A microsatellite map of white clover

The white clover (Trifolium repens) nuclear genome (n=2x=16) is an important yet under-characterised genetic environment. We have developed simple sequence repeat (SSR) genetic markers for the white clover genome by mining an expressed sequence tag (EST) database and by isolation from enriched genomic libraries. A total of 2,086 EST-derived SSRs (EST-SSRs) were identified among 26,480 database accessions. Evaluation of 792 EST-SSR primer pairs resulted in 566 usable EST-SSRs. Of these, 335 polymorphic EST-SSRs, used in concert with 30 genomic SSRs, detected 493 loci in the white clover genome using 92 F₁ progeny from a pair cross between two highly heterozygous genotypes—364/7 and 6525/5. Map length, as estimated using the joinmap algorithm, was 1,144 cM and spanned all 16 homologues. The R (red leaf) locus was mapped to linkage group B1 and is tightly linked to the microsatellite locus prs318c. The eight homoeologous pairs of linkage groups within the white clover genome were identified using 96 homoeologous loci. Segregation distortion was detected in four areas (groups A1, D1, D2 and H2). Marker locus density varied among and within linkage groups. This is the first time EST-SSRs have been used to build a whole-genome functional map and to describe subgenome organisation in an allopolyploid species, and T. repens is the only Trifolieae species to date to be mapped exclusively with SSRs. This gene-based microsatellite map will enable the resolution of quantitative traits into Mendelian characters, the characterisation of syntenic relationships with other genomes and acceleration of white clover improvement programmes.     

Characterization of the Soybean Genome Using EST-derived Microsatellite Markers

We generated a high-density genetic linkage map of soybean usingexpressed sequence tag (EST)-derived microsatellite markers.A total of 6920 primer pairs (10.9%) were designed to amplifysimple sequence repeats (SSRs) from 63 676 publicly availablenon-redundant soybean ESTs. The polymorphism of two parent plants,the Japanese cultivar ‘Misuzudaizu’ and the Chineseline ‘Moshidou Gong 503’, were examined using 10%polyacrylamide gel electrophoresis. Primer pairs showing polymorphismwere then used for genotyping 94 recombinant inbred lines (RILs)derived from a cross between the parents. In addition to previouslyreported markers, 680 EST-derived microsatellite markers wereselected and subjected to linkage analysis. As a result, 935marker loci were mapped successfully onto 20 linkage groups,which totaled 2700.3 cM in length; 693 loci were detected usingthe 668 EST-derived microsatellite markers developed in thisstudy, the other 242 loci were detected with 105 RFLP markers,136 genome-derived microsatellite markers, and one phenotypicmarker. We examined allelic variation among 23 soybean cultivars/linesand a wild soybean line using 668 mapped EST-derived microsatellitemarkers (corresponding to 686 marker loci), in order to determinethe transferability of the markers among soybean germplasms.A limited degree of macrosynteny was observed at the segmentallevel between the genomes of soybean and the model legume Lotusjaponicus, which suggests that considerable genome shufflingoccurred after separation of the species and during establishmentof the paleopolyploid soybean genome.     

Genome Wide Characterization of Short Tandem Repeat Markers in Sweet Orange (Citrus sinensis)

Sweet orange (Citrus sinensis) is one of the major cultivated and most-consumed citrus species. With the goal of enhancing the genomic resources in citrus, we surveyed, developed and characterized microsatellite markers in the ≈347 Mb sequence assembly of the sweet orange genome. A total of 50,846 SSRs were identified with a frequency of 146.4 SSRs/Mbp. Dinucleotide repeats are the most frequent repeat class and the highest density of SSRs was found in chromosome 4. SSRs are non-randomly distributed in the genome and most of the SSRs (62.02%) are located in the intergenic regions. We found that AT-rich SSRs are more frequent than GC-rich SSRs. A total number of 21,248 SSR primers were successfully developed, which represents 89 SSR markers per Mb of the genome. A subset of 950 developed SSR primer pairs were synthesized and tested by wet lab experiments on a set of 16 citrus accessions. In total we identified 534 (56.21%) polymorphic SSR markers that will be useful in citrus improvement. The number of amplified alleles ranges from 2 to 12 with an average of 4 alleles per marker and an average PIC value of 0.75. The newly developed sweet orange primer sequences, their in silico PCR products, exact position in the genome assembly and putative function are made publicly available. We present the largest number of SSR markers ever developed for a citrus species. Almost two thirds of the markers are transferable to 16 citrus relatives and may be used for constructing a high density linkage map. In addition, they are valuable for marker-assisted selection studies, population structure analyses and comparative genomic studies of C. sinensis with other citrus related species. Altogether, these markers provide a significant contribution to the citrus research community.     

