky 2000). The identified SSR motifs, repeat numbers of the motif, PCR primers, and expected amplicon sizes are also available from the database. In addition, allele information and/or gel images are also available for the SSR markers, which were tested in molecular experiments. This database also includes information on SNPs, which are the most abundant source of variation in the genome for both intragenic and intergenic regions. While the SNP data are limited to tomato at present, the number of crops with SNP data is expected to increase through the use of NGSs. The details for other markers are described in the next section for each crop.

Table 1 Types and numbers of DNA markers registered in the Kazusa Marker DataBase

Binomial nomenclature	Common name	Marker type	Abbreviation of marker name	No. of markers	Reference
Solanum lycopersicum	Tomato	Genome-SSR	TGS	13,501	Shirasawa et al. 2010a
		EST-SSR	TES	7,599	Shirasawa et al. 2010a
		Genome-SNP		1,473,798	Shirasawa et al. 2013b
		EST-SNP		5,607	Shirasawa et al. 2010b
		Intron-SNP	TEI	169	Shirasawa et al. 2010a
Capsicum annuum	Capsicum	EST-SSR	CaES	5,751	Shirasawa et al. 2013c
Fragaria × ananassa	Strawberry	EST-SSR	FAES	603	Isobe et al. 2013
		SSR dervied from F. vesca	FVES	3,746	Isobe et al. 2013
		Transcriptome-SSR	FATS	125	Isobe et al. 2013
Raphanus sativus	Radish	EST-SSR	RRS	3,811	Shirasawa et al. 2011
Lotus japonicus		dCAPS	BM, TM	82	Sato et al. 2001
		SSR	BM, TM	1,073	Sato et al. 2001
Glycine max	Soybean	EST-SSR	GMES	7,020	Hisano et al. 2007
Arachis hypogaea	Peanut	EST-SSR	AHS	3,187	Koilkonda et al. 2012
		Genome-SSR	AHGS	6,706	Shirasawa et al. 2012b
		Transposable element	AhTE	1,039	Shirasawa et al. 2012a, 2012b
Trifolium pratense	Red clover	SSR	RCS, TPSSR	7,262	Isobe et al. 2009
Trifolium repens	White clover	EST-SSR	WCS	1,993	Isobe et al. 2012
Eucalyptus camaldulensis	Eucaly	Genome-SSR	EcGAS	4,656	Hirakawa et al. 2011
		EST-SSR	EcES	1,028	Hirakawa et al. 2011

Genetic linkage maps, which were constructed with the DNA markers for seven crops, i.e., tomato, peanut, radish, soybean, red clover, L. japonicus, and strawberry, are also registered in this database (Table 2). The information provided on the genetic linkage maps includes the mapped positions of the various markers. For tomato, pepper, and radish, the DNA markers were mapped on the reference sequences themselves or their relatives.

Table 2 Genetic linkage maps registered in the Kazusa Marker DataBase

Binomial nomenclature	Common name	Map name	No. of linkage groups	No. of mapped loci	Total length (cM)	Mean marker density (cM/loci)	Reference
Solanum lycopersicum	Tomato	Tomato-EXPEN2000	12	2,116	1,503	0.7	Shirasawa et al. 2010a
		AMF2	12	990	1,468	1.5	Shirasawa et al. 2010b
		MMF2	13	637	1,230	2.0	Shirasawa et al. 2010b
Fragaria × ananassa	Strawberry	Integrated map	28	1,861	1,967	1.1	Isobe et al. 2013
Raphanus sativus	Radish	GHRI	9	843	1,129	1.4	Shirasawa et al. 2011
Lotus japonicus		MG-20 × B-129	6	1,155	421	0.4	Hayashi et al. 2001
Glycine max	Soybean	Map1	20	693	2,688	4.0	Hisano et al. 2007
Arachis hypogaea	Peanut	SKF2	21	1,114	2,226	2.0	Shirasawa et al. 2012b
		NYF2	19	326	1,333	4.3	Shirasawa et al. 2012b
		AF5	10	597	544	0.9	Shirasawa et al. 2013a
		BF5	10	798	461	0.6	Shirasawa et al. 2013a
		TF6	20	1,469	1,442	1.0	Shirasawa et al. 2013a
		Integrated map	20	3,693	2,651	0.7	Shirasawa et al. 2013a
Trifolium pratense	Red clover	HR × R130	7	1,714	834	0.5	Isobe et al. 2009

