Development and mapping of a public reference set of SSR markers in Lolium perenne L.

We report on the characterization and mapping of 76 simple sequence repeat (SSR) markers for Lolium perenne. These markers are publicly available or obtained either from genomic libraries enriched for SSR motifs or L. perenne expressed sequence tag (EST) clones. Four L. perenne mapping populations were used to map the SSR markers. A consensus linkage map of the four mapping populations containing 65 of the SSR markers is presented, together with primer information and a quality score indicating the usefulness of the SSR marker in different populations. The SSR markers identified all seven L. perenne linkage groups.     

Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus

Microsatellite or simple sequence repeat (SSR) markers are routinely used for tagging genes and assessing genetic diversity. In spite of their importance, there are limited numbers of SSR markers available for Brassica crops. A total of 627 new SSR markers (designated BnGMS) were developed based on publicly available genome survey sequences and used to survey polymorphisms among six B. napus cultivars that serve as parents for established populations. Among these SSR markers, 591 (94.3%) successfully amplified at least one fragment and 434 (73.4%) detected polymorphism among the six B. napus cultivars. No correlation was observed between SSR motifs, repeat number or repeat length with polymorphism levels. A linkage map was constructed using 163 newly developed BnGMS marker loci and anchored with 164 public SSRs in a doubled haploid population. These new markers are evenly distributed over all linkage groups (LGs). Given that the majority of these SSRs are derived from bacterial artificial chromosome (BAC) end sequences, they will be useful in the assignment of their cognate BACs to LGs and facilitate the integration of physical maps with genetic maps for genome sequencing in B. napus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow between sea beet (Beta vulgaris ssp. maritima) populations

Molecular markers can be used to estimate gene flow indirectly by monitoring the relative frequency of alleles in adjacent populations. Sea beet (Beta vulgaris ssp. maritima) is a wild plant species found along the coastlines of many European countries and is closely related to cultivated beets. A set of six simple sequence repeat (SSR) markers that are polymorphic in UK populations have been developed for sea beet to assess the problems of indirect measurement of gene flow in these populations.     

Comparison of Linkage Disequilibrium in Elite European Maize Inbred Lines using AFLP and SSR Markers

Application of association mapping to plant breeding populations has the potential to revolutionize plant genetics. The main objectives of this study were to (i) investigate the extent and genomic distribution of linkage disequilibrium (LD) between pairs of amplified fragment length polymorphism (AFLP) markers, (ii) compare these results with those obtained with simple sequence repeat (SSR) markers, and (iii) compare the usefulness of AFLP and SSR markers for genomewide association mapping in plant breeding populations. We examined LD in a cross-section of 72 European elite inbred lines genotyped with 452 AFLP and 93 SSR markers. LD was significant (p < 0.05) for about 15% of the AFLP marker pairs and for about 49% of the SSR marker pairs in each of the two germplasm groups, flint and dent. In both germplasm groups the ratio of linked to unlinked loci pairs in LD was higher for AFLPs than for SSRs. The observation of LD due to linkage for both marker types suggested that genome-wide association mapping should be possible using either AFLPs or SSRs. The results of our study indicated that SSRs should be favored over AFLPs but the opposite applies to populations with a long history of recombination.     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

Long‐term isolation of the coastal plant Calystegia soldanella (Convolvulaceae) in ancient freshwater Lake Biwa,Japan

Lake Biwa is an ancient freshwater lake that was formed approximately 4 Mya and harbours many coastal plants that commonly inhabit the seashore. We used chloroplast DNA haplotype analysis using two spacer sequences and simple sequence repeat (SSR) analysis using eight nuclear microsatellite markers to detect genomic signatures indicating long‐term isolation of inland populations of Calystegia soldanella in Lake Biwa from coastal populations. We used 348 samples from 63 populations for haplotype analysis and 478 samples from 27 populations for SSR analysis covering the inland and coastal distribution of the species. We detected seven haplotypes, and the distribution pattern of these haplotypes was geographically highly structured between Lake Biwa and the coast. Nuclear SSR analysis also supported genetic differentiation between Lake Biwa and coastal populations (analyses of molecular variance, 43%), and the grouping of Lake Biwa and coastal populations by a Neighbour‐joining tree. In addition, genetic diversity of the inland populations (mean H_E = 0.153) was significantly lower than that of coastal populations (mean H_E = 0.328). These results suggested that inland populations at Lake Biwa have been isolated from coastal populations for a very long time. The inland populations most likely experienced a bottleneck effect, resulting in sufficient in situ genetic divergence to clearly distinguish them from coastal populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 51–66.     