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.     

Distribution and characterization of simple sequence repeats in Gossypium raimondii genome

Simple sequence repeats (SSRs) can be derived from the complete genome sequence. These markers are important for gene mapping as well as marker-assisted selection (MAS). To develop SSRs for cotton gene mapping, we selected the complete genome sequence of Gossypium raimondii, which consisted of 4447 non-redundant scaffolds. Out of 775.2 Mb sequence examined, a total of 136,345 microsatellites were identified with a density of 5.69 kb per SSR in the G. raimondii genome leading to development of 112,177 primer pairs. The distributions of SSRs in the genome were non-random. Among the different motifs ranging from 1 to 6 bp, penta-nucleotide repeats were most abundant (30.5%), followed by tetra-nucleotide repeats (18.2%) and di-nucleotide repeats (16.9%). Among all identified 457 motif types, the most frequently occurring repeat motifs were poly-AT/TA, which accounted for 79.8% of the total di-nt SSRs, followed by AAAT/TTTA with 51.5% of the total tetra-nucleotede. Further, 18,834 microsatellites were detected from the protein-coding genes, and the frequency of gene containing SSRs was 46.0% in 40,976 genes of G. raimondii. These genome-based SSRs developed in the present study will lay the groundwork for developing large numbers of SSR markers for genetic mapping, gene discovery, genetic diversity analysis, and MAS breeding in cotton.     

First RFLP linkage map of red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) based on cDNA probes and its transferability to other red clover germplasm

We constructed a genetic linkage map of red clover (Trifolium pratense L., 2n=2x=14) using RFLP markers from cDNA probes of a backcrossed mapping population, and investigated the transferability of the markers to other red clover germplasm. The map contains 157 RFLP markers and one morphological marker on seven linkage groups. The total map distance was 535.7 cM and the average distance between two markers was 3.4 cM. All of the cDNA probes of the map were hybridized to the fragments of genomic DNA from 12 plants derived from three varieties, and 87% of the cDNA probes detected polymorphic bands that corresponded to those of mapping parents. This result indicated that RFLP markers on the present map were transferable to the genome analysis of other red clover germplasm. This is the first report to construct a linkage map of Trifolium species; it should provide fundamental and useful genetic information relevant to the breeding of red clover and genus Trifolium.Communicated by H.C. Becker     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

Mapping quantitative trait loci controlling early growth in a (longleaf pine × slash pine) × slash pine BC<Subscript>1</Subscript> family

Random amplified polymorphic DNA (RAPD) markers were employed to map the genome and quantitative trait loci controlling the early growth of a pine hybrid F₁ tree (Pinus palustris Mill. 2 P. elliottii Engl.) and a recurrent slash pine tree (P. elliottii Engl.) in a (longleaf pine 2 slash pine) 2 slash pine BC₁ family consisting of 258 progeny. Of the 150 hybrid F₁ parent-specific RAPD markers, 133 were mapped into 17 linkage groups covering a genetic distance of 1,338.2 cM. Of the 116 slash pine parent-specific RAPD markers, 83 were mapped into 19 linkage groups covering a genetic distance of 994.6 cM. A total of 11 different marker intervals were found to be significantly associated with 13 of the 20 traits on height and diameter growth using MAPMAKER/QTL. Nine of the eleven marker intervals were unique to the hybrid parent 488 genome, and two were unique to the recurrent parent 18-27 genome. The amount of phenotypic variance explained by the putative QTLs ranged from 3.6% to 11.0%. Different QTLs were detected at different ages. Two marker intervals from the hybrid parent 488 were found to have QTL by environment interactions.     

