Construction of a linkage map of Lentinula edodes (shiitake) with the HEGS (high-efficiency genome scanning) system: use of versatile AFLP and PCR-based gene markers

An improved linkage map of Lentinula edodes (shiitake) was constructed with an HEGS (high-efficiency genome scanning) system. Two hundred twenty-one HEGS-derived amplified fragment length polymorphism (AFLP-H) markers and 21 gene markers were developed and combined with 203 previously developed sequencer-derived AFLP markers (AFLP-S markers) and 3 mating factor loci (A, Bα, and Bβ) to construct a comprehensive linkage analysis. As a result, a novel linkage map with 166 markers including 2 mating factors (A and B), 10 HEGS-derived gene markers, 72 AFLP-H markers, and 82 AFLP-S markers was obtained. Of the total 448 markers, 273 could not be located on a linear map and thus were assigned to linkage groups as accessory markers. The map covers a total length of 1398.4 centimorgans (cM) with an average marker interval distance of 8.4 cM. The map consists of 11 linkage groups (LGs) in agreement with our previous map, and 7 LGs among them were found to contain branched linkages, which may be the result of reciprocal translocations representing dynamic reorganization of the shiitake genome. The previously reported linkage map was improved in terms of number of markers, marker density, linear order of markers, and total map length. Contribution no. 384 of the Tottori Mycological Institute     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

A second‐generation genetic linkage map for bighead carp (Aristichthys nobilis) based on microsatellite markers

Bighead carp (Aristichthys nobilis) is an important aquaculture fish worldwide. Genetic linkage maps for the species were previously reported, but map resolution remained to be improved. In this study, a second‐generation genetic linkage map was constructed for bighead carp through a pseudo‐testcross strategy using interspecific hybrids between bighead carp and silver carp. Of the 754 microsatellites genotyped in two interspecific mapping families (with 77 progenies for each family), 659 markers were assigned to 24 linkage groups, which were equal to the chromosome numbers of the haploid genome. The consensus map spanned 1917.3 cM covering 92.8% of the estimated bighead carp genome with an average marker interval of 2.9 cM. The length of linkage groups ranged from 52.2 to 133.5 cM with an average of 79.9 cM. The number of markers per linkage group varied from 11 to 55 with an average of 27.5 per linkage group. Normality tests on interval distances of the map showed a non‐normal marker distribution; however, significant correlation was found between the length of linkage group and the number of markers below the 0.01 significance level (two‐tailed). The length of the female map was 1.12 times that of the male map, and the average recombination ratio of female to male was 1.10:1. Visual inspection showed that distorted markers gathered in some linkage groups and in certain regions of the male and female maps. This well‐defined genetic linkage map will provide a basic framework for further genome mapping of quantitative traits, comparative mapping and marker‐assisted breeding in bighead carp.     

A microsatellite marker based framework linkage map of<Emphasis Type="Italic"> Vitis vinifera</Emphasis> L.

We have constructed a framework linkage map based on microsatellite markers for Vitis vinifera L., the European wine grape. The mapping population consisted of 153 progeny plants from a cross of Vitis vinifera cvs. Riesling × Cabernet Sauvignon. One hundred fifty-two microsatellite markers and one polymorphic EST marker have been mapped to 20 linkage groups (2n=38). The map covers 1,728 cM with an average distance between markers of 11.0 cM. Estimates of genome size, expected genome coverage, and observed genome coverage were determined with 135–140 markers. Genome length estimates differed between paternal and maternal data sets. Observed approximate genome coverage was 65% versus an expected coverage of 90%. Meiotic recombination rates were not significantly different between maternal and paternal parents. This map has been adopted as a reference map for the International Grape Genome Program.Communicated by C. Möllers     

Construction of a genetic linkage map for silver carp (Hypophthalmichthys molitrix)

