Development of SSR markers and construction of a linkage map in jute

Jute is an important natural fibre crop, which is only second to cotton in its importance at the global level. It is mostly grown in Indian subcontinent and has been recently used for the development of genomics resources.We recently initiated a programme to develop simple sequence repeat markers and reported a set of 2469 SSR that were developed using four SSR-enriched libraries (Mir et al. 2009). In this communication, we report an additional set of 607 novel SSR in 393 SSR containing sequences. However, primers could be designed for only 417 potentially useful SSR. Polymorphism survey was carried out for 374 primer pairs using two parental genotypes (JRO 524 and PPO4) of a mapping population developed for fibre fineness; only 66 SSR were polymorphic. Owing to a low level of polymorphism between the parental genotypes and a high degree of segregation distortion in recombinant inbred lines, genotypic data of only 53 polymorphic SSR on the mapping population consisting of 120 RIL could be used for the construction of a linkage map; 36 SSR loci were mapped on six linkage groups that covered a total genetic distance of 784.3 cM. Hopefully, this map will be enriched with more SSR loci in future and will prove useful for identification of quantitative trait loci/genes for molecular breeding involving improvement of fibre fineness and other related traits in jute.     

An interspecific backcross linkage map of mouse chromosome 8

We have established a 67-cM molecular genetic linkage map of mouse chromosome 8 by interspecific backcross analysis. Genes that were mapped in this study include Act-6, Aprt, Aprt-ps1, Emv-2, Es-N, Hp, Insr, Mt-1, Plat, Psx-8, Ucp, and Zfp-4. New regions of homology were established between mouse chromosome 8 and human chromosomes 8 and 19. A conserved linkage group was identified between mouse chromosome 8 and human chromosome 16. The map will be useful for establishing linkage of other markers to mouse chromosome 8.     

An AFLP-based interspecific linkage map of sympatric, hybridizing Colias butterflies

Colias eurytheme and C. philodice are sister species with broad sympatry in North America. They hybridize frequently and likely share a significant portion of their genomes through introgression. Both taxa have been ecologically well characterized and exploited to address a broad spectrum of evolutionary issues. Using AFLP markers, we constructed the first linkage map of Colias butterflies. The map is composed of 452 markers spanning 2541.7 cM distributed over 51 linkage groups (40 major groups and 11 small groups with 2-4 markers). Statistical tests indicate that these AFLP markers tend to cluster over the map, with the coefficient of variation of interval sizes being 1.236 (95% C.I. is 1.234-1.240). This nonrandom marker distribution can account for the nonequivalence between the number of linkage groups and the actual haploid chromosome number (N = 31). This study presents the initial step for further marker-assisted research on Colias butterflies, including QTL and introgression analyses. Further investigation of the genomes will help us understand better the roles of introgression and natural selection in the evolution of hybridizing species and devise more appropriate strategies to control these pests.     

Development of a molecular linkage map of pearl millet integrating DArT and SSR markers

Pearl millet is an important component of food security in the semi-arid tropics and is assuming greater importance in the context of changing climate and increasing demand for highly nutritious food and feed. Molecular tools have been developed and applied for pearl millet on a limited scale. However, the existing tool kit needs to be strengthened further for its routine use in applied breeding programs. Here, we report enrichment of the pearl millet molecular linkage map by exploiting low-cost and high-throughput Diversity Arrays Technology (DArT) markers. Genomic representation from 95 diverse genotypes was used to develop a DArT array with circa 7,000 clones following PstI/BanII complexity reduction. This array was used to genotype a set of 24 diverse pearl millet inbreds and 574 polymorphic DArT markers were identified. The genetic relationships among the inbred lines as revealed by DArT genotyping were in complete agreement with the available pedigree data. Further, a mapping population of 140 F₇ Recombinant Inbred Lines (RILs) from cross H 77/833-2 × PRLT 2/89-33 was genotyped and an improved linkage map was constructed by integrating DArT and SSR marker data. This map contains 321 loci (258 DArTs and 63 SSRs) and spans 1148 cM with an average adjacent-marker interval length of 3.7 cM. The length of individual linkage groups (LGs) ranged from 78 cM (LG 3) to 370 cM (LG 2). This better-saturated map provides improved genome coverage and will be useful for genetic analyses of important quantitative traits. This DArT platform will also permit cost-effective background selection in marker-assisted backcrossing programs as well as facilitate comparative genomics and genome organization studies once DNA sequences of polymorphic DArT clones are available.     

