Unique Simple Sequence Repeat Research Articles

SSR-Marker Analysis of the Intracultivar Phenotypic Variation Discovered within 3 Soybean Cultivars

Genetic variation within homogeneous gene pools in various crops is assumed to be very limited. One objective of this study was to use 144 simple sequence repeat (SSR) markers to determine if the single-plant lines selected at ultra-low plant density in honeycomb designs within the soybean cultivars Benning, Haskell, and Cook had unique SSR genetic fingerprints. Another objective was to investigate if the variation found was the result of residual genetic heterozygosity that could be detected in the original gene pool where selection initiated. Our results showed that the phenotypic variation for seed protein content and seed weight has a genotypic component identified by the SSR band variation. The 7 lines from Haskell had a total of 63 variant alleles, the 5 lines from Benning had 34 variant alleles, and the 7 lines from Cook had 34 variant alleles, therefore, possessing unique genetic fingerprints. Most of the intracultivar SSR band variation discovered was the result of residual heterozygosity in the initial plant selected to become the cultivar. More specifically, 82% of the SSR variant alleles were traced in the Benning Foundation seed source, 93% in the Haskell seed source, and 82% in the Cook seed source. The remaining variant bands (18% for Benning, 7% for Haskell, and 18% for Cook) could not be detected in the Foundation seed source and were likely the result of mutation or some other mechanism generating de novo variation. These results provide evidence that genetic variation among individual plants is present even in homogeneous gene pools and can be further utilized in breeding programs.

SSR FINGERPRINTING OF BLACK RASPBERRY CULTIVARS SHOWS DISCREPANCIES IN IDENTIFICATION

Breeding progress in black raspberry (Rubus occidentalis L.) has been limited by a lack of genetic diversity in elite germplasm. Black raspberry cultivars are noted for showing very few differences, and seedlings for a lack of segregation for important traits. Genetic fingerprinting using microsatellite, or simple sequence repeat (SSR) markers, can reliably identify unique clones and evaluate diversity in black raspberry cultivars. Twenty-one black raspberry cultivars were sampled from the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon. Black raspberry clones were also sampled from nurseries, grower’s fields, and other black raspberry breeding programs for comparison. These genotypes were compared using 18 polymorphic SSR primer pairs. The black raspberries 'Bristol,' 'Jewel,' and 'Mac Black' had consistent SSR fingerprints between sources. However, plants being sold as 'Black Hawk' and 'Cumberland' had the same fingerprint as 'Jewel'. Plants of 'Bristol', 'Cumberland', 'Munger', 'New Logan', 'Plum Farmer' and 'Shuttleworth' in the NCGR collection had identical fingerprints. Eleven unique SSR fingerprints were found among plants being grown or sold as ‘Munger’, though there was one predominant fingerprint for this cultivar. 'Allen' and 'John Robertson' were each represented by three different fingerprints from three different sources, and 'Earlysweet' and 'Jewel' had SSR alleles at multiple loci that cannot be explained by their reported pedigrees. While overall genetic diversity in black raspberry cultivars is low, discrepancies in the naming of clones appear to be widespread in commercial and research plantings. Future work in this area should focus on sampling additional independent sources of plant material and evaluating clones to determine the extent of performance differences. Further SSR development in black raspberry may be needed to fingerprint some unique clones.

Microsatellite fingerprinting of maize cultivars (Zea maysL.)

Maize cultivars (4 composites, 14 inbreds and 7 hybrids) cultivated in Punjab were characterized using a set of 70 Simple Sequence Repeat (SSR) markers with a total of 199 alleles. Out of these, 67 markers produced polymorphic profiles, 3 were monomorphic. Eight SSR primers detected eight unique alleles in three genotypes. These unique SSR alleles provide an opportunity for unambiguous differentiation of the respective genotypes. SSR profiles were analyzed by using computer softwares NTSYS-pc and DARwin 5.0. Both of them classified the genotypes in four distinct groups.

