A molecular marker linkage map of tetraploid alfalfa (Medicago sativa L.)

A genetic linkage map was constructed for an F₁ genotype of auto-tetraploid alfalfa (Medicago sativa L.) using two backcross populations of 101 individuals each and 82 single-dose restriction fragments segregating in each population. The percentages of marker loci deviating from Mendelian ratios were considerably less than reported for inbred diploid mapping populations (4–9% compared to 18–54%), probably due to the greater buffering capacity of autotetraploids against the effects of deleterious recessive alleles. Four homologous coupling-phase cosegregation groups were detected for seven of the eight linkage groups of diploid alfalfa and aligned using probes in common. No cosegregation groups were found for linkage group 7 due to the lack of polymorphisms in this cross. A composite map was generated by integrating the four homologous cosegregation groups and consisted of 88 loci on seven linkage groups covering 443 cM. The locus map-orders and distances were in general agreement with those found in diploid alfalfa. The mapping population segregates for winterhardiness, fall dormancy, and freezing tolerance; and the map will be used to locate genomic regions affecting these traits. Received: 9 December 1998 / Accepted: 22 June 1999     

Construction of an improved linkage map of diploid alfalfa (Medicago sativa)

An improved genetic map of diploid (2n=2x=16) alfalfa has been developed by analyzing the inheritance of more than 800 genetic markers on the F₂ population of 137 plant individuals. The F₂ segregating population derived from a self-pollinated F₁ hybrid individual of the cross Medicago sativa ssp. quasifalcata ×Medicago sativa ssp. coerulea. This mapping population was the same one which had been used for the construction of our previous alfalfa genetic map. The genetic analyses were performed by using maximum-likelihood equations and related computer programs. The improved genetic map of alfalfa in its present form contains 868 markers (four morphological, 12 isozyme, 26 seed protein, 216 RFLP, 608 RAPD and two specific PCR markers) in eight linkage groups. Of the markers 80 are known genes, including 2 previously cytologically localized genes, the rDNA and the β-tubulin loci. The genetic map covers 754 centimorgans (cM) with an average marker density of 0.8/cM. The correlation between the physical and genetic distances is about 1000–1300 kilobase pairs per centiMorgan. In this map, the linkage relationships of some markers on linkage groups 6, 7, and 8 are different from the previously published one. The cause of this discrepancy was that the genetic linkage of markers displaying distorted segregation (characterized by an overwhelming number of heterozygous individuals) had artificially linked genetic regions that turned out to be unlinked. To overcome the disadvantageous influence of the excess number of heterozygous genotypes on the recombination fractions, we used recently described maximum-likelihood formulas and colormapping, which allowed us to exclude the misleading linkages and to estimate the genetic distances more precisely. Received: 19 October 1998 / Accepted: 15 April 1999     

Genome mapping of white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and comparative analysis within the Trifolieae using cross-species SSR markers

Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F₁ population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F₁s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC₁F₁ plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.     

Development of a composite map in Vicia faba,breeding applications and future prospects

A composite map of the Vicia faba genome based on morphological markers, isozymes, RAPDs, seed protein genes and microsatellites was constructed. The map incorporates data from 11 F₂ families for a total of 654 individuals all sharing the common female parent Vf 6. The integrated map is arranged in 14 major linkage groups (five of which were located in specific chromosomes). These linkage groups include 192 loci and cover 1,559 cM with an overall average marker interval of 8 cM. By joining data of a new F₂ population segregating for resistance to ascochyta, broomrape and others traits of agronomic interest, have been saturated new areas of the genome. The combination of trisomic segregation, linkage analysis among loci from different families with a recurrent parent, and the analysis of new physically located markers, has allowed the establishment of the present status of the V. faba map with a wide coverage. This map provides an efficient tool in breeding applications such as disease-resistance mapping, QTL analyses and marker-assisted selection.Communicated by J.W. Snape     

Comparative mapping between<Emphasis Type="Italic"> Medicago sativa</Emphasis> and<Emphasis Type="Italic"> Pisum sativum</Emphasis>