Mapping of or, a gene conferring orange color on the inner leaf of the Chinese cabbage (Brassica rapa L. ssp. pekinensis)

The orange inner leaf of the Chinese cabbage is controlled by a single recessive gene (or), which causes abnormal accumulation of carotene. In the present study, an F2 population consisting of 600 individuals was used for mapping or and developing new markers closely linked to this gene. Bulked segregant analysis was performed by screening 435 simple sequence repeat (SSR) markers well-distributed on 10 linkage groups and 16 SSR primers derived from nine bacterial artificial chromosome (BAC) clones. On the basis of linkage analysis, the or gene was mapped in a region covering a total interval of 4.6 centimorgans (cM) between two SSR markers derived from BAC clones AC172873 and AC189246 at the end of linkage group 9, which matches with chromosome 1 of A genome in Chinese cabbage. A genetic map of the or locus was constructed by using five SSR markers and two morphological markers. Three SSR markers were tightly linked to or and two of them, sau (C) 586 and syau19, were located on the same side at distances of 1.6 and 1.3 cM, respectively. The other marker, syau15, was located on the other side at a distance of 3.3 cM. The two morphological markers, orange flower and orange cotyledon (before cotyledon turns green during the germination period), were obtained by visual determination and screening of the differences in the morphological traits between parents and the two segregated F2 populations; the two markers were designated as or-f (orange flower) and or-c (orange cotyledon). It was suggested that these two markers co-segregate with orange inner leaf trait or that the three characters, namely orange inner leaf, orange flower, and orange cotyledon, are determined by the same gene. These markers could be very helpful for marker-assisted selection in Chinese cabbage hybrid breeding programs.     

Mapping 245 SSR markers on the <Emphasis Type="Italic">Vitis vinifera</Emphasis> genome: a tool for grape genetics

The aim of the present work was to develop a microsatellite marker-based map of the Vitis vinifera genome (n=19), useful for genetic studies in this perennial heterozygous species, as SSR markers are highly transferable co-dominant markers. A total of 346 primer pairs were tested on the two parents (Syrah and Grenache) of a full sib population of 96 individuals (S × G population), successfully amplifying 310 markers. Of these, 88.4% markers were heterozygous for at least one of the two parents. A total of 292 primer pairs were then tested on Riesling, the parent of the RS₁ population derived from selfing (96 individuals), successfully amplifying 299 markers among which 207 (62.9%) were heterozygous. Only 6.7% of the markers were homozygous in all three genotypes, stressing the interest of such markers in grape genetics. Four maps were constructed based on the segregation of 245 SSR markers in the two populations. The Syrah map was constructed from the segregations of 177 markers that could be ordered into 19 linkage groups (total length 1,172.2 cM). The Grenache map was constructed with the segregations of 178 markers that could be ordered into 18 linkage groups (total length 1,360.6 cM). The consensus S × G map was constructed with the segregations of 220 markers that were ordered into 19 linkage groups (total length 1,406.1 cM). One hundred and eleven markers were scored on the RS₁ population, among them 27 that were not mapped using the S × G map. Out of these 111 markers, 110 allowed to us to construct a map of a total length of 1,191.7 cM. Using these four maps, the genome length of V. vinifera was estimated to be around 2,200 cM. The present work allowed us to map 123 new SSR markers on the V. vinifera genome that had not been ordered in a previous SSR-based map (Riaz et al. 2004), representing an average of 6.5 new markers per linkage group. Any new SSR marker mapped is of great potential usefulness for many applications such as the transfer of well-scattered markers to other maps for QTL detection, the use of markers in specific regions for the fine mapping of genes/QTL, or for the choice of markers for MAS.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Mapping of a broad-spectrum brown planthopper resistance gene, <Emphasis Type="Italic">Bph3</Emphasis>, on rice chromosome 6