Usage instruction for the Kazusa Marker DataBase

The top page of the Kazusa Marker DataBase represents crops registered in this database. Users can click either “Images”, “Scientific names” in the table, or icons below the table to access pages of each crop, which include “Keyword Search”, “Marker List”, “Reference list”, “Linkage Map”, “Physical Map”, and “Markers on the Genome” depending on crops (see below section). Thorough the “Keyword Search”, marker names, sequence names used for marker designing, and descriptions in comment boxes can be searched. The “Marker List” contained marker types, e.g., genome-SSR and EST-SSR, as described in the below section and Table 1. By selecting the marker type, lists of the markers comprised of “Marker Name”, “Marker Type”, and primer sequences are available. Then, clicking marker names enables users to obtain all information on the markers, e.g., sequence name corresponding the markers with a hyperlink to public DNA sequence databases, map positions (if available), PCR fragment size estimated from the sequence, experimental conditions such as methods on PCR and detections, SSR motif and the repeated number (if SSRs), gel images (if available), and reference articles. The “Markers on the Genome” is available to presume physical genome positions of the markers, if the genome sequences of the crop itself or its relatives are released. Although bulk data download is not supported in the current version, it is available upon request to [e-mail] as well as to us.

Plant species registered in the Kazusa Marker DataBase

Tomato

Tomato (S. lycopersicum), an important fruit crop throughout the world and a model for fresh fruit research, is an autogamous diploid species (2n = 2x = 24) with a genome of 900 Mb, and its sequences have been published by a multinational project consortium (The Tomato Genome Consortium 2012). The database contains information on DNA markers, as well as genetic linkage and physical maps. The DNA markers include both SSR and SNP markers. The SSR markers, 7,599 EST-SSR (TES) and 13,501 genome-SSR (TGS) designed from EST and BAC-end sequences available from a public database (http://solgenomics.net), respectively, were developed to construct an interspecific high-density genetic linkage map (Shirasawa et al. 2010a), on which totals of 648 TES and 634 TGS were mapped. In addition, 674 EST-derived intronic polymorphism markers (TEI) were developed and 151 TEI markers were mapped (Shirasawa et al. 2010a). The SNP markers were also developed from EST and genome sequence data. Each of the EST-derived SNPs was developed from the alignment data of EST sequences derived from at least two tomato lines. From this analysis, 5,607 SNPs were identified in 2,634 contigs, and 793 were mapped on the two genetic linkage maps based on intraspecific crossings (Shirasawa et al. 2010b). On the other hand, the genome-SNPs were discovered by the re-sequencing strategy (Shirasawa et al. 2013b), in which sequence reads for six tomato lines by the ABI-5500xl SOLiD (DRA accession numbers: DRA001017 to DRA001022) were mapped onto the tomato reference genome, SL2.40 (The Tomato Genome Consortium 2012). A total of 1,473,798 genome-SNPs were identified and 1,536 SNPs were employed for genotyping of 663 tomato accessions stocked in gene banks (Shirasawa et al. 2013b). The SNPs are searchable by accession names, the genome positions, or seven categories depending on their effects on gene function, e.g., intergenic, intron, splice site, untranscribed regions, synonymous, missense, and nonsense SNPs. Furthermore, 170,173 of the SNP-derived cleaved amplified polymorphic sequence (CAPS) markers, in which 19 restriction enzymes are employed, are also available. The positions of the DNA markers developed in this study were identified on the published tomato genome (The Tomato Genome Consortium 2012). Then, the DNA markers and the predicted genes in the tomato genome were ordered in parallel based on the physical positions of the reference genome, from which the users can obtain information on the DNA markers and the predicted genes. This tool is very useful to search for DNA markers or loci in genes of interest.

In addition, we have established a portal website for tomato genomics, KatomicsDB (Shirasawa and Hirakawa 2013: http://www.kazusa.or.jp/tomato/), because our group provides not only information on the DNA markers and genetic maps as described above but also inferences of SNP effects on gene functions and sequence data of gene-rich regions in the tomato genome. The KatomicsDB contains links to the marker database described above, a functional SNP database (Hirakawa et al. 2013a: http://plant1.kazusa.or.jp/tomato/), and a database for genome sequences of selected BAC clone mixtures in gene-rich regions (http://www.kazusa.or.jp/tomato_sbm/).