A high-density genetic linkage map of bronze loquat based on SSR and RAPD markers

We constructed a high-density genetic linkage map of bronze loquat (Eriobotrya deflexa) by using a three-way cross of loquat (Eriobotrya japonica) × (loquat × bronze loquat) and simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers. The positions of the SSR loci used in this study were previously identified on reference maps of pears (Pyrus spp.) and apples (Malus spp.). The map of bronze loquat (‘Taiwan loquat No. 1’) consisted of 308 loci including 167 SSRs (8 loquat, 57 pear, and 102 apple SSRs), 140 RAPDs, and the loquat canker resistance gene Pse-a on 19 linkage groups covering a genetic distance of 1036 cM. Almost all loquat linkage groups were aligned to the pear consensus map by using at least two pear or apple SSRs, suggesting that positions and linkages of SSR loci were well conserved between loquat and pear and between loquat and apple. The constructed map may be used to determine the location of genes and quantitative trait loci of interest and to analyze genome synteny in the tribe Pyreae, subfamily Spiraeoideae of the family Rosaceae.     

Development of a second generation linkage map for almond using RAPD and SSR markers.

Fifty-four RAPD (random amplified polymorphic DNA) markers and 6 SSRs (simple sequence repeats) were included in a molecular marker map with 120 RFLPs (restriction fragment length polymorphisms) and 7 isozyme genes previously constructed using the offspring of a cross between the almond (Prunus amygdalus) cultivars 'Ferragnès' and 'Tuono'. Only highly reproducible RAPDs segregating 1:1 were used. To identify these markers, a total of 325 primers were screened, from which 41 produced RAPDs useful for mapping. Polymorphism was detected in six of the eight Prunus SSRs (simple sequence repeats) studied, thus enabling these to be mapped. All markers were placed on the 8 linkage groups previously identified. The number of new markers included in the map of 'Ferragnès' was 33 for a total of 126, and 30 in the map of 'Tuono' for a total of 99. The sizes of the maps of 'Ferragnès' (415 cM) and 'Tuono' (416 cM) were similar, representing a 5% increase over the maps constructed solely with isozymes and RFLPs. The estimated total size of the almond map was of 457 cM. Some markers were placed in zones with low density of markers and others in the extreme of linkage groups. The use of RAPD markers to complete genetic maps constructed with transferable markers is discussed.     

An interspecific backcross linkage map of the proximal half of mouse chromosome 14

We have generated a 30-cM molecular genetic linkage map of the proximal half of mouse chromosome 14 by interspecific backcross analysis. Loci that were mapped in this study include Bmp-1, Ctla-1, Hap, hr, Plau, Psp-2, Rib-1, and Tcra. A region of homology between mouse chromosome 14 and human chromosome 10 was identified by the localization of Plau to chromosome 14. This interspecific backcross map will be valuable for establishing linkage relationships of additional loci to mouse chromosome 14.     

Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus

Background and Aims

Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus.

Methods

For simple sequence repeat (SSR) isolation, a genomic library enriched for (GA)_n repeats was constructed from C. roseus ‘Nirmal’ (CrN1). In addition, GTMs were also designed from 12 genes of the TIA (terpenoid indole alkaloid) pathway – the medicinally most significant pathway in C. roseus. An F₂ mapping population was also generated by crossing two diverse accessions of C. roseus CrN1 (Nirmal)×CrN82 (Kew).

Key Results

A new set of 314 STMS markers and 64 GTMs were developed in this study. A segregating F₂ mapping population consisting of 111 F₂ individuals was generated. For generating the linkage map, a set of 423 co-dominant markers (378 newly developed and 45 published earlier) were screened for polymorphism between the parental genotypes, of which 134 were identified to be polymorphic. A total of 114 markers were mapped on eight linkage groups that spanned a 632·7 cM region of the genome with an average marker distance of 5·55 cM. Further, the mechanism of hypervariability at the gene-targeted loci was investigated at the sequence level.

Conclusions

For the first time, a large array of STMS markers and GTMs was generated in the model medicinal plant C. roseus. Moreover, the first microsatellite marker-based linkage map was described in this study. Together, these will serve as a foundation for future genomics studies related to quantitative trait loci analysis and molecular breeding in C. roseus.     