For silver carp (Hypophthalmichthys molitrix), a combined microsatellite (or simple sequence repeat) and amplified fragment length polymorphism (AFLP) sex average linkage map was constructed. A total of 483 markers (245 microsatellites and 238 AFLPs) were assigned to 33 linkage groups. The map spanned 1352.2 cM, covering 86.4% of the estimated genome size of silver carp. The maximum and average spaces between 420 loci were 21.5 cM and 3.2 cM, respectively. The length of linkage groups ranged from 3.6 cM to 98.5 cM with an average of 41.0 cM. The number of markers per group varied from 2 to 44 with an average of 14.6. The AFLP markers significantly improved the integrity of microsatellite-based linkage groups and increased the genome coverage and marker evenness. A genome-wide recombination suppression was observed in male. In an extreme case, six microsatellites co-segregated in male, but spanned a 45.1 cM region in female.     

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F₂ cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.     

RFLP inheritance and linkage in walnut

Thirty-two low-copy-number genomic DNA clones from a walnut (Juglans sp.) Pst I genomic library were used to establish a molecular-marker linkage map for walnut. The clones were hybridized to restriction-endonuclease-digested DNA from parent walnut trees involved in an interspecific backcross of (J. hindsii x J. regia) x J. regia in order to identify parental polymorphism. Sixty-three backcross progeny were analyzed to determine the inheritance and linkage of 48 RFLP loci. Sixty-six percent of the walnut cloned sequences detected duplicated, but unlinked, loci. Twelve linkage groups were identified by 42 of the RFLP loci. A Poisson probability method for estimating genome size was utilized to calculate the approximate walnut genome length as 1660 cM and to estimate that 138 markers would be needed to cover 95% of the walnut genome within 20 cM of each marker.     

A genetic linkage map for azuki bean [Vigna angularis (Willd.) Ohwi & Ohashi]

To make progress in genome analysis of azuki bean (Vigna angularis) a genetic linkage map was constructed from a backcross population of (V. nepalensis x V. angularis) x V.angularis consisting of 187 individuals. A total of 486 markers—205 simple sequence repeats (SSRs), 187 amplified fragment length polymorphisms (AFLPs) and 94 restriction fragment length polymorphisms (RFLPs) —were mapped onto 11 linkage groups corresponding to the haploid chromosome number of azuki bean. This map spans a total length of 832.1 cM with an average marker distance of 1.85 cM and is the most saturated map for a Vigna species to date. In addition, RFLP markers from other legumes facilitated finding several orthologous linkage groups based on previously published RFLP linkage maps. Most SSR primers that have been developed from SSR-enriched libraries detected a single locus. The SSR loci identified are distributed throughout the azuki bean genome. This moderately dense linkage map equipped with many SSR markers will be useful for mapping a range of useful traits such as those related to domestication and stress resistance. The mapping population will be used to develop advanced backcross lines for high resolution QTL mapping of these traits. O.K. Han, A. Kaga, T. Isemura have contributed equally to this paper.     

Genetic Mapping and QTL Analysis of Growth-Related Traits in the Pacific Oyster

The Pacific oyster (Crassostrea gigas) is one of the most important oysters cultured worldwide. To analyze the oyster genome and dissect growth-related traits, we constructed a sex-averaged linkage map by combining 64 genomic simple sequence repeats, 42 expressed sequence tag-derived SSRs, and 320 amplified fragment length polymorphism markers in an F₁ full-sib family. A total of 426 markers were assigned to 11 linkage groups, spanning 558.2 cM with an average interval of 1.3 cM and 94.7% of genome coverage. Segregation distortion was significant for 18.8% of the markers (P < 0.05), and distorted markers tended to occur on some genetic regions or linkage groups. Most growth-related quantitative traits were highly significantly (P < 0.01) correlated, and principal component analysis obtained four principal components. Quantitative trait locus (QTL) analysis identified three significant QTLs for two principal components, which explained 0.6–13.9% of the phenotypic variation. One QTL for sex was detected on linkage group 6, and the inheritabilities of sex for parental alleles and maternal alleles on that locus C15 are 39.8% and 0.01%, respectively. The constructed linkage map and determined QTLs can provide a tool for further genetic analysis of the traits and be potential for marker-assisted selection in C. gigas breeding.     