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by W. R. McCombie     

RAPD linkage mapping in a longleaf pine x slash pine F1 family

Random amplified polymorphic DNAs (RAPDs) were used to construct linkage maps of the parent of a longleaf pine (Pinus palustris Mill.) slash pine (Pinus elliottii Englm.) F₁ family. A total of 247 segregating loci [233 (1∶1), 14 (3∶1)] and 87 polymorphic (between parents), but non-segregating, loci were identified. The 233 loci segregating 1∶1 (testcross configuration) were used to construct parent-specific linkage maps, 132 for the longleaf-pine parent and 101 for the slash-pine parent. The resulting linkage maps consisted of 122 marker loci in 18 groups (three or more loci) and three pairs (1367.5 cM) for longleaf pine, and 91 marker loci in 13 groups and six pairs for slash pine (952.9 cM). Genome size estimates based on two-point linkage data ranged from 2348 to 2392 cM for longleaf pine, and from 2292 to 2372 cM for slash pine. Linkage of 3∶1 loci to testcross loci in each of the parental maps was used to infer further linkages within maps, as well as potentially homologous counterparts between maps. Three of the longleaf-pine linkage groups appear to be potentially homologous counterparts to four different slash-pine linkage groups. The number of heterozygous loci (previously testcross in parents) per F₁ individual, ranged from 96 to 130. With the 87 polymorphic, but non-segregating, loci that should also be heterozygous in the F₁ progeny, a maximum of 183–217 heterozygous loci could be available for mapping early height growth (EHG) loci and for applying genomic selection in backcross populations.     

An integrated SSR map of grapevine based on five mapping populations

A grapevine (mainly Vitis vinifera L., 2n = 38) composite genetic map was constructed with CarthaGene using segregation data from five full-sib populations of 46, 95, 114, 139 and 153 individuals, to determine the relative position of a large set of molecular markers. This consensus map comprised 515 loci (502 SSRs and 13 other type PCR-based markers), amplified using 439 primer pairs (426 SSRs and 13 others) with 50.1% common markers shared by at least two crosses. Out of all loci, 257, 85, 74, 69 and 30 were mapped in 1, 2, 3, 4 and 5 individual mapping populations, respectively. Marker order was generally well conserved between maps of individual populations, with only a few significant differences in the recombination rate of marker pairs between two or more populations. The total length of the integrated map was 1,647 cM Kosambi covering 19 linkage groups, with a mean distance between neighbour loci of 3.3 cM. A framework-integrated map was also built, with marker order supported by a LOD of 2.0. It included 257 loci spanning 1,485 cM Kosambi with a mean inter-locus distance of 6.2 cM over 19 linkage groups. These integrated maps are the most comprehensive SSR-based maps available so far in grapevine and will serve either for choosing markers evenly scattered over the whole genome or for selecting markers that cover particular regions of interest. The framework map is also a useful starting point for the integration of the V. vinifera physical and genetic maps.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Extensive macrosynteny between Medicago truncatula and Lens culinaris ssp. culinaris

The first predominantly gene-based genetic linkage map of lentil (Lens culinaris ssp. culinaris) was constructed using an F₅ population developed from a cross between the cultivars Digger (ILL5722) and Northfield (ILL5588) using 79 intron-targeted amplified polymorphic (ITAP) and 18 genomic simple sequence repeat (SSR) markers. Linkage analysis revealed seven linkage groups (LGs) comprised of 5–25 markers that varied in length from 80.2 to 274.6 cM. The genome map spanned a total length of 928.4 cM. Clear evidence of a simple and direct macrosyntenic relationship between lentil and Medicago truncatula was observed. Sixty-six out of the 71 gene-based markers, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the Lens c. ssp. culinaris and M. truncatula genomes. However, there was evidence of moderate chromosomal rearrangements which may account for the difference in chromosome numbers between these two legume species. Eighteen common SSR markers were used to connect the current map with the most comprehensive and recent map that exists for lentil, providing the syntenic context of four important domestication traits. The composite map presented, anchored with orthologous markers mapped in M. truncatula, provides a strong foundation for the future use of genomic and genetic information in lentil genetic analysis and breeding. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Development of an RFLP map in diploid alfalfa

