A microsatellite linkage map for the cultivated strawberry (Fragaria?×?ananassa) suggests extensive regions of homozygosity in the genome that may have resulted from breeding and selection

The linkage maps of the cultivated strawberry, Fragaria × ananassa (2n = 8x = 56) that have been reported to date have been developed predominantly from AFLPs, along with supplementation with transferrable microsatellite (SSR) markers. For the investigation of the inheritance of morphological characters in the cultivated strawberry and for the development of tools for marker-assisted breeding and selection, it is desirable to populate maps of the genome with an abundance of transferrable molecular markers such as microsatellites (SSRs) and gene-specific markers. Exploiting the recent release of the genome sequence of the diploid F. vesca, and the publication of an extensive number of polymorphic SSR markers for the genus Fragaria, we have extended the linkage map of the ‘Redgauntlet’ × ‘Hapil’ (RG × H) mapping population to include a further 330 loci, generated from 160 primer pairs, to create a linkage map for F. × ananassa containing 549 loci, 490 of which are transferrable SSR or gene-specific markers. The map covers 2140.3 cM in the expected 28 linkage groups for an integrated map (where one group is composed of two separate male and female maps), which represents an estimated 91% of the cultivated strawberry genome. Despite the relative saturation of the linkage map on the majority of linkage groups, regions of apparent extensive homozygosity were identified in the genomes of ‘Redgauntlet’ and ‘Hapil’ which may be indicative of allele fixation during the breeding and selection of modern F. × ananassa cultivars. The genomes of the octoploid and diploid Fragaria are largely collinear, but through comparison of mapped markers on the RG × H linkage map to their positions on the genome sequence of F. vesca, a number of inversions were identified that may have occurred before the polyploidisation event that led to the evolution of the modern octoploid strawberry species.     

A genetic linkage map of the cultivated strawberry (Fragaria × ananassa) and its comparison to the diploid Fragaria reference map

The cultivated strawberry, Fragaria × ananassa, is the most economically-important soft-fruit species, but few practical molecular tools for the purpose of marker assisted selection currently exist. As a precursor to the development of such tools, a genetic linkage map was developed from a F₁ population comprising 174 seedlings derived from a cross between two F. × ananassa cultivars, ‘Redgauntlet’ × ‘Hapil’. The resultant map is composed of 315 molecular markers—218 microsatellites, 11 gene-specific markers and 86 AFLP and RAPD markers—and spans 3,116 cM. In total, 69 linkage group fragments were recovered, more than the 56 linkage groups expected for the cultivated strawberry, however, all fragments contained a transferable marker that could be associated with one of 56 linkage group scaffolds. The female (Redgauntlet) and male (Hapil) linkage maps are composed, respectively of 170 loci in 32 linkage groups covering 1,675.3 cM and 182 loci in 37 linkage groups covering 1,440.7 cM, with 37 markers common to both maps. The maximum number of markers in one linkage group was 15, the minimum was two. All linkage groups resolved contained at least one transferable marker (SSR or gene-specific) that had been mapped on the diploid Fragaria reference map (FV × FB), and therefore all linkage groups could be identified as homologous to one of the seven diploid Fragaria linkage groups. When marker order was compared to the diploid Fragaria reference map, effectively complete colinearity was observed. However, the occurrence of duplicated loci on homologues of linkage groups FG1 and FG6 provided evidence of a putative chromosomal duplication or translocation event in Fragaria. The development of this linkage map will facilitate the study and dissection of QTL associated with traits of economic importance such as disease resistance and fruit quality, and provides a foundation for the development of markers for the purpose of marker assisted breeding and selection in the cultivated strawberry, F. × ananassa.     