A computer simulation study on the number of loci and trees required to estimate genetic variability in cacao (Theobroma cacao L.)

Current methods for measures of genetic diversity of populations and germplasm collections are often based on statistics calculated from molecular markers. The objective of this study was to investigate the precision and accuracy of the most common estimators of genetic variability and population structure, as calculated from simple sequence repeat (SSR) marker data from cacao (Theobroma cacao L.). Computer simulated genomes of replicate populations were generated from initial allele frequencies estimated using SSR data from cacao accessions in a collection. The simulated genomes consisted of ten linkage groups of 100 cM in length each. Heterozygosity, gene diversity and the F statistics were studied as a function of number of loci and trees sampled. The results showed that relatively small random samples of trees were needed to achieve consistency in the observed estimations. In contrast, very large random samples of loci per linkage group were required to enable reliable inferences on the whole genome. Precision of estimates was increased by more than 50% with an increase in sample size from one to five loci per linkage group or 50 per genome, and up to 70% with ten loci per linkage group, or equivalently, 100 loci per genome. The use of fewer, highly polymorphic loci to analyze genetic variability led to estimates with substantially smaller variance but with an upward bias. Nevertheless, the relative differences of estimates among populations were generally consistent for the different levels of polymorphism considered.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.     

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.     

Development and Characterization of a Genetic Linkage Map of Cryptococcus neoformans var. neoformans Using Amplified Fragment Length Polymorphisms and Other Markers

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.     

RAPD markers linked to a gene for resistance to pine needle gall midge in Japanese black pine (Pinus thunbergii)

Linkage of RAPD markers to a single dominant gene for resistance to pine needle gall midge was investigated in Japanese black pine (Pinus thunbergii). Three primers that generated linked markers were found after 1160 primers were screened by bulked segregant analysis. The distances between the resistance gene, R, and the marker genes OPC06580, OPD01700, and OPAX192100 were 5.1 cM, 6.7 cM and 13.6 cM, respectively. OPC06580 was in coupling phase to R, whereas OPD01700 and OPAX192100 were in repulsion phase to R. A linkage map for a resistant tree was constructed using 96 macrogametophytes. In linkage analysis, 98 out of 127 polymorphic markers were assigned to 17 linkage groups and six linked pairs. The total length of this map was 1469.8 cM, with an average marker density of 15.6 cM. The genome length was estimated to be 2138.3 cM, and the derived linkage map covered 67.5% of the genome. Although the linked markers OPC06580, OPAX192100, and OPD01700, belonged to the same linkage group, no precise positions were found for OPC06580 or OPD01700. Received: 15 May 1999 / Accepted: 29 July 1999     

An ultradense genetic recombination map for <Emphasis Type="Italic">Brassica napus</Emphasis>, consisting of 13551 SRAP markers

Sequence related amplified polymorphism (SRAP) was used to construct an ultradense genetic recombination map for a doubled haploid (DH) population in B. napus. A total of 1,634 primer combinations including 12 fluorescently labeled primers and 442 unlabeled ones produced 13,551 mapped SRAP markers. All these SRAPs were assembled in 1,055 bins that were placed onto 19 linkage groups. Ten of the nineteen linkage groups were assigned to the A genome and the remaining nine to the C genome on the basis of the differential SRAP PCR amplification in two DH lines of B. rapa and B. oleracea. Furthermore, all 19 linkage groups were assigned to their corresponding N1–N19 groups of B. napus by comparison with 55 SSR markers used to construct previous maps in this species. In total, 1,663 crossovers were detected, resulting in a map length span of 1604.8 cM. The marker density is 8.45 SRAPs per cM, and there could be more than one marker in 100 kb physical distance. There are four linkage groups in the A genome with more than 800 SRAP markers each, and three linkage groups in the C genome with more 1,000 SRAP markers each. Our studies suggest that a single SRAP map might be applicable to the three Brassica species, B. napus, B. oleracea and B. rapa. The use of this ultra high-density genetic recombination map in marker development and map-based gene cloning is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

