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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of 1,030 genomic SSR markers in switchgrass

Switchgrass, Panicum virgatum L., a native to the tall grass prairies in North America, has been grown for soil conservation and herbage production in the USA and recently widely recognized as a promising dedicated cellulosic bioenergy crop. A large amount of codominant molecular markers including simple sequence repeats (SSRs) are required for the construction of linkage maps and implementation of molecular breeding strategies to develop superior switchgrass cultivars. The objectives of this study were (1) to identify SSR-containing clones and to design PCR primer pairs (PPs) in SSR-enriched genomic libraries, and (2) to validate and characterize the designed SSR PPs. Five genomic SSR enriched libraries were constructed using genomic DNA of ‘SL93 7 × 15’, a switchgrass genotype selected in an Oklahoma State University (OSU) southern lowland breeding population. A total of 3,046 clones from four libraries enriched in (CA/TG)n, (GA/TC)n, (CAG/CTG)n and (AAG/CTT)n SSR repeats were sequenced at the OSU Core Facility. From the sequences, we isolated 1,300 unique SSR-containing clones, from which we designed 1,398 PPs using SSR Locator V.1 software. Among the designed PPs, 1,030 (73.7%) amplified reproducible and strong bands with expected fragment size, and 802 detected polymorphic alleles, in SL93 7 × 15 and ‘NL94 16 × 13’, two parents of one mapping population. All of the four libraries contained a high rate of perfect SSR repeat types, ranging from 62.7 to 76.2%. Polymorphism of the effective SSR markers was also tested in two lowland and two upland switchgrass cultivars, encompassing ‘Alamo’ and ‘Kanlow’, and ‘Blackwell’ and ‘Dacotah’, respectively. The developed SSR markers should be useful in genetic and breeding research in switchgrass.     

An EST-SSR-based linkage map for Persea americana Mill. (avocado)

Recent enhancement of the pool of known molecular markers for avocado has allowed the construction of the first moderately dense genetic map for this species. Over 300 SSR markers have been characterized and 163 of these were used to construct a map from the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage'. One hundred thirty-five primer pairs amplified 163 usable loci with 20 primer pairs amplifying more than one locus. 'Tonnage' was heterozygous for 152 (93%) loci, whereas 'Simmonds' was heterozygous for 64 (39%). Null alleles were identified at several loci. Linkage maps were produced for both reciprocal crosses and combined to generate a composite linkage map for the F₁ population of 715 individuals. The composite map contains 12 linkage groups. Linkage groups ranged in size from 157.3 cM (LG2) to 2.4 cM (LG12) and the number of loci mapped per group ranged from 29 (LG1) to two (LG12). The total map length was 1,087.4 cM. Only seven markers were observed to have segregation distortion (α ≤ 0.05) across both sub-composite (reciprocal) maps. Phenotypic data from traits of horticultural interest are currently being collected on this population with the ultimate goal of identifying useful quantitative trait loci and the development of a marker-assisted selection program.     

A linkage map of the pea (Pisum sativum L.) genome containing cloned sequences of known function and expressed sequence tags (ESTs)

 A linkage map of the pea (Pisum sativum L.) genome is presented which is based on F₂ plants produced by crossing the marrowfat cultivar ‘Primo’ and the blue-pea breeding line ‘OSU442-15’. This linkage map consists of 209 markers and covers 1330 cM (Kosambi units) and includes RFLP, RAPD and AFLP markers. By mapping a number of anchor loci, the ‘Primo’בOSU442-15’ map has been related to other pea linkage maps. A feature of the map is the incorporation of 29 loci representing genes of known function, obtained from other laboratories. The map also contains RFLP loci detected using sequence-characterized cDNA clones developed in our laboratory. The putative identities of 38 of these cDNA clones were assigned by examining public-sequence databases for protein or nucleotide-sequence similarities. The conversion of sequence-characterized pea cDNAs into PCR-amplifiable and polymorphic sequence-tagged sites (STSs) was investigated using 18 pairs of primers designed for single-copy sequences. Eleven polymorphic STSs were developed. Received: 18 June 1997 / Accepted: 11 August 1997     

In silico polymorphic novel SSR marker development and the first SSR-based genetic linkage map in pistachio