Development and bin mapping of strawberry genic-SSRs in diploid <Emphasis Type="Italic">Fragaria</Emphasis> and their transferability across the Rosoideae subfamily

Cultivated strawberry (Fragaria × ananassa) together with other economically important genera such as Rosa (roses) and Rubus (raspberry and blackberry) belongs to the subfamily Rosoideae. There is increasing interest in the development of transferable markers to allow genome comparisons within the Rosaceae family. In this report, 122 new genic microsatellite (SSR) markers have been developed from cultivated strawberry and its diploid ancestor Fragaria vesca. More than 77% of the sequences from which the markers were developed show significant homology to known or predicted proteins and more than 92% were polymorphic among strawberry cultivars, representing valuable markers in transcribed regions of the genome. Sixty-three SSRs were polymorphic in the diploid Fragaria reference population and were bin-mapped together with another five previously reported but unmapped markers. In total, 72 loci were distributed across the seven linkage groups. In addition, the transferability of 174 Fragaria SSRs to the related Rosa and Rubus genera was investigated, ranging from 28.7% for genic-SSRs in rose to 16.1% for genomic-SSRs in raspberry. Among these markers, 33 and 16 were both localized in the diploid Fragaria reference map and cross-amplified in rose and raspberry, respectively. These results indicate that transferability of SSRs across the Rosoideae subfamily is limited. However, we have identified a set of Fragaria markers, polymorphic in the diploid reference population, which cross-amplified in both Rosa and Rubus, which represents a valuable tool for comparative mapping and genetic diversity analyses within the Rosoideae subfamily.     

Genetic linkage maps of rose constructed with new microsatellite markers and locating QTL controlling flowering traits

New microsatellites markers [simple sequence repeat (SSR)] have been isolated from rose and integrated into an existing amplified fragment-length polymorphism genetic map. This new map was used to identify quantitative trait locus (QTL) controlling date of flowering and number of petals. From a rose bud expressed sequence tag (EST) database of 2,556 unigenes and a rose genomic library, 44 EST-SSRs and 20 genomic-SSR markers were developed, respectively. These new rose SSRs were used to expand genetic maps of the rose interspecific F₁ progeny. In addition, SSRs from other Rosaceae genera were also tested in the mapping progeny. Genetic maps for the two parents of the progeny were constructed using pseudo-testcross mapping strategy. The maps consist of seven linkage groups of 105 markers covering 432 cM for the maternal map and 136 markers covering 438 cM for the paternal map. Homologous relationships among linkage groups between the maternal and paternal maps were established using SSR markers. Loci controlling flowering traits were localised on genetic maps as a major gene and QTL for the number of petals and a QTL for the blooming date. New SSR markers developed in this study will provide tools for the establishment of a consensus linkage map for roses that combine traits and markers in various rose genetic maps.     

Genic Microsatellite Markers in Brassica rapa: Development, Characterization, Mapping, and Their Utility in Other Cultivated and Wild Brassica Relatives

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.     

Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and GenBank ESTs to map the sugar beet genome

Sugar beet (Beta vulgaris) is an important root crop for sucrose production. A study was conducted to find a new abundant source of microsatellite (SSR) markers in order to develop marker assistance for breeding. Different sources of existing microsatellites were used and new ones were developed to compare their efficiency to reveal diversity in mapping population and mapping coverage. Forty-one microsatellite markers were isolated from a B. vulgaris ssp maritima genomic library and 201 SSRs were extracted from a B. vulgaris ssp vulgaris library. Data mining was applied on GenBank B. vulgaris expressed sequence tags (ESTs), 803 EST-SSRs were identified over 19,709 ESTs. Characteristics, polymorphism and cross-species transferability of these microsatellites were compared. Based on these markers, a high density genetic map was constructed using 92 F₂ individuals from a cross between a sugar and a table beet. The map contains 284 markers, spans over 555 cM and covers the nine chromosomes of the species with an average markers density of one marker every 2.2 cM. A set of markers for assignation to the nine chromosomes of sugar beet is provided.     