Analysis of molecular diversity and fingerprinting of commercially grown Indian rice hybrids

Twenty-five commercially grown Indian rice hybrids developed by both the public and private sectors, were analysed for molecular diversity and identification of simple sequence repeat (SSR) marker(s) that can distinguish them from each other. For diversity analysis, a total of fifty eight SSR markers providing genome wide coverage, were used. Forty out of fifty eight SSR markers were polymorphic amplifying a total of 121 alleles with molecular weight ranging from 70 bp – 280 bp. Further, characterisation of these markers was carried out generating parameters of heterozygosity (0.42), polymorphism information content (0.31), probability of identity (4.2 × 10−8) and probability of exclusion (99.99%). Cluster analysis based on a set of fourty highly polymorphic SSR markers generated three groups with dissimilarity index values ranging from 0.0 to 0.8. The hybrids based on common female parent IR58025A grouped together indicating a narrow genetic base of hybrid breeding programme. By combining the rapid and simple method of utilising these unique SSR markers alone or in combination, as molecular tags, identification of all the hybrids was possible even without having their parental lines. Twenty SSR loci produced hybrid specific unique alleles, which will be useful in establishing hybrid’s identity. The results have wide prospective in diversifying the genetic base of hybrid breeding programme, identification of rice hybrids, authentication of genetic purity of hybrid seed and protection of IPR.

Evaluation of Anonymous and Expressed Sequence Tag-Derived Polymorphic Microsatellite Markers in the Tobacco Budworm,Heliothis virescens(Lepidoptera: Noctuidae)

Abstract. Polymorphic genetic markers were identified and characterized using a partial genomic library of the tobacco budworm, Heliothis virescens (Fabricius), enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 unique SSRs while EST evaluations identified 280 SSR-containing sequences. All anonymous SSRs and 192 EST-SSRs were evaluated to select loci that produced the best quality peaks without stutter peaks. Six anonymous markers and nine EST-derived markers were selected to evaluate a sample of 96 insects collected from Stoneville, MS. The observed number of alleles ranged from two to eight, with an average of 5.87 (SE ± 0.53). The polymorphic information content (PIC) value averaged 0.446 (SE ± 0.052). Significant deviations from Hardy-Weinberg expectations were detected at eight loci. We speculate that these loci are under selection in the population of tobacco budworm use...

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 132cM in length, and 4 smaller linkage groups between 7 and 40cM. The total effective size of the map is 1658cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75cM. 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 100,000bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.

Application of SSR markers and real-time PCR for variety identification in azuki-bean [Vigna angularis (Willd.) Ohwi and Ohashi

The azuki bean in Korea consists of seven domestic varieties which have been developed and registered for the public during last 25 years. Here, we present a simple but reliable method to screen and identify Korean azuki bean varieties. A method based on simple sequence repeat (SSR) markers is widely used for prominent gene identification and variety discrimination. In molecular biology, real-time polymerase chain reaction (PCR) is a laboratory technique based on the polymerase chain reaction that is used to amplify and simultaneously quantify a targeted DNA molecule. It enables easy detection of a specific sequence in a DNA sample without performing electrophoresis and further processes. For separation of seven Korean azuki bean varieties, 110 unique azuki bean SSR markers from an (AG)n-enriched library were selected, synthesized and used for polymerase chain reaction (PCR). Data were taken through acrylamide gel electrophoresis and automated multi-capillary electrophoresis system for selection of specific markers and then changed into proper formats for data mining analysis. Ten primer pairs that showed high polymorphism were chosen for the indepth study. These ten primers were re-amplified with real-time PCR and checked the cycle threshold (Ct) and temperature (Tm) for comparison of amplification sequence in seven varieties. Consequently, a total of 20 alleles and 6 SSR primers were detected from the standard PCR amplification. Within these 6 primers, 7 alleles of 3 SSR primers were isolated for variety identification. From real-time PCR results, 3 SSR primers were selected as efficient markers for discrimination of seven Korean azuki bean varieties. The approach described here could be applied in monitoring our varieties and can be adapted in the azuki bean breeding program.