Simple sequence repeats (SSRs) are co-dominant markers, and are very useful in constructing consensus maps in heterozygous perennial plant species like pistachio. Pistacia vera L. is the only cultivated species in the genus Pistacia. It is dioecious with a haploid chromosome count of n =?15. Saturated genetic linkage maps can be a reference to identify markers linked to economically important phenotypic traits that could be useful for early breeding and selection programs. Therefore, this study aimed to develop polymorphic SSR markers in silico and to construct the first SSR-based genetic linkage map in pistachio. The DNA sequences of three cultivars (Siirt, Ohadi, and Bagyolu) of P. vera and one genotype belonging to P. atlantica (Pa-18) were obtained by next-generation sequencing, and 625 polymorphic SSR loci were identified from 750 screened in silico polymorphic SSR primer pairs. The novel SSRs were used to construct SSR-based genetic linkage maps in pistachio along with published SSRs in Siirt × Bagyolu F1 population. Most (71.4%) of the SSRs were common markers that were used to construct consensus and parental maps spanning 15 linkage groups (LGs). A total of 384, 317, and 341 markers were mapped in the consensus, female, and male genetic maps with total lengths of 1511.3, 1427.0, and 1453.4 cM, respectively. The large number of SSR markers discovered and the first SSR-based genetic linkage map constructed in this study will be useful for anchoring loci for map integration, and will facilitate marker-assisted selection efforts for important horticultural traits in the genus Pistacia.     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

Simple sequence repeat (SSR) markers survey of the cassava (Manihot esculenta Crantz) genome: towards an SSR-based molecular genetic map of cassava

The development of PCR-based, easily automated molecular genetic markers, such as SSR markers, are required for realistic cost-effective marker-assisted selection schemes. This paper describes the development and characterization of 172 new SSR markers for the cassava genome. The placement of 36 of these markers on the existing RFLP framework map of cassava is also reported. Two similar enrichment methods were employed. The first method yielded 35 SSR loci, for which primers could be designed, out of 148 putative DNA clones. A total of 137 primer pairs could be designed from 544 putative clones sequenced for the second enrichment. Most of the SSRs (95%) were di-nucleotide repeats, and 21% were compound repeats. A major drawback of these methods of SSR discovery is the redundancy – 20% duplication; in addition, primers could not be designed for many SSR loci that were too close to the cloning site – 45% of the total. All 172 SSRs amplified the corresponding loci in the parents of the mapping progeny, with 66% of them revealing a unique allele in at least one of the parents, and 26% having unique alleles in both of the parents. Of the 36 SSRs that have been mapped, at least 1 was placed on 16 out of the 18 linkage groups of the framework map, indicating a broad coverage of the cassava genome. This preliminary mapping of the 36 markers has led to the joining of a few small groups and the creation of one new group. The abundance of allelic bridges as shown by these markers will lead to the development of a consensus map of the male- and female-derived linkage groups. In addition, the relatively higher number of these allelic bridges, 30% as against 10% for RFLPs in cassava, underscores SSR as the marker of choice for cassava. The 100% primer amplification obtained for this set of primers also confirms the appropriateness of SSR markers for use in cassava genome analysis and the transferability of the technology as a low-cost approach to increasing the efficiency of cassava breeding. Current efforts are geared towards the generation of more SSR markers to attain a goal of 200 SSR markers, or 1 SSR marker every 10 cM. Received: 15 November 1999 / Accepted: 14 April 2000     

Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes

 Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms. Received: 13 August 1998 / Accepted: 13 October 1998     

Development of two loquat [Eriobotrya japonica (Thunb.) Lindl.] linkage maps based on AFLPs and SSR markers from different Rosaceae species