Pepper

Capsicum spp., including C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens, belong to the Solanaceae family, and are widely cultivated for use as vegetables and spices. Like tomato, all these species are autogamous diploids (2n = 2x = 24), while the size of their genomes (~ 3.3 Gb: Moscone et al. 2003) is more than three times larger than that of the other members of Solanaceae, e.g., tomato, potato, and eggplant. The pepper marker database includes mainly Capsicum EST-SSR (CaES) information. A total of 5,751 CaES markers were designed from the 118,060 EST sequences for Capsicum annuum obtained from a public DNA database, GenBank (http://www.ncbi.nlm.nih.gov). The CaES markers were mapped on the tomato genome by in silico analysis based on sequence similarity search, which is recognized as a model for the Solanaceae, because the genome sequence of pepper has not been reported (at the time of writing). However, the genome structures between tomato and pepper are conserved and exhibit a macrosynteny relationship (Wu et al. 2010). Therefore, the positions of the pepper DNA markers and genes on the pepper genome can be speculated by mapping them onto the tomato genome (Shirasawa et al. 2013c). As a result, the positions of 2,245 of the CaES markers were identified on the tomato genome. Among the 2,245 markers, 96 CaES markers were subjected to genotyping analysis of 192 Capsicum accessions, which have been stocked at the Kihara Institute for Biological Research of the Yokohama City University, Japan, to reveal their genetic diversity. The polymorphism information content (PIC) values and allele sizes for the 192 accessions are also available from this database. As additional markers, SNPs detected in the matK and rbcL genes coded in the chloroplast genome, which are known as “barcode” sequences for the identification of species (CBOL Plant Working Group 2009), are also available for the 192 accessions (Accession numbers: AB721552 to AB721935).

Strawberry

Strawberry (F. × ananassa) is a popular fruit cultivated throughout the world, and possesses a complex genome structure due to its octoploid nature (2n = 8x = 56) and its allogamous reproductive system. The genome size of strawberry is estimated to be 692 Mb (Hirakawa et al. 2013b). A wild diploid species, F. vesca, is one of the probable ancestral species, and 240 Mb of its genome has been sequenced (Shulaev et al. 2011). This database includes three types of SSR markers and an integrated map. The SSR markers were designed from EST sequences of not only F. × ananassa but also F. vesca, because a larger number of EST sequences for F. vesca were available from public DNA databases than for F. × ananassa. A total of 3,746 SSRs derived from ESTs for F. vesca, 603 SSRs derived from ESTs for F. × ananassa, and 125 SSRs derived from transcriptomes for F. × ananassa markers were developed and subjected to map constructions (Isobe et al. 2013). Three genetic linkage maps were established using three mapping populations, and integrated into a consensus map consisting of 28 linkage groups with 1,856 loci, the number of which corresponded to the haploid chromosome number of F. × ananassa. In addition to the map constructions, the SSR markers were employed for the genetic diversity analysis of 129 strawberry cultivars. A total of 45 SSR markers were determined to be sufficient to distinguish 129 F. × ananassa lines except for four lines.

Radish

Radish (R. sativus), or Japanese daikon, is an allogamous species due to its self-incompatibility system, and has a diploid genome (2n = 2x = 18), sizing of 526 Mb (Arumuganathan and Earle 1991). The radish is a vegetable crop and a member of the Brassicaceae, to which the genera Arabidopsis and Brassica also belong, but the genomic research on radish has not been progressed as far as for members of the Brassicaceae. The daikon marker database includes mainly EST-SSR markers. A total of 3,800 radish EST-SSR markers (RSS) were developed from 26,606 EST sequences (Accession numbers: FY428055 to FY454660) (Shirasawa et al. 2011). Genetic linkage maps of 630 RSS markers and 213 previously reported markers were obtained from this database. Subsequent comparative analysis of the Raphanus map with the Arabidopsis and B. rapa genomes (The Arabidopsis Genome Initiative 2000, The Brassica rapa Genome Sequencing Project Consortium 2011) revealed the genomic synteny between the two species. Therefore, the radish DNA markers were in silico mapped on the genomes of Arabidopsis and B. rapa to speculate on the positions of the radish DNA markers and genes on the radish genome. This analysis revealed the positions of 3,234 and 3,730 SSR markers on the Arabidopsis and B. rapa genomes, respectively.

Lotus japonicus

L. japonicus is not a crop but is recognized as a model for legume crops and symbiosis research because of its rapid life cycle, fixed genotypes due to autogamous reproduction, simple and compact genome (2n = 2x = 12, 472 Mb), and easy transformability (Handberg and Stougaard 1992). The marker database for Lotus japonicus consists of DNA markers and their linkage map. A total of 1,073 SSR and 82 derived CAPS (dCAPS) markers were developed by comparative analysis of the genome sequences from two L. japonicus strains, Miyakojima MG-20 and Gifu B-129 (Sato et al. 2001). A genetic linkage map of the SSR and dCAPS markers, which consisted of six linkage groups covering 1,155 cM in total, were generated by using an F₂ mapping population derived from a cross between the MG-20 and B-129 (Hayashi et al. 2001). By using this linkage map as a reference, the genome sequences of the MG-20 were anchored to the chromosomes of L. japonicus (Sato et al. 2008: http://www.kazusa.or.jp/lotus/).