A genetic map of 1,000 SSR and DArT markers in a wide barley cross

A high-density genetic map was developed from an F1-derived doubled haploid population generated from a cross between cultivated barley (Hordeum vulgare) and the subspecies H. vulgare ssp. spontaneum. The map comprises 1,000 loci, amplified using 536 SSR (558 loci) and 442 DArT markers. Of the SSRs, 149 markers (153 loci) were derived from barley ESTs, and 7 from wheat ESTs. A high level of polymorphism (∼70%) was observed, which facilitated the mapping of 197 SSRs for which genetic assignments had not been previously reported. Comparison with a published composite map showed a high level of co-linearity and telomeric coverage on all seven chromosomes. This map provides access to previously unmapped SSRs, improved genome coverage due to the integration of DArT and EST-SSRs and overcomes locus order issues of composite maps constructed from the alignment of several genetic maps. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural characterization and mapping of functional EST-SSR markers in Theobroma cacao

Theobroma cacao L. is a major cash crop for tropical countries, providing incomes for 14 million small farmers. Establishing sustainable disease resistance and maintaining cocoa qualities are among the major objectives of breeding programs. To enrich the high-density genetic map, useful for all cocoa genetic studies, with gene-based markers, a recently produced large EST resource was mined to develop expressed sequence tag-based simple sequence repeat markers (EST-SSRs) defined in genes with a putative known function. A set of 174 polymorphic EST-SSRs was identified from a selection of 314 non-redundant EST-SSRs with a putative known function. Of them, 115 loci were mapped on the cocoa reference map. This new map contains 582 codominant markers arranged in ten linkage groups corresponding to the haploid number of chromosomes. An average interval between markers of 1.3?cM was found, with approximately one SSR every 2?cM. This new set of EST-SSRs includes 14 candidate genes for plant resistance or cocoa qualities. The percentage of polymorphic SSRs varied depending on the different gene regions from which they originated, with respectively 54%, 69%, and 82% of polymorphic EST-SSRs originating from coding sequences, and from the non-coding untranslated 5??UTR and 3??UTR regions. This new map contains a set of 384 SSR markers that are easily transferable across different mapping populations and useful for all genetic analyses in T. cacao. The new set of EST-SSRs will be a useful tool for studying the functional diversity of populations and for carrying out association mapping studies.     

A ddRAD Based Linkage Map of the Cultivated Strawberry,Fragaria xananassa

The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array’s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F₁ hybrid population raised from the cross between the cultivars Sonata (♀) and Babette (♂). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. ×ananassa genome. Here, we have developed the first linkage map for F. ×ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

The development and mapping of functional markers in Fragaria and their transferability and potential for mapping in other genera

We have developed 46 primer pairs from exon sequences flanking polymorphic introns of 23 Fragaria gene sequences and one Malus sequence deposited in the EMBL database. Sequencing of a set of the PCR products amplified with the novel primer pairs in diploid Fragaria showed the products to be homologous to the sequences from which the primers were originally designed. By scoring the segregation of the 24 genes in two diploid Fragaria progenies FV × FN (F. vesca × F. nubicola F₂) and 815 × 903BC (F. vesca × F. viridis BC₁) 29 genetic loci at discrete positions on the seven linkage groups previously characterised could be mapped, bringing to 35 the total number of known function genes mapped in Fragaria. Twenty primer pairs, representing 14 genes, amplified a product of the expected size in both Malus and Prunus. To demonstrate the applicability of these gene-specific loci to comparative mapping in Rosaceae, five markers that displayed clear polymorphism between the parents of a Malus and a Prunus mapping population were selected. The markers were then scored and mapped in at least one of the two additional progenies.     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait

Black poplar (Populus nigra L.) is a tree of ecological and economic interest. A better knowledge of P. nigra genome is needed for an effective protection and use of its genetic resources. The main objective of this study is the construction of a highly informative genetic map of P. nigra species including genes of adaptive and economic interest. Two genotypes originated from contrasted natural Italian populations were crossed to generate a F₁ mapping pedigree of 165 individuals. Amplification fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers were used to genotype 92 F₁ individuals, and the pseudo-test-cross strategy was applied for linkage analysis. The female parent map included 368 markers (274 AFLPs, 91 SSRs, and 3 SNPs) and spanned 2,104 cM with 20 linkage groups, and the male parent map, including 317 markers (205 AFLPs, 106 SSRs, 5 SNPs, and sex trait), spanned 2,453 cM with 23 main linkage groups. The sex, as morphological trait, was mapped on the linkage group XIX of the male parent map. The generated maps are among the most informative in SSRs when compared to the Populus maps published so far and allow a complete alignment with the 19 haploid chromosomes of Populus sequence genome. These genetic maps provide informative tools for a better understanding of P. nigra genome structure and genetic improvement of this ecologically and economically important European tree species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

Background

Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.