An integrated genetic linkage map for silkworms with three parental combinations and its application to the mapping of single genes and QTL

BackgroundBombyx mori, the domesticated silkworm, is a well-studied model insect with great economic and scientific significance. Although more than 400 mutations have been described in silkworms, most have not been identified, especially those affecting economically-important traits. Simple sequence repeats (SSRs) are effective and economical tools for mapping traits and genetic improvement. The current SSR linkage map is of low density and contains few polymorphisms. The purpose of this work was to develop a dense and informative linkage map that would assist in the preliminary mapping and dissection of quantitative trait loci (QTL) in a variety of silkworm strains.ResultsThrough an analysis of > 50,000 genotypes across new mapping populations, we constructed two new linkage maps covering 27 assigned chromosomes and merged the data with previously reported data sets. The integrated consensus map contains 692 unique SSR sites, improving the density from 6.3 cM in the previous map to 4.8 cM. We also developed 497 confirmed neighboring markers for corresponding low-polymorphism sites, with 244 having polymorphisms. Large-scale statistics on the SSR type were suggestive of highly efficient markers, based upon which we searched 16,462 available genomic scaffolds for SSR loci. With the newly constructed map, we mapped single-gene traits, the QTL of filaments, and a number of ribosomal protein genes.ConclusionThe integrated map produced in this study is a highly efficient genetic tool for the high-throughput mapping of single genes and QTL. Compared to previous maps, the current map offers a greater number of markers and polymorphisms; thus, it may be used as a resource for marker-assisted breeding.

Development of Novel EST-SSRs from Sacred Lotus (Nelumbo nucifera Gaertn) and Their Utilization for the Genetic Diversity Analysis of N. nucifera

Expressed sequence tags (ESTs) provide a valuable resource for the development of simple sequence repeat (SSR) or microsatellite markers. This study identified SSRs within ESTs from Nelumbo nucifera (lotus or sacred lotus), developed markers from them, and assessed the potential of those markers for diversity analysis. Within 2207 ESTs from N. nucifera downloaded from GenBank, 1483 unigenes (303 contigs and 1180 singletons) were identified. After eliminating for redundancy, 125 SSR-containing ESTs were derived, and 71 unique SSRs were detected with an average density of one SSR per 13.04 kb. Dinucleotide repeats were the dominant motif in N. nucifera, whereas the sequences AG/TC/GA/CT, AAG/TTC/GAT/AGA, and AAAGCC were the most frequent of di-, tri-, and hexanucleotide motifs, respectively. The AG/TC (40.85%) and AAG (5.63%) motifs were predominant for the di- and trinucleotide repeats, respectively. Sixty-two SSR-containing ESTs were suitable for primer design. From these sequences, 23 EST-SSR markers were developed and were applied to 39 cultivated varieties of N. nucifera, 10 accessions of wild N. nucifera, and 1 accession of Nelumbo lutea (American lotus). Genetic diversity and genetic relationships were examined by constructing unweighted pair-group method with arithmetic average dendrograms and principal coordinates analysis plots based on SSR polymorphisms. Results indicated genetic differentiation between cultivated and wild lotus and between seed lotus cultivars and rhizome lotus cultivars. These EST-SSR markers will be useful for further studies of the evolution and diversity of Nelumbo.

Development and Characterization of Expressed Sequence Tag-Derived Microsatellite Markers for the Wheat Stem Rust Fungus Puccinia graminis f. sp. tritici

Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated di- and tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis.

New Insight into Wild Red Raspberry Populations using Simple Sequence Repeat Markers