Loquat [Eriobotrya japonica (Thunb.) Lindl.] is a Rosaceae fruit species of growing interest as an alternative to the main fruit crops. However, only a few genetic studies have been carried out on this species. This paper reports the construction of the first genetic maps of two loquat cultivars based on AFLP and microsatellite markers from Malus, Eriobotrya, Pyrus and Prunus genera. An F₁ population consisting of 81 individuals, derived from the cross between ‘Algerie’ and ‘Zaozhong-6’ cultivars, was used to construct both maps. A total of 111 scorable simple sequence repeat (SSR) loci resulted from the testing of 440 SSR primer pairs in the analyzed progeny and the SSR transferability to Eriobotrya was found to be 74% from apple, 58% from pear and 49% from Prunus spp. In addition, 183 AFLP polymorphic bands were produced using 42 primer combinations. The ‘Algerie’ map was organized in 17 linkage groups covering a distance of 900 cM and comprising 177 loci (83 SSRs and 94 AFLPs) with an average marker distance of 5.1 cM. Self-incompatibility trait was mapped at the distal part of the LG17 linkage group, as previously reported in Malus and Pyrus. The ‘Zaozhong-6’ map covered 870 cM comprising 146 loci (64 SSRs and 82 AFLPs) with an average marker distance of 5.9 cM. The 44 SSRs and the 48 AFLPs share in common by both maps were essentially collinear and, moreover, the order of the 75% of apple and pear SSRs mapped in Eriobotrya was shown to be consistent across the Maloideae subfamily. As a whole, these maps represent a useful tool to facilitate loquat breeding and an interesting framework for map comparison in the Rosaceae.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Development and incorporation of microsatellite markers into the linkage map of sugar beet (Beta vulgaris spp.)

A set of informative simple sequence repeat markers has been identified for use in the marker-assisted breeding of Beta vulgaris. Highly enriched small insert genomic libraries were constructed, consisting of 1536 clones (with inserts of between 250–900 bp). Screening the clones with CA, CT, CAA, CATA and GATA nucleotide-repeat probes revealed positive hybridisation to over 50% of the clones. Of these 340 were sequenced. Primer pairs were designed for sequences flanking the repeats and, of these, 57 pairs revealed length polymorphism with 12 Beta accessions. Heterozygosity levels of the SSR loci ranged from 0.069 to 0.809. Heterozygosity levels were found to be similar to those detected employing RFLP probes with the same accessions. Phenetic analysis using the markers, indicated relationships in accordance with known pedigrees. Twenty three of the SSR markers were polymorphic in one or both of two F₂ mapping populations, and were placed relative to a framework of RFLP probes. The markers are distributed over all nine linkage groups of sugar beet. Received: 14 July 1999 / Accepted: 27 October 1999     

An integrated SSR and RFLP linkage map of Sorghum bicolor (L.) Moench.

We report the development, testing, and use (for genetic mapping) of a large number of polymerase chain reaction (PCR) primer sets that amplify DNA simple sequence repeat (SSR) loci of Sorghum bicolor (L.) Moench. Most of the primer sets were developed from clones isolated from two sorghum bacterial artificial chromosome (BAC) libraries and three enriched sorghum genomic-DNA (gDNA) libraries. A few were developed from sorghum DNA sequences present in public databases. The libraries were probed with radiolabeled di- and trinucleotide oligomers, the BAC libraries with four and six oligomers, respectively, and the enriched gDNA libraries with four and three oligomers, respectively. Both types of libraries were markedly enriched for SSRs relative to a size-fractionated gDNA library studied earlier. However, only 2% of the sequenced clones obtained from the size-fractionated gDNA library lacked a SSR, whereas 13% and 17% of the sequenced clones obtained from the BAC and enriched gDNA libraries, respectively, lacked a SSR. Primer sets were produced for 313 SSR loci. Two-hundred sixty-six (85%) of the loci were amplified and 165 (53%) of the loci were found to be polymorphic in a population composed of 18 diverse sorghum lines. (AG/TC)n and (AC/TG)n repeats comprised 91% of the dinucleotide SSRs and 52% of all of the SSRs at the polymorphic loci, whereas four types of repeats comprised 66% of the trinucleotide SSRs at the loci. Primer sequences are reported for the 165 polymorphic loci and for eight monomorphic loci that have a high degree of homology to genes. Also reported are the genetic map locations of 113 novel SSR loci (including four SSR-containing gene loci) and a linkage map composed of 147 SSR loci and 323 RFLP (restriction fragment length polymorphism) loci. The number of SSR loci per linkage group ranges from 8 to 30. The SSR loci are distributed relatively evenly throughout approximately 75% of the 1406-cM linkage map, but segments of five linkage groups comprising about 25% of the map either lack or contain few SSR loci. Mapping of SSR loci isolated from BAC clones located to these segments is likely to be the most efficient method for placing SSR loci in the segments.     

