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     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

An RFLP linkage map for loblolly pine based on a three-generation outbred pedigree

A genetic linkage map for loblolly pine (Pinus taeda L.) was constructed using segregation data from a three-generation outbred pedigree consisting of four grandparents, two parents, and 95 F₂ progeny. The map was based predominantly on restriction fragment length polymorphism (RFLP) loci detected by cDNA probes. Sixty-five cDNA and three genomic DNA probes revealed 90 RFLP loci. Six polymorphic isozyme loci were also scored. One-fourth (24%) of the cDNA probes detected more than 1 segregating locus, an indication that multigene families are common in pines. As many as six alleles were observed at a single segregating locus among grandparents and it was not unusual for the progeny to segregate for three or four alleles per locus. Multipoint linkage analysis placed 73 RFLP and 2 isozyme loci into 20 linkage groups; the remaining 17 RFLP and 4 isozyme loci were unlinked. The mapped RFLP probes provide a new set of codominant markers for genetic analyses in loblolly pine.     

Analysis of a Detailed Genetic Linkage Map of Lactuca Sativa (Lettuce) Constructed from RFLP and Rapd Markers

A detailed genetic map has been constructed from the F(2) population of a single intraspecific cross of Lactuca sativa (n = 9). It comprises 319 loci, including 152 restriction fragment length polymorphism (RFLP), 130 random amplified polymorphic DNA (RAPD), 7 isozyme, 19 disease resistance, and 11 morphological markers. Thirteen major, four minor linkage groups and several unlinked markers are identified for this genome which is estimated to be approximately 1950 cM. RFLP and RAPD markers show similar distributions throughout the genome and identified similar levels of polymorphism. RAPD loci were much quicker to identify but more difficult to order. Procedures for generating accurate genetic maps and their limitations are described.     

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.     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP,STS, and RAPD markers

We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F₁ produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F₂ generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400–2800 cM.Abbreviations RFLP restriction fragment length polymorphism - STS sequence-tagged site - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome.

We have constructed a tomato genetic linkage map based on an intraspecific cross between two inbred lines of Lycopersicon esculentum and L. esculentum var. cerasiforme. The segregating population was composed of 153 recombinant inbred lines. This map is comprised of one morphological, 132 RFLP (restriction fragment length polymorphism, including 16 known-function genes), 33 RAPD (random amplified polymorphic DNA), and 211 AFLP (amplified fragment length polymorphism) loci. We compared the 3 types of markers for their polymorphism, segregation, and distribution over the genome. RFLP, RAPD, and AFLP methods revealed 8.7%, 15.8%, and 14.5% informative bands, respectively. This corresponded to polymorphism in 30% of RFLP probes, 32% of RAPD primers, and 100% of AFLP primer combinations. Less deviation from the 1:1 expected ratio was obtained with RFLP than with AFLP loci (8% and 18%, respectively). RAPD and AFLP markers were not randomly distributed over the genome. Most of them (60% and 80%, respectively) were grouped in clusters located around putative centromeric regions. This intraspecific map spans 965 cM with an average distance of 8.3 cM between markers (of the framework map). It was compared to other published interspecific maps of tomato. Despite the intraspecific origin of this map, it did not show any increase in length when compared to the high-density interspecific map of tomato.     

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L.

An F₂ population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including 3 morphological character genes, 4 disease resistance genes, 56 RFLP loci, 4 microsatellite loci and 2 RAPD loci. Molecular markers linked to each resistance gene were found: Fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er) and pea common Mosaic virus (15 cM from mo). QTLs (quantitative traits loci) for Ascochyta pisi race C resistance were mapped, with most of the variation explained by only three chromosomal regions. The QTL with the largest effect, on chromosome 4, was also mapped using a qualitative, Mendelian approach. Another QTL displayed a transgressive segregation, i.e. the parental line that was susceptible to Ascochyta blight had a resistance allele at this QTL. Analysis of correlations between developmental traits in terms of QTL effects and positions suggested a common genetic control of the number of nodes and earliness, and a loose relationship between these traits and height.     

Construction of a genetic linkage map in celery using DNA-based markers.

A F2 population of two celery cultivated types (Apium graveolens L. var. rapaceum and A. graveolens L. var. secalinum) was used to construct a linkage map consisting of 29 RFLP (restriction fragment length polymorphism), 100 RAPD (random amplified polymorphic DNA), four isozyme, one disease resistance, and one growth habit markers. The map contains 11 major groups and 9 small groups and has a total length of 803 cM with an average distance of 6.4 cM between two adjacent loci. Ten percent of the RAPDs segregated as codominant markers and their allelic homologies were tested by Southern hybridization. One-quarter of the dominant RAPDs were linked in repulsion phase, whereas the majority of them were linked to either codominant or dominant markers in coupling phase. About 10% of the markers showed significant segregation distortion. The detectable level of duplications in the celery genome was relatively low.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Linkage relationships among molecular markers and storage root traits of carrot (Daucus carota L. ssp. sativus)

 A 109-point linkage map consisting of three phenotypic loci (P ₁, Y ₂, and Rs), six restriction fragment length polymorphisms (RFLPs), two random amplified polymorphic DNAs (RAPDs), 96 amplified fragment length polymorphisms (AFLPs), and two selective amplification of microsatellite polymorphic loci (SAMPL) was constructed for carrot (Daucus carota L. ssp. sativus; 2n=2x=18). The incidence of polymorphism was 36% for RFLP probes, 20% for RAPD primers, and 42% for AFLP primers. The overall incidence of disturbed segregation was 18%. Linkage relationships at a LOD score of 4.0 and θ=0.25 indicated 11 linkage groups. The total map length was 534.4 cM and the map was clearly unsaturated with markers spaced at 4.9 cM. AFLP P6B15 was 1.7 cM from P ₁, AFLP P1B34 was 2.2 cM from Y ₂, and AFLP P3B30XA was 8.1 cM from Rs. Received: 2 September 1998 / Accepted: 28 November 1998     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

A linkage map of hexaploid oat based on grass anchor DNA clones and its relationship to other oat maps.

A cultivated oat linkage map was developed using a recombinant inbred population of 136 F6:7 lines from the cross 'Ogle' x 'TAM O-301'. A total of 441 marker loci, including 355 restriction fragment length polymorphism (RFLP) markers, 40 amplified fragment length polymorphisms (AFLPs), 22 random amplified polymorphic DNAs (RAPDs), 7 sequence-tagged sites (STSs), 1 simple sequence repeat (SSR), 12 isozyme loci, and 4 discrete morphological traits, was mapped. Fifteen loci remained unlinked, and 426 loci produced 34 linkage groups (with 2-43 loci each) spanning 2049 cM of the oat genome (from 4.2 to 174.0 cM per group). Comparisons with other Avena maps revealed 35 genome regions syntenic between hexaploid maps and 16-34 regions conserved between diploid and hexaploid maps. Those portions of hexaploid oat maps that could be compared were completely conserved. Considerable conservation of diploid genome regions on the hexaploid map also was observed (89-95%); however, at the whole-chromosome level, colinearity was much lower. Comparisons among linkage groups, both within and among Avena mapping populations, revealed several putative homoeologous linkage group sets as well as some linkage groups composed of segments from different homoeologous groups. The relationships between many Avena linkage groups remain uncertain, however, due to incomplete coverage by comparative markers and to complications introduced by genomic duplications and rearrangements.