The brown planthopper (BPH) is one of the most destructive insect pests of rice in Thailand. We performed a cluster analysis that revealed the existence of four groups corresponding to the variation of virulence against BPH resistance genes in 45 BPH populations collected in Thailand. Rice cultivars Rathu Heenati and PTB33, which carry Bph3, showed a broad-spectrum resistance against all BPH populations used in this study. The resistant gene Bph3 has been extensively studied and used in rice breeding programs against BPH; however, the chromosomal location of Bph3 in the rice genome has not yet been determined. In this study, a simple sequence repeat (SSR) analysis was performed to identify and localize the Bph3 gene derived from cvs. Rathu Heenati and PTB33. For mapping of the Bph3 locus, we developed two backcross populations, BC₁F₂ and BC₃F₂, from crosses of PTB33 × RD6 and Rathu Heenati × KDML105, respectively, and evaluated these for BPH resistance. Thirty-six polymorphic SSR markers on chromosomes 4, 6 and 10 were used to survey 15 resistant (R) and 15 susceptible (S) individuals from the backcross populations. One SSR marker, RM190, on chromosome 6 was associated with resistance and susceptibility in both backcross populations. Additional SSR markers surrounding the RM190 locus were also examined to define the location of Bph3. Based on the linkage analysis of 208 BC₁F₂ and 333 BC₃F₂ individuals, we were able to map the Bph3 locus between two flanking SSR markers, RM589 and RM588, on the short arm of chromosome 6 within 0.9 and 1.4 cM, respectively. This study confirms both the location of Bph3 and the allelic relationship between Bph3 and bph4 on chromosome 6 that have been previously reported. The tightly linked SSR markers will facilitate marker-assisted gene pyramiding and provide the basis for map-based cloning of the resistant gene.     

Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers

Molecular‐marker‐aided evaluation of germplasm plays an important role in defining the genetic diversity of plant genotypes for genetic and population improvement studies. A collection of African cassava landraces and elite cultivars was analysed for genetic diversity using 20 amplified fragment length polymorphic (AFLP) DNA primer combinations and 50 simple sequence repeat (SSR) markers. Within‐population diversity estimates obtained with both markers were correlated, showing little variation in their fixation index. The amount of within‐population variation was higher for landraces as illustrated by both markers, allowing discrimination among accessions along their geographical origins, with some overlap indicating the pattern of germplasm movement between countries. Elite cultivars were grouped in most cases in agreement with their pedigree and showed a narrow genetic variation. Both SSR and AFLP markers showed some similarity in results for the landraces, although SSR provided better genetic differentiation estimates. Genetic differentiation (Fst) in the landrace population was 0.746 for SSR and 0.656 for AFLP. The molecular variance among cultivars in both populations accounted for up to 83% of the overall variation, while 17% was found within populations. Gene diversity (H_e) estimated within each population varied with an average value of 0.607 for the landraces and 0.594 for the elite lines. Analyses of SSR data using ordination techniques identified additional cluster groups not detected by AFLP and also captured maximum variation within and between both populations. Our results indicate the importance of SSR and AFLP as efficient markers for the analysis of genetic diversity and population structure in cassava. Genetic differentiation analysis of the evaluated populations provides high prospects for identifying diverse parental combinations for the development of segregating populations for genetic studies and the introgression of desirable genes from diverse sources into the existing genetic base.     

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål)

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens St?l) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.     

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.     

Isolation, characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)

The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

Exploiting rice–sorghum synteny for targeted development of EST-SSRs to enrich the sorghum genetic linkage map

The sequencing and detailed comparative functional analysis of genomes of a number of select botanical models open new doors into comparative genomics among the angiosperms, with potential benefits for improvement of many orphan crops that feed large populations. In this study, a set of simple sequence repeat (SSR) markers was developed by mining the expressed sequence tag (EST) database of sorghum. Among the SSR-containing sequences, only those sharing considerable homology with rice genomic sequences across the lengths of the 12 rice chromosomes were selected. Thus, 600 SSR-containing sorghum EST sequences (50 homologous sequences on each of the 12 rice chromosomes) were selected, with the intention of providing coverage for corresponding homologous regions of the sorghum genome. Primer pairs were designed and polymorphism detection ability was assessed using parental pairs of two existing sorghum mapping populations. About 28% of these new markers detected polymorphism in this 4-entry panel. A subset of 55 polymorphic EST-derived SSR markers were mapped onto the existing skeleton map of a recombinant inbred population derived from cross N13 × E 36-1, which is segregating for Striga resistance and the stay-green component of terminal drought tolerance. These new EST-derived SSR markers mapped across all 10 sorghum linkage groups, mostly to regions expected based on prior knowledge of rice–sorghum synteny. The ESTs from which these markers were derived were then mapped in silico onto the aligned sorghum genome sequence, and 88% of the best hits corresponded to linkage-based positions. This study demonstrates the utility of comparative genomic information in targeted development of markers to fill gaps in linkage maps of related crop species for which sufficient genomic tools are not available.     