An extended genetic linkage map of markers for human chromosome 10

We have extended, in both directions, our recently published genetic map of markers for human chromosome 10 by the addition of 10 newly defined arbitrary loci. The map now covers 230 cM in males and 329 cM in females. In addition, three new markers, one of them a new RFLP at the IRBP gene locus, have been mapped in the vicinity of the locus responsible for multiple endocrine neoplasia type 2A (MEN2A). A significantly higher frequency of recombination in males than in females was observed near both ends of the new map.     

A genetic linkage map based on AFLP and SSR markers and mapping of QTL for dry-matter content in sweetpotato

We developed a genetic linkage map of sweetpotato using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers and a mapping population consisting of 202 individuals derived from a broad cross between Xushu 18 and Xu 781, and mapped quantitative trait loci (QTL) for the storage root dry-matter content. The linkage map for Xushu 18 included 90 linkage groups with 2077 markers (1936 AFLP and 141 SSR) and covered 8,184.5 cM with an average marker distance of 3.9 cM, and the map for Xu 781 contained 90 linkage groups with 1954 markers (1824 AFLP and 130 SSR) and covered 8,151.7 cM with an average marker distance of 4.2 cM. The maps described herein have the best coverage of the sweetpotato genome and the highest marker density reported to date. These are the first maps developed that have 90 complete linkage groups, which is in agreement with the actual number of chromosomes. Duplex and triplex markers were used to detect the homologous groups, and 13 and 14 homologous groups were identified in Xushu 18 and Xu 781 maps, respectively. Interval mapping was performed first and, subsequently, a multiple QTL model was used to refine the position and magnitude of the QTL. A total of 27 QTL for dry-matter content were mapped, explaining 9.0–45.1 % of the variation; 77.8 % of the QTL had a positive effect on the variation. This work represents an important step forward in genomics and marker-assisted breeding of sweetpotato.     

An EST-derived SNP and SSR genetic linkage map of cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is one of the most important food security crops in the tropics and increasingly being adopted for agro-industrial processing. Genetic improvement of cassava can be enhanced through marker-assisted breeding. For this, appropriate genomic tools are required to dissect the genetic architecture of economically important traits. Here, a genome-wide SNP-based genetic map of cassava anchored in SSRs is presented. An outbreeder full-sib (F1) family was genotyped on two independent SNP assay platforms: an array of 1,536 SNPs on Illumina's GoldenGate platform was used to genotype a first batch of 60 F1. Of the 1,358 successfully converted SNPs, 600 which were polymorphic in at least one of the parents and was subsequently converted to KBiosciences' KASPar assay platform for genotyping 70 additional F1. High-precision genotyping of 163 informative SSRs using capillary electrophoresis was also carried out. Linkage analysis resulted in a final linkage map of 1,837 centi-Morgans (cM) containing 568 markers (434 SNPs and 134 SSRs) distributed across 19 linkage groups. The average distance between adjacent markers was 3.4?cM. About 94.2% of the mapped SNPs and SSRs have also been localized on scaffolds of version 4.1 assembly of the cassava draft genome sequence. This more saturated genetic linkage map of cassava that combines SSR and SNP markers should find several applications in the improvement of cassava including aligning scaffolds of the cassava genome sequence, genetic analyses of important agro-morphological traits, studying the linkage disequilibrium landscape and comparative genomics.     

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.     

A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers.

A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 x OSU 414.062 was used. RAPD markers in testcross configuration, segregating 1:1, were used to construct separate maps for each parent. Fifty additional RAPD loci were assigned to linkage groups as accessory markers whose exact location could not be determined. Markers in intercross configuration, segregating 3:1, were used to pair groups in one parent with their homologues in the other. Eleven groups were identified for each parent, corresponding to the haploid chromosome number of hazelnut (n = x = 11). Thirty of the 31 SSR loci were able to be assigned to a linkage group. The maternal map included 249 RAPD and 20 SSR markers and spanned a distance of 661 cM. The paternal map included 271 RAPD and 28 SSR markers and spanned a distance of 812 cM. The maps are quite dense, with an average of 2.6 cM between adjacent markers. The S-locus, which controls pollen-stigma incompatibility, was placed on chromosome 5S where 6 markers linked within a distance of 10 cM were identified. A locus for resistance to eastern filbert blight, caused by Anisogramma anomala, was placed on chromosome 6R for which two additional markers tightly linked to the dominant allele were identified and sequenced. These maps will serve as a starting point for future studies of the hazelnut genome, including map-based cloning of important genes. The inclusion of SSR loci on the map will make it useful in other populations.     