A genetic linkage map of red drum,Sciaenops ocellatus

Second‐generation, sex‐specific genetic linkage maps were generated for the economically important estuarine‐dependent marine fish Sciaenops ocellatus (red drum). The maps were based on F₁ progeny from each of two single‐pair mating families. A total of 237 nuclear‐encoded microsatellite markers were mapped to 25 linkage groups. The female map contained 226 markers, with a total length of 1270.9 centiMorgans (cM) and an average inter‐marker interval of 6.53 cM; the male map contained 201 markers, with a total length of 1122.9 cM and an average inter‐marker interval of 6.03 cM. The overall recombination rate was approximately equal in the two sexes (♀:♂ = 1.03:1). Recombination rates in a number of linkage intervals, however, differed significantly between the same sex in both families and between sexes within families. The former occurred in 2.4% of mapped intervals, while the latter occurred in 51.2% of mapped intervals. Sex‐specific recombination rates varied within chromosomes, with regions of both female‐biased and male‐biased recombination. Original clones from which the microsatellite markers were generated were compared with genome sequence data for the spotted green puffer, Tetraodon nigroviridis; a total of 43 matches were located in 17 of 21 chromosomes of T. nigroviridis, while seven matches were in unknown portions of the T. nigroviridis genome. The map for red drum provides a new, useful tool for aquaculture, population genetics, and comparative genomics of this economically important marine species.     

The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny with Medicago truncatula

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorised users.     

A genetic map of citrus based on the segregation of isozymes and RFLPs in an intergeneric cross

Summary Isozymes and restriction fragment length polymorphisms were used as markers in the construction of a genetic map of the citrus nuclear genome. The map was based on the segregation of 8 isozyme, 1 protein, and 37 RFLP loci in 60 progeny of a cross of two intergeneric hybrids, Sacaton citrumelo (Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) and Troyer citrange (C. sinensis (L.) Osbeck x P. trifoliata), often used as rootstocks. The map contains 38 of 46 studied loci distributed on ten linkage groups. A genome size of 1,700 cM was estimated from partial linkage data. Approximately 35% of the genome should be within 10 cM and 58% within 20 cM of the mapped markers. Eight loci in three linkage groups and 1 unlinked locus deviated significantly from Mendelian segregation.     

Development and genetic mapping of microsatellite markers from whole genome shotgun sequences in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The availability of whole genome shotgun sequences (WGSs) in Brassica oleracea provides an unprecedented opportunity for development of microsatellite or simple sequence repeat (SSR) markers for genome analysis and genetic improvement in Brassica species. In this study, a total of 56,465 non-redundant SSRs were identified from the WGSs in B. oleracea, with dinucleotide repeats being the most abundant, followed by tri-, tetra- and pentanucleotide repeats. From these, 1,398 new SSR markers (designated as BoGMS) with repeat length ≥25 bp were developed and used to survey polymorphisms with a panel of six rapeseed varieties, which is the largest number of SSR markers developed for the C genome in a single study. Of these SSR markers, 752 (69.5%) showed polymorphism among the six varieties. Of these, 266 markers that showed clear scorable polymorphisms between B. napus varieties No. 2127 and ZY821 were integrated into an existing B. napus genetic linkage map. These new markers are preferentially distributed on the linkage groups in the C genome, and significantly increased the number of SSR markers in the C genome. These SSR markers will be very useful for gene mapping and marker-assisted selection of important agronomic traits in Brassica species.