Scottish wild red raspberry (Rubus idaeus) plants at 12 sites were re-examined 10 years after initial studies had been carried out to examine the decline in population size and to address an earlier finding that demonstrated significant population differentiation over a small scale. Ten simple sequence repeat (SSR) loci were screened on the plants and a total of 80 alleles were detected, half of which were unique to a particular population, with all populations containing unique SSR alleles. Only 18 of the 80 alleles present in the wild were found in cultivated raspberries, highlighting the genetic diversity available for future breeding. This finding makes the decline in population number observed a concern, as this unique diversity is being eroded, primarily due to human impact. An additional 17 unique alleles were identified in the cultivars that were not present in the wild individuals studied. Gene flow into one lowland site was identified by the gain of one new allele into progeny at the site, but three alleles were also lost from parents to progeny. SSR markers were used to estimate the levels of outcrossing in wild red raspberry for the first time, and confirmed that the populations studied were outcrossing, intermating populations. The nonsignificant global F IS value indicates that red raspberry is an outbreeder (global F IS = −0.117), but significant population differentiation was observed [global F ST = 0.348 (P < 0.001)]. Diversity in this crop's wild relative and the population differentiation observed may have use in the future for breeding aimed at addressing climate change scenarios, and consideration should be given to means of conserving the diversity revealed by these studies.

Development and characterization of simple sequence repeats for flowering dogwood (Cornus florida L.)

Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences. The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and ‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and gene tagging of flowering dogwood.

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.

Development of SSR markers located in the G1 linkage group of apricot (Prunus armeniaca L.) using a bacterial artificial chromosome library

AbstractSixteen simple sequence repeats (SSRs) of apricot (Prunus armeniaca L.) were isolated from a bacterial artificial chromosome (BAC) library. Twelve restriction fragment length polymorphism (RFLP) probes mapped on LG1 of the Prunus general map were hybridized to the BAC library in order to select clones belonging to G1 linkage group of apricot. Selected BACs were digested, subcloned and hybridized with probes containing repeat motifs (GA)10 and (TA)10. Sequencing of the positive subclones revealed 18 unique SSR sequences of which 16 allowed the design of primers flanking the SSR. From the 16 primer pairs, 10 amplified polymorphic markers with an average of observed and expected heterozygosities of 0.44 and 0.68, respectively. The procedure described here proves to be a useful technique for obtaining markers in target areas of a genome.

Microsatellite Marker Development in Rose and its Application in Tetraploid Mapping

Microsatellite or simple sequence repeat (SSR) markers were developed from Rosa wichurana Crépin to combine two previously constructed tetraploid rose (Rosa hybrida L.) genetic maps. To isolate SSR-containing sequences from rose a small-insert genomic library was constructed from diploid Rosa wichurana and screened with several SSR probes. Specific primers were designed for 43 unique SSR regions, of which 30 primer pairs gave rise to clear PCR products. Seventeen SSR primer pairs (57%) produced polymorphism in the tetraploid rose 90-69 mapping family. These markers were incorporated into existing maps of the parents 86-7 and 82-1134, which were constructed primarily with AFLP markers. The current map of the male parent, amphidiploid 86-7, consists of 286 markers assigned to 14 linkage groups and covering 770 cm. The map of the female tetraploid parent, 82-1134, consists of 256 markers assigned to 20 linkage groups and covering 920 cm. Nineteen rose SSR loci were mapped on the 86-7 map and 11 on the 82-1134 map. Several homeologous linkage groups within maps were identified based on SSR markers. In addition, some of the SSR markers provided anchoring points between the two parental maps. SSR markers were also useful for joining small linkage groups. Based on shared SSR markers, consensus orders for four rose linkage groups between parental maps were generated. Microsatellite markers developed in this study will provide valuable tools for many aspects of rose research including future consolidation of diploid and tetraploid rose genetic linkage maps, genetic, phylogenetic and population analyses, cultivar identification, and marker-assisted selection.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

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.