Estimation of population structure in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] using allozyme and microsatellite markers

Characterizing population structure using neutral markers is an important first step in association genetic studies in order to avoid false associations between phenotypes and genotypes that may arise from non-selective demographic factors. Population structure was studied in a wide sample of ∼1,300 coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] trees from Washington and Oregon. This sample is being used for association mapping between cold hardiness and phenology phenotypes and single-nucleotide polymorphisms in adaptive-trait candidate genes. All trees were genotyped for 25 allozyme and six simple sequence repeat (SSR) markers using individual megagametophytes. Population structure analysis was done separately for allozyme and SSR markers, as well as for both datasets combined. The parameter of genetic differentiation (θ or F _ST) was standardized to take into account high within-population variation in the SSR loci and to allow comparison with allozyme loci. Genetic distance between populations was positively and significantly correlated with geographic distance, and weak but significant clinal variation was found for a few alleles. Although the STRUCTURE simulation analysis inferred the same number of populations as used in this study and as based on previous analysis of quantitative adaptive trait variation, clustering among populations was not significant. In general, results indicated weak differentiation among populations for both allozyme and SSR loci (θ _s = 0.006–0.059). The lack of pronounced population subdivision in the studied area should facilitate association mapping in this experimental population, but we recommend taking the STRUCTURE analysis and population assignments for individual trees (Q-matrix) into account in association mapping.     

Identification of simple sequence repeat markers for utilizing wide-compatibility genes in inter-subspecific hybrids in rice (Oryza sativa L.)

Although pronounced heterosis in inter-subspecific hybrids was known in rice for a long time, its utilization for hybrid rice breeding has been limited due to their hybrid sterility (HS). For the last two decades, however, a few inter-subspecific hybrids have been developed by incorporating wide-compatibility genes (WCG) that resolve HS, into parental lines of these inter-subspecific hybrids. For effective use of WCG, it is necessary to find convenient markers linked to WCG of practical importance. In this paper, initially a set of simple sequence repeat (SSR) markers in the vicinity of known WCG loci identified based on comparative linkage maps have been surveyed in a population derived from the three-way cross- IR36/Dular//Akihikari, where a known donor of WCG Dular was crossed to a representative indica and japonica cultivar. Of the five parental polymorphic markers, RM253 and RM276 were found to be closely linked to the WCG locus S5 at a distance of 3.0 and 2.8 cM, respectively. Later, loci for HS were examined in three F₂ populations derived from inter-subspecific crosses, with same set of SSR markers. The locus S8 was confirmed to have major influence on HS in the F₂ population derived from CHMRF-1/Taichung65 since two SSR markers in its vicinity, RM412 and RM141, co-segregated with HS at a map distance of 7.6 and 4.8 cM, respectively. In the F₂ population derived from the cross BPT5204/Taipei309, three SSR markers in the vicinity of S5, RM50, RM276 and RM136 co-segregated with HS at a map distance of 4.2, 3.2 and 7.8 cM, respectively. In the third F₂ population derived from Swarna/Taipei309, the SSR markers in the vicinity of S5, RM225, RM253, RM50, RM276 and RM136 were identified to co-segregate with HS at a map distance of 3.2, 2.6, 3.4, 2.6 and 6.6 cM, respectively. These results indicated a clear picture of WCG in Dular as well as the predominant role of HS alleles at S5 locus. The identified SSR markers are expected to be used for incorporation of WCG into parental lines in hybrid rice breeding to solve HS in inter-subspecific hybrids.S.P. Singh , R.M. Sundaram contributed equally     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.