Construction of a genetic linkage map using simple sequence repeat markers from expressed sequence tags for cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

Cassava (Manihot esculenta) is an economically important crop that is grown in tropical and sub-tropical regions. Use of molecular technology for genetic improvement of cassava has been limited by the lack of a large set of DNA markers and a genetic map. Therefore, the aims here were to develop additional simple sequence repeat (SSR) markers from the public expressed sequence tags (ESTs), and to construct a genetic linkage map. In this study, we designed 425 EST-SSR markers from sequences obtained from the cassava EST database in GenBank, and integrated them with 667 SSR markers from a microsatellite-enriched genomic sequence received from the International Center for Tropical Agriculture (CIAT). Of these, 107 EST-SSR and 500 genomic SSR primer pairs showed polymorphic patterns when screened in two cassava varieties, Hauy Bong 60 and Hanatee, which were used as female and male parental lines, respectively. Within the 107 and 500 primer pairs, 81 and 226 EST-SSR and SSR primer pairs were successfully genotyped with 100 samples of F₁ progeny, respectively. The results showed 20 linkage groups consisting of 211 markers—56 EST-SSR and 155 SSR markers—spanning 1,178 cM, with an average distance between markers of 5.6 cM and about 11 markers per linkage group. These novel EST-SSR markers provided genic PCR-based co-dominant markers that were useful, reliable and economical. The EST-SSRs were used together with SSR markers to construct the cassava genetic linkage map which will be useful for the identification of quantitative trait loci controlling the traits of interest in cassava breeding programs.     

Functional integrated genetic linkage map based on EST-markers for a sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp.) commercial cross

The growing availability of ESTs provides a potentially valuable source of new DNA markers. The authors examined the SUCEST database and developed EST-derived markers. Thus to enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in a sugarcane commercial cross, 149 EST-SSRs and 10 EST-RFLPs were screened in the SP80-180 × SP80-4966 mapping population. With the markers already analyzed in the previous map, 2303 polymorphic markers were generated, of which 1669 (72.5%) were single-dose (SD) markers. Out of these 1669 SD markers, 664 (40%) were scattered onto 192 co-segregation groups (CGs) with a total estimated length of 6.261,1 cM. Using both genomic and EST-derived SSR and RFLP markers, 120 out of the 192 CGs were formed into fourteen putative homology groups (HGs). The EST-derived markers were subjected to BLASTX search in the SUCEST database, of which putative function was assigned to 113 EST-SSRs and six EST-RFLPs based on high nucleotide homology to previously studied genes. The integration of EST-derived markers improved the map, making it possible to consider additional fine mapping of the genome, and providing the means for developing ‘perfect markers’ associated with key QTL. To summarize, this paper deals with the construction of a genetic linkage map of sugarcane that is populated by functionally associated markers.     

Limited variability of CTG/CAG repeats in <Emphasis Type="Italic">Lycopersicon</Emphasis> nuclear DNA

Seven clones containing (CTG)_n/(CAG)_n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.     

A first linkage map of olive (Olea europaea L.) cultivars using RAPD,AFLP, RFLP and SSR markers

The first linkage map of the olive (Olea europaea L.) genome has been constructed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) as dominant markers and a few restriction fragment length polymorphisms (RFLP) and simple-sequence repeats (SSR) as codominant markers. Ninety-five individuals of a cross progeny derived from two highly heterozygous olive cultivars, Leccino and Dolce Agogia, were used by applying the pseudo test-cross strategy. From 61 RAPD primers 279 markers were obtained - 158 were scored for Leccino and 121 for Dolce Agogia. Twenty-one AFLP primer combinations gave 304 useful markers - 160 heterozygous in Leccino and 144 heterozygous in Dolce Agogia. In the Leccino map 249 markers (110 RAPD, 127 AFLP, 8 RFLP and 3 SSR) were linked. This resulted in 22 major linkage groups and 17 minor groups with fewer than four markers. In the Dolce Agogia map, 236 markers (93 RAPD, 133 AFLP, 6 RFLP and 4 SSR) were linked; 27 major linkage groups and three minor groups were obtained. Codominant RFLPs and SSRs, as well as few RAPDs in heteroduplex configuration, were used to establish homologies between linkage groups of both parents. The total distance covered was 2,765 cM and 2,445 cM in the Leccino and Dolce Agogia maps, respectively. The mean map distance between adjacent markers was 13.2 cM in Leccino and 11.9 cM in Dolce Agogia, respectively. Both AFLP and RAPD markers were homogeneously distributed in all of the linkage groups reported. The stearoyl-ACP desaturase gene was mapped on linkage group 4 of cv. Leccino.     

Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis

 Genes cloned from diverse plants for resistance to different pathogens have sequence similarities in domains presumably involved in pathogen recognition and signal transduction in triggering the defense response. Primers based on the conserved regions of resistance genes often amplify multiple fragments that may not be separable in an agarose gel. We used denaturing polyacrylamide-gel electrophoresis to detect PCR products of plant genomic DNA amplified with primers based on conserved regions of resistance genes. Depending upon the primer pairs used, 30–130 bands were detected in wheat, rice, and barley. As high as 47%, 40%, and 27% of the polymorphic bands were detected in rice, barley, and wheat, respectively, and as high as 12.5% of the polymorphic bands were detected by certain primers in progeny from a cross of the wheat cultivars ‘Stephens’ and ‘Michigan Amber’. Using F₆ recombinant inbred lines from the ‘Stephens’בMichigan Amber’ cross, we demonstrated that polymorphic bands amplified with primers based on leucine-rich repeats, nucleotide-binding sites and protein kinase genes, were inherited as single loci. Linkages between molecular markers and stripe rust resistance genes were detected. This technique provides a new way to develop molecular markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species. Received : 5 September 1997 / Accepted : 6 November 1997     

Molecular characterization and genetic mapping of random amplified microsatellite polymorphism in barley

 This study has analyzed the molecular basis and genetic behaviour of the polymorphism generated by the amplification of barley genomic DNA with primers complementary to microsatellites. Primers anchored at the 5′ end, used alone or in combination with arbitrary sequence primers, generated random amplified microsatellite polymorphisms (RAMPs). Unanchored primers were also used as single primers in a microsatellite primed-PCR (MP-PCR). Twenty six randomly selected RAMP DNA fragments which showed polymorphism between the cultivars Steptoe and Morex were cloned and sequenced. All sequences showed the expected repeated motif at the end of the insert, with the number of repeats ranging from five to ten. Genomic sequences containing low numbers of microsatellite motifs were preferentially amplified; therefore, only a fraction of the polymorphism could be attributed to variation in the number of microsatellite motifs at the priming site. Some sequences contained either cryptic simple sequences or members of families of repeated DNA. Polymorphism at the internal cryptic simple sequences was detected by RAMP bands inherited as co-dominant markers. Four MP-PCR bands were cloned and sequenced. A number of repeats identical to the primer itself were found at each end of the insert. Two allelic bands were polymorphic for an internal microsatellite. The potential use of cloned bands as fingerprinting tools was investigated by employing them as hybridization probes in Southern blots containing digested barley DNA from a sample of cultivars. RAMP probes produced complex hybridization band patterns. MP-PCR probes produced either a highly variable single locus or low-copy number loci. Segregations for 31 RAMPs and three MP-PCR bands were studied in a population of 70 doubled-haploids from the Steptoe/Morex cross. One third of all markers were co-dominantly inherited. Markers were positioned on an RFLP map and found to be distributed in all barley chromosomes. The new markers enlarged the overall length of the map to 1408 cM. Received: 6 May 1998 / Accepted: 20 July 1998     

Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers

 Linkage maps for the apple cultivars ‘Prima’ and ‘Fiesta’ were constructed using RFLP, RAPD, isozyme, AFLP, SCAR and microsatellite markers in a ‘Prima’בFiesta’ progeny of 152 individuals. Seventeen linkage groups, putatively corresponding to the seventeen haploid apple chromosomes, were obtained for each parent. These maps were aligned using 67 multi-allelic markers that were heterozygous in both parents. A large number of duplicate RFLP loci was observed and, in several instances, linked RFLP markers in one linkage group showed corresponding linkage in another linkage group. Distorted segregation was observed mainly in two regions of the genome, especially in the male parent alleles. Map positions were provided for resistance genes to scab and rosy leaf curling aphid (Vf and Sd ₁, respectively) for the fruit acidity gene Ma and for the self-incompatibility locus S. The high marker density and large number of mapped codominant RFLPs and some microsatellite markers make this map an ideal reference map for use in other progenies also and a valuable tool for the mapping of quantitative trait loci. Received: 17 November 1997 / Accepted: 9 December 1997     

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.