Summary We have developed a restriction fragment length polymorphism (RFLP) linkage map in diploid alfalfa (Medicago sativa L.) to be used as a tool in alfalfa improvement programs. An F₂ mapping population of 86 individuals was produced from a cross between a plant of the W2xiso population (M. sativa ssp. sativa) and a plant from USDA PI440501 (M. sativa ssp. coerulea). The current map contains 108 cDNA markers covering 467.5 centimorgans. The short length of the map is probably due to low recombination in this cross. Marker order may be maintained in other populations even though the distance between clones may change. About 50% of the mapped loci showed segregation distortion, mostly toward excess heterozygotes. This is circumstantial evidence supporting the maximum heterozygote theory which states that relative vigor is dependent on maximizing the number of loci with multiple alleles. The application of the map to tetraploid populations is discussed.     

Microsatellite and AFLP markers in the Prunus persica [L. (Batsch)]×P. ferganensis BC1linkage map: saturation and coverage improvement

A set of 146 single sequence repeats (SSRs) and 14 amplified fragment length polymorphism (AFLP) primer combinations were used to enrich a previously developed linkage map obtained from a (Prunus persica×P. ferganensis)×P. persica BC₁ progeny. Forty-one SSR primer pairs gave polymorphic patterns detecting 42 loci. The restriction/selective primer AFLP combinations produced a total of 79 segregating fragments. The resulting map is composed of 216 loci covering 665 cM with an average distance of 3.1 cM. Novel regions were covered by the newly mapped loci for a total of 159 cM. Eight linkage groups were assembled instead of the earlier 10 as two small groups (G1a and G8b), previously independent, were joined to their respective major groups (G1b and G8a). Several gaps were also reduced resulting in an improved saturation of the map. Twelve gaps ≥10 cm are still present. A comparative analysis against the Prunus reference map (71 anchor loci) pointed out an almost complete synteny and colinearity. Six loci were not syntenic and only two were not colinear. Genetic distances were significantly longer in our map than in the reference one.     

Constructing a Genetic Linkage Map Using an F<Subscript>1</Subscript> Population of Non-inbred Parents in Cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz)

A genetic linkage map of cassava has been generated with total of 355 molecular markers, 231 amplified fragment length polymorphisms (AFLPs), 41 simple sequence repeats (SSRs), 48 sequence-related amplified polymorphisms (SRAPs), and 35 expressed sequence tag (EST)-SSRs segregating from an F₁ population of an intraspecific cross between SC6, as female parent, and Mianbao, as male parent. The genetic map consisted of 18 linkage groups and spanned a 1,707.9 cM genetic distance, with the average marker interval being 4.81 cM. Thirty-five EST-SSR markers that were developed by our laboratory were mapped onto this map. The genetic map generated in this study will enrich the information of structural genomics in cassava, facilitating to identify quantitative trait loci (QTL) of interest and to serve as a useful complement for construction of an integrated map in the future.     

Interspecific genetic linkage map, segregation distortion and genetic conversion in coffee (Coffea sp.)

An interspecific partial genetic linkage map of Coffea sp. based on 62 backcross hybrids is presented. F₁ hybrids were generated by a cross between the wild C. pseudozanguebariae and the anciently cultivated C. liberica var. dewevrei (DEW); progeny were then derived from a backcross between F₁ hybrid and DEW. The map construction consisted of a two-step strategy using 5.5 and 3.1 LOD scores revealed by simulation file. The map consisted of 181 loci: 167 amplified fragment length polymorphism (AFLP) and 13 random fragment length polymorphism (RFLP) loci. The markers were assembled into 14 linkage groups, each with 4–31 markers covering 1,144 cM. Segregation distortion was observed for 30% of all loci, in particular 3:1 and 1:3 ratios equally favouring each of the two parents. The existence of such ratios suggests genetic conversion events. This map also represents an initial step towards the detection of quantitative trait loci. Received: 4 Janaury 2000 / Accepted: 17 January 2000     