Results

A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.

Conclusions

The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.     

Genetic mapping of EST-derived simple sequence repeats (EST-SSRs) to identify QTL for leaf morphological characters in a Quercus robur full-sib family

The availability of genomic resources such as expressed sequence tag-derived simple sequence repeat (EST-SSR) markers in adaptive genes with high transferability across related species allows the construction of genetic maps and the comparison of genome structure and quantitative trait loci (QTL) positions. In the present study, genetic linkage maps were constructed for both parents of a Quercus robur × Q. robur ssp. slavonica full-sib pedigree. A total of 182 markers (61 AFLPs, 23 nuclear SSRs, 98 EST-SSRs) and 172 markers (49 AFLPs, 21 nSSRs, 101 EST-SSRs, 1 isozyme) were mapped on the female and male linkage maps, respectively. The total map length and average marker spacing were 1,038 and 5.7 cM for the female map and 998.5 and 5.8 cM for the male map. A total of 68 nuclear SSRs and EST-SSRs segregating in both parents allowed to define homologous linkage groups (LG) between both parental maps. QTL for leaf morphological traits were mapped on all 12 LG at a chromosome-wide level and on 6 LG at a genome-wide level. The phenotypic effects explained by each single QTL ranged from 4.0 % for leaf area to 15.8 % for the number of intercalary veins. QTL clusters for leaf characters that discriminate between Q. robur and Quercus petraea were mapped reproducibly on three LG, and some putative candidate genes among potentially many others were identified on LG3 and LG5. Genetic linkage maps based on EST-SSRs can be valuable tools for the identification of genes involved in adaptive trait variation and for comparative mapping.     

Constructing a genetic linkage map and mapping quantitative trait loci for skeletal traits in Japanese flounder

A genetic linkage map of Japanese flounder was constructed using 165 doubled haploids (DHs) derived from a single female. A total of 574 genomic microsatellites (type II SSRs) and expressed sequence tag (EST)-derived markers (EST-SSRs) were mapped to 24 linkage groups. The length of linkage map was estimated as 1270.9 centiMorgans (cM), with an average distance between markers of 2.2 cM. The EST-SSRs were used together with type II SSR markers to construct the Japanese flounder genetic linkage map which will facilitate identify quantitative trait locus (QTL) controlling important economic traits in Japanese flounder. Thus, twelve skeletal traits at 2 years of age were measured for all DHs. Forty-one QTLs were detected on 14 linkage groups and totally account for a small proportion of phenotypic variation (4.5 to 17.3%). Most of QTLs detected distribute on linkage groups 5 (9 QTLs), 8 (9 QTLs), 9 (5 QTLs) and 20 (4 QTLs), in which, some QTLs perform the pleiotropy.     

SSR and EST-SSR-based genetic linkage map of cassava (Manihot esculenta Crantz)

Simple sequence repeat (SSR) markers provide a powerful tool for genetic linkage map construction that can be applied for identification of quantitative trait loci (QTL). In this study, a total of 640 new SSR markers were developed from an enriched genomic DNA library of the cassava variety 'Huay Bong 60' and 1,500 novel expressed sequence tag-simple sequence repeat (EST-SSR) loci were developed from the Genbank database. To construct a genetic linkage map of cassava, a 100 F(1) line mapping population was developed from the cross Huay Bong 60 by 'Hanatee'. Polymorphism screening between the parental lines revealed that 199 SSRs and 168 EST-SSRs were identified as novel polymorphic markers. Combining with previously developed SSRs, we report a linkage map consisted of 510 markers encompassing 1,420.3?cM, distributed on 23 linkage groups with a mean distance between markers of 4.54?cM. Comparison analysis of the SSR order on the cassava linkage map and the cassava genome sequences allowed us to locate 284 scaffolds on the genetic map. Although the number of linkage groups reported here revealed that this F(1) genetic linkage map is not yet a saturated map, it encompassed around 88% of the cassava genome indicating that the map was almost complete. Therefore, sufficient markers now exist to encompass most of the genomes and efficiently map traits in cassava.