The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding

This study reports on the construction of the first genetic maps of subterranean clover (Trifolium subterraneum L.), a diploid, inbreeding annual pasture legume, and alignment of its linkage groups with those of red clover (T. pratense L.) and Medicago truncatula Gaertn. Transferability of red and white clover (T. repens L.) simple sequence repeat (SSR) markers to subterranean clover was observed. A total of 343 SSR loci were mapped into eight subterranean clover linkage groups, with 6?C31 loci per linkage group and 27 loci with similar locations between two distinct F ₂ mapping populations. Phenotypic data obtained for flowering time, content of three isoflavonoids (formononetin, genistein and biochanin A), hardseededness, leaf markings, calyx pigmentation and hairiness of stems were analyzed, together with genotypic data. Genomic intervals influencing each trait were assigned to one to three chromosome regions, accounting for 5.5?C59.8% of the phenotypic variance. Syntenic relationships were observed among subterranean clover, red clover and Medicago truncatula genomes. Comparisons of loci shared between the three species indicated that at least two chromosomal regions have undergone duplications in the subterranean clover genome. Candidate genes for isoflavone content were identified using M. truncatula as a reference genome. Synteny-based segmentation observed in Brassicaceae chromosomes helped to account for the apparent segmental-based relationship between the clover genomes, particularly within the subterranean clover lines. The proposed segmental nature of clover genome could account for the extensive variation observed between the parental genotypes, while not preventing production of fertile intercrosses.     

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese Bayberry (Myrica rubra)

ABSTRACT: BACKGROUND: Chinese bayberry (Myrica rubra Sieb. et Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. RESULTS: The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs ([greater than or equal to]5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. CONCLUSION: Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.     

Characterization of genome-wide simple sequence repeats and application in interspecific genetic map integration in kiwifruit

Simple sequence repeats (SSRs) have been widely used in the construction of linkage maps, quantitative trait loci (QTLs) mapping, and marker-assisted selection (MAS). The availability of the sequenced Actinidia chinensis (kiwifruit) genome allows for the inexpensive and efficient development of microsatellite markers. In this study, a total of 49,067 SSRs were identified and characterized in the genome sequences of kiwifruit. Dinucleotide repeats are the most abundant SSRs, with the AG/TC motif accounting for 44.2 % of all SSRs in the genome. Fifty-five newly derived SSRs, together with 46 previously available SSRs, were integrated into linkage maps of an interspecific kiwifruit population. In addition, eight sex-linked SSR markers (including one previously published SSR) were mapped in the sex-related region on the LG25, suggesting that recombination is partially suppressed to maintain dioecy in kiwifruit. The SSRs developed from this study are a valuable resource for kiwifruit genetics and will contribute to the use of MAS in early sex determination of dioecious plant breeding.     

Discovery and experimental analysis of microsatellites in an oil woody plant Camellia chekiangoleosa

Oil camellia trees are important woody plants for the production of high-quality cooking oil. On the contrary to their economic importance, their genetic and genomic resources are very limited, which greatly hamper the genetic studies on oil camellia trees. Microsatellites or simple sequence repeats (SSRs) have great value in many aspects of genetic analyses due to their high polymorphism and codominant inheritance. In this study, we report the large-scale development and characterization of SSR markers derived from genomic sequences of Camellia chekiangoleosa by high-throughput pyrosequencing technology. A total of 1,091,393 genomic shotgun reads were generated using Roche 454 FLX sequencer, the average read length was 319 bp, and the total sequence throughput was 347.9 Mb. These sequences were assembled into 35,315 contigs with total length of 14.8 Mb and the N50 contig size of 770 bp. By analyzing with microsatellite (MISA), a total of 5,844 perfect microsatellites were detected from the assembled sequences. Among them, tetranucleotide repeats were found to be the most frequent microsatellites in the genome of C. chekiangoleosa, and all the dominant repeat motifs for different types of SSRs were detected to be rich in A/T. Experimental analysis with 900 SSR primer pairs revealed that 66 % of them succeeded in PCR amplification. Further investigation with 345 SSR primer pairs showed that a relatively high percentage of primers amplified polymorphic loci (31.9 %). Experimental data also revealed that, overall, long microsatellite repeats (>20 bp) were more variable than the short ones (<20 bp) in the genome of oil camellia tree.     

Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing

Opium poppy (Papaver somniferum L.) is an important pharmaceutical crop with very few genetic marker resources. To expand these resources, we sequenced genomic DNA using pyrosequencing technology and examined the DNA sequences for simple sequence repeats (SSRs). A total of 1,244,412 sequence reads were obtained covering 474 Mb. Approximately half of the reads (52 %) were assembled into 166,724 contigs representing 105 Mb of the opium poppy genome. A total of 23,283 non-redundant SSRs were identified in 18,944 contigs (11.3 % of total contigs). Trinucleotide and tetranucleotide repeats were the most abundant SSR repeats, accounting for 49.0 and 27.9 % of all SSRs, respectively. The AAG/TTC repeat was the most abundant trinucleotide repeat, representing 19.7 % of trinucleotide repeats. Other SSR repeat types were AT-rich. A total of 23,126 primer pairs (98.7 % of total SSRs) were designed to amplify SSRs. Fifty-three genomic SSR markers were tested in 37 opium poppy accessions and seven Papaver species for determination of polymorphism and transferability. Intraspecific polymorphism information content (PIC) values of the genomic SSR markers were intermediate, with an average 0.17, while the interspecific average PIC value was slightly higher, 0.19. All markers showed at least 88 % transferability among related species. This study increases sequence coverage of the opium poppy genome by sevenfold and the number of opium poppy-specific SSR markers by sixfold. This is the first report of the development of genomic SSR markers in opium poppy, and the genomic SSR markers developed in this study will be useful in diversity, identification, mapping and breeding studies in opium poppy.     