Towards a Saturated Molecular Genetic Linkage Map for Cultivated Sunflower

The density and utility of the molecular genetic linkage map of cultivated sunflower (Helianthus annuus L.) has been greatly increased by the development and mapping of several hundred simple sequence repeat (SSR) markers. Of 1089 public SSR markers described thus far, 408 have been mapped in a recombinant inbred line (RIL) mapping population (RHA280 × RHA801). The goal of the present research was to increase the density of the sunflower map by constructing a new RIL map (PHA × PHB) based on SSRs, adding loci for newly developed SSR markers to the RHA280 × RHA801 RIL map, and integrating the restriction fragment length polymorphism (RFLP) and SSR maps of sunflower. The latter was accomplished by adding 120 SSR marker loci to a backbone of 80 RFLP marker loci on the HA370 × HA372 F2 map. The map spanned 1275.4 centimorgans (cM) and had a mean density of 6.3 cM per locus. The PHA × PHB SSR map was constructed from 264 SSR marker loci, spanned 1199.4 cM, and had a mean density of 4.5 cM per locus. The RHA280 × RHA801 map was constructed by adding 118 new SSR and insertion–deletion (INDEL) marker loci to 459 previously mapped SSR marker loci. The 577‐locus map spanned 1423.0 cM and had a mean density of 2.5 cM per locus. The three maps were constructed from 1044 DNA marker loci (701 unique SSR and 89 unique RFLP or INDEL marker loci) and supply a dense genome‐wide framework of sequence‐based DNA markers for molecular breeding and genomics research in sunflower.

Towards a Saturated Molecular Genetic Linkage Map for Cultivated Sunflower

The density and utility of the molecular genetic linkage map of cultivated sunflower (Helianthus annuus L.) has been greatly increased by the development and mapping of several hundred simple sequence repeat (SSR) markers. Of 1089 public SSR markers described thus far, 408 have been mapped in a recombinant inbred line (RIL) mapping population (RHA280 × RHA801). The goal of the present research was to increase the density of the sunflower map by constructing a new RIL map (PHA × PHB) based on SSRs, adding loci for newly developed SSR markers to the RHA280 × RHA801 RIL map, and integrating the restriction fragment length polymorphism (RFLP) and SSR maps of sunflower. The latter was accomplished by adding 120 SSR marker loci to a backbone of 80 RFLP marker loci on the HA370 × HA372 F2 map. The map spanned 1275.4 centimorgans (cM) and had a mean density of 6.3 cM per locus. The PHA × PHB SSR map was constructed from 264 SSR marker loci, spanned 1199.4 cM, and had a mean density of 4.5 cM per locus. The RHA280 × RHA801 map was constructed by adding 118 new SSR and insertion–deletion (INDEL) marker loci to 459 previously mapped SSR marker loci. The 577-locus map spanned 1423.0 cM and had a mean density of 2.5 cM per locus. The three maps were constructed from 1044 DNA marker loci (701 unique SSR and 89 unique RFLP or INDEL marker loci) and supply a dense genome-wide framework of sequence-based DNA markers for molecular breeding and genomics research in sunflower.

Simple sequence repeat map of the sunflower genome.

Several independent molecular genetic linkage maps of varying density and completeness have been constructed for cultivated sunflower ( Helianthus annuus L.). Because of the dearth of sequence and probe-specific DNA markers in the public domain, the various genetic maps of sunflower have not been integrated and a single reference map has not emerged. Moreover, comparisons between maps have been confounded by multiple linkage group nomenclatures and the lack of common DNA markers. The goal of the present research was to construct a dense molecular genetic linkage map for sunflower using simple sequence repeat (SSR) markers. First, 879 SSR markers were developed by identifying 1,093 unique SSR sequences in the DNA sequences of 2,033 clones isolated from genomic DNA libraries enriched for (AC)(n) or (AG)(n) and screening 1,000 SSR primer pairs; 579 of the newly developed SSR markers (65.9% of the total) were polymorphic among four elite inbred lines (RHA280, RHA801, PHA and PHB). The genetic map was constructed using 94 RHA280 x RHA801 F(7) recombinant inbred lines (RILs) and 408 polymorphic SSR markers (462 SSR marker loci segregated in the mapping population). Of the latter, 459 coalesced into 17 linkage groups presumably corresponding to the 17 chromosomes in the haploid sunflower genome ( x = 17). The map was 1,368.3-cM long and had a mean density of 3.1 cM per locus. The SSR markers described herein supply a critical mass of DNA markers for constructing genetic maps of sunflower and create the basis for unifying and cross-referencing the multitude of genetic maps developed for wild and cultivated sunflowers.