An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. sativa × L. saligna F2 populations

AFLP markers were obtained with 12 EcoRI/ MseI primer combinations on two independent F₂ populations of Lactuca sativa ×Lactuca saligna. The polymorphism rates of the AFLP products between the two different L. saligna lines was 39%, between the two different L. sativa cultivars 13% and between the L. sativa and L. saligna parents on average 81%. In both F₂ populations segregation distortion was found, but only Chromosome 5 showed skewness that was similar for both populations. Two independent genetic maps of the two F₂ populations were constructed that could be integrated due to the high similarity in marker order and map distances of 124 markers common to both populations. The integrated map consisted of 476 AFLP markers and 12 SSRs on nine linkage groups spanning 854 cM. The AFLP markers on the integrated map were randomly distributed with an average spacing between markers of 1.8 cM and a maximal distance of 16 cM. Furthermore, the AFLP markers did not show severe clustering. This AFLP map provides good opportunities for use in QTL mapping and marker-assisted selection. Received: 13 July 2000 / Accepted: 19 January 2001     

An AFLP linkage map for Douglas-fir based upon multiple full-sib families

An amplified fragment length polymorphism (AFLP) linkage map for coastal Douglas-fir (Pseudotsuga menziesii) was constructed from eight full-sib families each consisting of 40 progeny. These families were part of the British Columbia Ministry of Forests second-generation progeny test program and represent typical family sizes used in progeny trials. For map construction, ten primer pairs using EcoRI+3 and MseI+4 were employed to identify and assay AFLP loci that segregated in backcross configurations. A new technique was used to obtain a single recombination rate for each pair of marker loci: for each locus pair, a recombination rate and log-odd value were estimated across all segregating families using a joint maximum likelihood function that considered the full dataset of segregating genotypes. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 19 linkage groups spanning 938.6 cM at an average distance of 9.3 cM between markers. The simultaneous integration of data from multiple families may provide an effective way to construct a linkage map, using the genetic resources inherent in most tree improvement programs, where progeny tests of small size are conducted. The statistical property of number of families used is briefly discussed. For our data, at least three to four families greatly increased the chance of obtaining an informative locus in at least one family. Families as small as ten are adequate for closely linked loci (<10 cM), while the size used in our study (40) is adequate for loci within 30 cM.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Mapping of sequence-specific markers and loci controlling preharvest sprouting and alpha-amylase activity in rye (Secale cereale L.) on the genetic map of an F2 (S120×S76) population

Location of the loci that control preharvest sprouting and alpha-amylase activity in rye was studied based on intercross S120×S76, consisting of 110 genotypes of F₂ and F₃ progenies. The genetic map currently consists of 141 loci distributed in 11 linkage groups, covering a distance of 506.4 cM, and was enriched during this study with 24 sequence-specific markers (7 SCARs, 7 SSRs, and 10 STSs). The extended map was applied for composite interval mapping of the loci controlling preharvest sprouting and α-amylase activity, revealing 3 significant QTLs for preharvest sprouting, located on chromosomes 3R, 5R and 6R (in 1999), and one QTL for α-amylase activity found on chromosome 2R (in 2000).     

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F₂ mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.     

Structure and organization of the B genome based on a linkage map in Brassica nigra

We constructed a genetic map on Brassica nigra based on a segregating population of 83 F₂ individuals. Three different types of molecular markers were used to build the map including isozymes, restriction fragment length polymorphisms (RFLP), and random amplified polymorphic DNA (RAPD). The final map contained 124 markers distributed in 11 linkage groups. The map covered a total distance of 677 cM with the markers distributed within a mean distance of 5.5cM. Of the sequences found in the B. nigra map, 40% were duplicated and organized into three different types of arrangements. They were either scattered throughout the genome, organized in tandem, or organized in blocks of duplicated loci conserved in more than 1 linkage group.