Development and genetic mapping of microsatellite markers from whole genome shotgun sequences in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The availability of whole genome shotgun sequences (WGSs) in Brassica oleracea provides an unprecedented opportunity for development of microsatellite or simple sequence repeat (SSR) markers for genome analysis and genetic improvement in Brassica species. In this study, a total of 56,465 non-redundant SSRs were identified from the WGSs in B. oleracea, with dinucleotide repeats being the most abundant, followed by tri-, tetra- and pentanucleotide repeats. From these, 1,398 new SSR markers (designated as BoGMS) with repeat length ≥25 bp were developed and used to survey polymorphisms with a panel of six rapeseed varieties, which is the largest number of SSR markers developed for the C genome in a single study. Of these SSR markers, 752 (69.5%) showed polymorphism among the six varieties. Of these, 266 markers that showed clear scorable polymorphisms between B. napus varieties No. 2127 and ZY821 were integrated into an existing B. napus genetic linkage map. These new markers are preferentially distributed on the linkage groups in the C genome, and significantly increased the number of SSR markers in the C genome. These SSR markers will be very useful for gene mapping and marker-assisted selection of important agronomic traits in Brassica species.     

Genome-Wide Comparative Analysis of Microsatellites in Pineapple

Pineapple (Ananas comosus (L.) Merrill) is the second most important tropical fruit in term of international trade. The availability of whole genomic sequences and expressed sequence tags (ESTs) offers an opportunity to identify and characterize microsatellite or simple sequence repeat (SSR) markers in pineapple. A total of 278,245 SSRs and 41,962 SSRs with an overall density of 728.57 SSRs/Mb and 619.37 SSRs/Mb were mined from genomic and ESTs sequences, respectively. 5′-untranslated regions (5′-UTRs) had the greatest amount of SSRs, 3.6–5.2 fold higher SSR density than other regions. For repeat length, 12 bp was the predominant repeat length in both assembled genome and ESTs. Class I SSRs were underrepresented compared with class II SSRs. For motif length, dinucleotide repeats were the most abundant in genomic sequences, whereas trinucleotides were the most common motif in ESTs. Tri- and hexanucleotides of total SSRs were more prevalent in ESTs than in the whole genome. The SSR frequency decreased dramatically as repeat times increased. AT was the most frequent single motif across the entire genome while AG was the most abundant motif in ESTs. Across six examined plant species, the pineapple genome displayed the highest density, substantially more than the second-place cucumber. Annotation and expression analyses were also conducted for genes containing SSRs. This thorough analysis of SSR markers in pineapple provided valuable information on the frequency and distribution of SSRs in the pineapple genome. This genomic resource will expedite genomic research and pineapple improvement.     

Genome-Wide Comparative Analyses of Microsatellites in Papaya

Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic and universally distributed in eukaryotes. SSRs have been used extensively as sequence tagged markers in genetic studies. Recently, the functional and evolutionary importance of SSRs has received considerable attention. Here we report the mining and characterization of the SSRs in papaya genome. We analyzed SSRs from 277.4 Mb of whole genome shotgun (WGS) sequences, 51.2 Mb bacterial artificial chromosome (BAC) end sequences (BES), and 13.4 Mb expressed sequence tag (EST) sequences. The papaya SSR density was one SSR per 0.7 kb of DNA sequence in the WGS, which was higher than that in BES and EST sequences. SSR abundance was dramatically reduced as the repeat length increased. According to SSR motif length, dinucleotide repeats were the most common motif in class I, whereas hexanucleotides were the most copious in class II SSRs. The tri- and hexanucleotide repeats of both classes were greater in EST sequences compared to genomic sequences. In class I SSR, AT and AAT were the most frequent motifs in BES and WGS sequences. By contrast, AG and AAG were the most abundant in EST sequences. For SSR marker development, 9,860 primer pairs were surveyed for amplification and polymorphism. Successful amplification and polymorphic rates were 66.6% and 17.6%, respectively. The highest polymorphic rates were achieved by AT, AG, and ATG motifs. The genome wide analysis of microsatellites revealed their frequency and distribution in papaya genome, which varies among plant genomes. This complete set of SSRs markers throughout the genome will assist diverse genetic studies in papaya and related species.     