Soybean

Because soybean (G. max) is a major crop that is important for oil and protein production, its genome (2n = 2x = 40, genome size of 1.1 Gb) was sequenced in spite of the complexity of its paleopolyploidy (Schmutz et al. 2010). The database includes EST-SSR markers and a genetic linkage map. A total of 6,920 EST-SSR markers were developed from 63,676 publicly available non-redundant soybean ESTs from public databases (Dana-Farber Center Institute; http://compbio.dfci.harvard.edu/tgi/). Among them, 693 SSR marker loci were combined with 242 RFLP, genome-SSR, and phenotypic markers. The resultant maps consisting 20 linkage groups covered 2,700.3 cM in a total length (Hisano et al. 2007). The transferability of the 686 mapped markers was investigated for 24 Glycine accessions. The EST-SSR markers were in silico mapped on the genome sequences to identify the positions of the EST-SSR markers on the soybean genome.

Peanut

Peanut (A. hypogaea), or groundnut, is an autogamous allotetraploid (2n = 4x = 40) legume species with a genome of approximately 2.8 Gb (Arumuganathan and Earle 1991). It is used for food and oil production, and its probable ancestral species have been identified as A. duranensis and A. ipaënsis. The database consists of information on DNA markers and genetic linkage maps. As the DNA markers, a total of 6,706 genome-SSR (AHGS), 3,187 EST-SSR (AHS), and 1,039 transposon insertion length polymorphism markers (AhTE) have been developed from the sequence data collected from SSR-enriched genomic libraries (accession numbers: DH964238 to DH968256) (Shirasawa et al. 2012b), cDNA libraries (accession numbers: FS960760 to FS988327) (Koilkonda et al. 2012), and transposon-enriched genomic libraries (accession numbers: DE998420 to DE998923 and DH968257 to DH968767) (Shirasawa et al. 2012a, 2012b), respectively. The genome- and EST-SSR and transposon markers were subjected to constructions of five genetic linkage maps in Arachis, SKF2 and NYF2 for cultivated peanut (A. hypogaea), AF5 and BF6 for wild diploid relatives (A. duranensis, A. stenosperma, A. ipaënsis, and A. duranensis), and TF6 or A. hypogaea and an artificial amphidiploid (A. ipaënsis × A. duranensis)^4x (Shirasawa et al. 2013a). In addition, the five genetic linkage maps were integrated with 11 published maps from other research groups under collaborations between Japan, Brazil, India, France, the US, and China (Shirasawa et al. 2013a). The EST-SSR markers were employed for the genetic diversity analysis of peanut accessions, including 17 Japanese, 4 American, 2 Indian, and 1 Chinese cultivated lines as well as 6 wild relatives (Koilkonda et al. 2012).

Red clover

Red clover (T. pratense) is an allogamous diploid legume (2n = 2x = 14, genome size of 468 Mb: Arumuganathan and Earle 1991) that is cultivated as a forage crop. The database for red clover consists of information on the DNA markers, RFLP and SSR markers, and genetic linkage map. The RFLP markers were developed to construct a genetic linkage map in red clover. The resultant map contains 157 RFLP markers and covers 535.7 cM in total (Isobe et al. 2003). Subsequently, 7,262 SSR markers were developed from 26,356 EST sequences (Accession numbers: BB902456 to BB928811), and employed to generate a genetic linkage map consisting of 1,434 marker loci covering 868.7 cM in total (Sato et al. 2005). Finally, additional new linkage maps together with the developed genetic linkage maps were integrated into the consensus map with 1,804 marker loci covering 836.6 cM in total (Isobe et al. 2009). The resultant genetic linkage map, i.e., HR × R130, is available from this database.

White clover

White clover (T. repens) is an allogamous allotetraploid legume (2n = 4x = 32, genome size of 999 Mb: Arumuganathan and Earle 1991) widely cultivated as a forage crop. We generated the white clover linkage maps using SSR markers in order to conduct comparative genomics analyses among legume species (Isobe et al. 2012). In this database, a total of 1,993 primers are available for the EST-derived SSR markers. A total of 15,214 EST sequences used for primer construction are also available through the accession numbers FY454661 to FY469874.