An expanded genetic linkage map of Prunus based on an interspecific cross between almond and peach.

The genetic linkage map of Prunus constructed earlier and based on an interspecific F2 population resulting from a cross between almond (Prunus dulcis D.A. Webb) and peach (Prunus persica L. Batsch) was extended to include 8 isozyme loci, 102 peach mesocarp cDNAs, 11 plum genomic clones, 19 almond genomic clones, 7 resistance gene analogs (RGAs), 1 RGA-related sequence marker, 4 morphological trait loci, 3 genes with known function, 4 simple sequence repeat (SSR) loci, 1 RAPD, and 1 cleaved amplified polymorphic sequence (CAP) marker. This map contains 161 markers placed in eight linkage groups that correspond to the basic chromosome number of the genus (x = n = 8) with a map distance of 1144 centimorgans (cM) and an average marker density of 6.8 cM. Four more trait loci (Y, Pcp, D, and SK) and one isozyme locus (Mdh1) were assigned to linkage groups based on known associations with linked markers. The linkage group identification numbers correspond to those for maps published by the Arús group in Spain and the Dirlewanger group in France. Forty-five percent of the loci showed segregation distortion most likely owing to the interspecific nature of the cross and mating system differences between almond (obligate outcrosser) and peach (selfer). The Cat1 locus, known to be linked to the D locus controlling fruit acidity, was mapped to linkage group 5. A gene or genes controlling polycarpel fruit development was placed on linkage group 3, and control of senesced leaf color (in late fall season) (LFCLR) was mapped to linkage group 1 at a putative location similar to where the Y locus has also been placed.     

Genetic linkage map in sour cherry using RFLP markers

 Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS) and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS×EB. The RS linkage map consists of 126 single-dose restriction fragment (SDRF, Wu et al. 1992) markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95 SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifty three markers mapped in both parents were used as bridges between both maps and 13 sets of homologous linkage groups were identified. Homoeologous relationships among the sour cherry linkage groups could not be determined because only 15 probes identified duplicate loci. Fifty nine of the markers on the linkage maps were detected with probes used in other Prunus genetic linkage maps. Four of the sour cherry linkage groups may be homologous with four of the eight genetic linkage groups identified in peach and almond. Twenty one fragments expected to segregate in a 1 : 1 ratio segregated in a 2 : 1 ratio. Three of these fragments were used in the final map construction because they all mapped to the same linkage group. Six fragments exhibited segregation consistent with the expectations of intergenomic pairing and/or recombination. Received: 1 April 1998 / Accepted: 9 June 1998     

A genetic map of an interspecific cross in Allium based on amplified fragment length polymorphism (AFLPTM) markers

Segregation of 692 polymorphic AFLP^TM (amplified fragment length polymorphism) fragments was determined in an F₂ of the interspecific cross A. roylei x A. cepa. Two different enzyme combinations were used, PstI/MseIand EcoRI/MseI; in the latter one extra selective nucleotide was added to the MseI primer. The map based on A. cepa markers consisted of eight linkage groups with 262 markers covering 694 cM of the expected 800 cM. The map based on A. roylei markers comprised 15 linkage groups with 243 markers and had a length of 626 cM. The two maps were not integrated, and 25% of the markers remained unlinked. One of the alliinase genes and a SCAR marker linked to the disease resistance gene to downy mildew are present on this map. Of the AFLP markers, 50—80% were polymorphic between A. cepa and A. roylei; the level of polymorphic markers between different A. cepa accessions was4-8%. Received: 28 August 1998 / Accepted: 31 March 1999     

A genetic linkage map of 41 restriction fragment length polymorphism markers for human chromosome 3.

A genetic linkage map for human chromosome 3 has been constructed using 41 polymorphic DNA markers genotyped in 40 CEPH reference families. The map spans a genetic distance of 261 cM in males and 413 cM in females; the ratio of these distances (approximately 1.6 in favor of female meioses) was fairly constant across the map. Frequency of recombination was relatively uniform throughout much of the chromosome, except that in both telomeric regions recombination was more frequent than the physical distances would predict. The genetic map was basically in agreement with physical localization of 24 loci that were mapped by fluorescent in situ hybridization. This map can be used for linkage studies for genetic diseases, and it will serve as a step toward a high-resolution map for human chromosome 3.     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.