Development of simple sequence repeat DNA markers and their integration into a barley linkage map

Simple sequence repeats (SSRs), or microsatellites, are a new class of PCR-based DNA markers for genetic mapping. The objectives of the present study were to develop SSR markers for barley and to integrate them into an existing barley linkage map. DNA sequences containing SSRs were isolated from a barley genomic library and from public databases. It is estimated that the barley genome contains one (GA)_n repeat every 330 kb and one (CA)_n repeat every 620 kb. A total of 45 SSRs were identified and mapped to seven barley chromosomes using doubled-haploid lines and/or wheat-barley addition-line assays. Segregation analysis for 39 of these SSRs identified 40 loci. These 40 markers were placed on a barley linkage map with respect to 160 restriction fragment length polymorphism (RFLP) and other markers. The results of this study demonstrate the value of SSRs as markers in genetic studies and breeding research in barley.     

Microsatellite mapping of <Emphasis Type="Italic">Ae. speltoides</Emphasis> and map-based comparative analysis of the S,G, and B genomes of Triticeae species

The first microsatellite linkage map of Ae. speltoides Tausch (2n = 2x = 14, SS), which is a wild species with a genome closely related to the B and G genomes of polyploid wheats, was developed based on two F₂ mapping populations using microsatellite (SSR) markers from Ae. speltoides, wheat genomic SSRs (g-SSRs) and EST-derived SSRs. A total of 144 different microsatellite loci were mapped in the Ae. speltoides genome. The transferability of the SSRs markers between the related S, B, and G genomes allowed possible integration of new markers into the T. timopheevii G genome chromosomal maps and map-based comparisons. Thirty-one new microsatellite loci assigned to the genetic framework of the T. timopheevii G genome maps were composed of wheat g-SSR (genomic SSR) markers. Most of the used Ae. speltoides SSRs were mapped onto chromosomes of the G genome supporting a close relationship between the G and S genomes. Comparative microsatellite mapping of the S, B, and G genomes demonstrated colinearity between the chromosomes within homoeologous groups, except for intergenomic T6A^tS.1G, T4AL.5AL.7BS translocations. A translocation between chromosomes 2 and 6 that is present in the T. aestivum B genome was found in neither Ae. speltoides nor in T. timopheevii. Although the marker order was generally conserved among the B, S, and G genomes, the total length of the Ae. speltoides chromosomal maps and the genetic distances between homoeologous loci located in the proximal regions of the S genome chromosomes were reduced compared with the B, and G genome chromosomes.     

Establishment of a microsatellite panel covering the sugi (Cryptomeria japonica) genome,and its application for localization of a male-sterile gene (ms-2)

A microsatellite (simple sequence repeat; SSR) panel for Cryptomeria japonica was established, using both newly developed and previously reported markers, to construct a frame of linkage map and facilitate localization of important genes in this species. In this study, 32 new expressed sequence tag SSRs (EST-SSRs) and 12 new genomic SSRs (gSSRs) were developed. Their average polymorphism information content (PIC) values were 0.549 and 0.776, respectively. The markers were mapped onto a high-density linkage map. The SSR panel that was established to cover the genome consisted of 46 gSSRs and 47 EST-SSRs. The number of SSR markers in each linkage group, the average map distance between loci within a linkage group, and the average PIC values in each linkage group ranged from 6 to 13, 6.77 to 19.88 and 0.475 to 0.712, respectively. The utility of the SSR panel was tested by using it to localize a male-sterile gene, ms-2. The ms-2 locus was successfully localized on the linkage group 5 using 33 SSR markers (three SSRs per linkage group) which were selected from the SSR panel based on the existence of polymorphisms and the absence of null alleles in the mapping population for ms-2. A partial linkage map surrounding the ms-2 locus was then constructed using a further 57 single nucleotide polymorphisms and three SSRs, to facilitate future development of markers tightly linked to the ms-2 locus for use in marker-assisted selection. The SSR panel covering the C. japonica genome will allow researchers to localize important genes efficiently.