Eucalyptus

E. camaldurensis is a diploid species (2n = 2x = 22, genome size of 650 Mb) that is used in the pulp industry. Therefore, the genome sequencing of E. camaldulensis and development of markers have been performed to survey the genetic information and accelerate the process of molecular breeding (Hirakawa et al. 2011: http://www.kazusa.or.jp/eucaly/). The eucalyptus marker database consists of information on 4,656 genome- and 1,028 EST-SSR markers, which were developed from the sequence data of the transcriptome and genome of E. camaldulensis, respectively. The SSR markers were employed for the genetic diversity analysis of six Eucalyptus species, i.e., E. camaldulensis, E. dunnii, E. globules, E. grandis, E. nitens and E. urophylla. The PIC values based on this analysis are also available from this database.

Marker densities in the genetic and physical maps

Marker density, or mean distances between any neighboring marker intervals, would be important information for gene mapping studies of a map-based cloning strategy or genome-wide association studies (GWAS), and for MAS in breeding programs. On the one hand, as for the linkage maps registered in the database, the marker densities between any neighboring loci were varied from 0.4 cM in L. japonicus to 4.3 cM in peanut, and 1.5 cM in average over the 14 genetic maps of the six species (Table 2). On the other hand, as for the physical maps among the ten species, tomato had the highest dense marker loci due to the massive SNP data from the re-sequencing analysis (Table 3). The markers were estimated to locate in every 600 bp interval in the tomato genome. In the remaining nine species, mean physical intervals of any neighboring two markers were ranging from 64 kb in red clover to 573 kb in Capsicum (Table 3). While availability of whole genome sequence data at present were limited to tomato (The Tomato Genome Consortium 2012), strawberry (Hirakawa et al. 2013b), L. japonicus (Sato et al. 2008), soybean (Schmutz et al. 2010), and eucalyptus (Hirakawa et al. 2011) among the plant species registered in the Kazusa Marker DataBase at the time of writing, genome sequences for the other species would be determined in near future with the cooperation of the NGSs as summarized in Genomes OnLine Database (http://www.genomesonline.org). The whole genome sequence data identify the physical genome positions of the DNA markers, and provide useful information for the gene mapping studies as well as MAS.

Table 3 The estimated genome sizes and marker densities of the plant species in the Kazusa Marker DataBase

Binomial nomenclature	Common name	Total no. of markers	The estimated genome size (Mb)	Mean marker density (kb/loci)
Solanum lycopersicum	Tomato	1,500,674	900	0.6
Capsicum annuum	Capsicum	5,751	3,300	573.8
Fragaria × ananassa	Strawberry	4,474	692	154.7
Raphanus sativus	Radish	3,811	526	138.0
Lotus japonicus		1,155	472	408.7
Glycine max	Soybean	7,020	1,115	158.8
Arachis hypogaea	Peanut	10,932	2,813	257.3
Trifolium pratense	Red clover	7,262	468	64.4
Trifolium repens	White clover	1,993	999	501.3
Eucalyptus camaldulensis	Eucaly	5,684	650	114.4

Future directions

Until now, the Kazusa Marker Database includes information on DNA markers, genetic linkage maps, and physical maps for 10 plant species comprised of mainly crops. Because our research groups have been working on more than 25 plant species, the contents of this database will increase when we publish papers on each project. Databases for the DNA markers have been globally established in each crop species, institutes, and countries, a situation which is considered to be undesirable for users. To overcome the problem, an integrated database of the plant genome-related information, i.e., PGDBj (http://pgdbj.jp), has been established (Asamizu et al. 2013), which includes parts of the marker and map information registered in the Kazusa Marker DataBase. In addition, we are planning to provide graphical views of the marker positions on genome sequences or linkage maps by using GBrowse (Stein et al. 2002) or CMap (Fang et al. 2003) from the Kazusa Marker DataBase. The user-friendly interfaces will accelerate comparative analysis of QTL and GWAS loci across the plant species, which will also contribute to gene isolation and molecular breeding.

Note added in proof:

A paper on faba bean (Vicia faba) entitled “Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)” by El-Rodeny et al. was published in Breeding Science. The information on the EST-SSRs and map were added to the Kazusa Marker DataBase.

Acknowledgements

We thank Ms. Mitsuyo Kohara for her technical assistance. This work was supported by the KAKENHI Grant-in-Aid for Scientific Research (C) (24510286), Japan Society for the Promotion of Science; and the Kazusa DNA Research Institute Foundation.

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