Construction Of High-density Genetic Map Research Articles

High-Density Genetic Mapping with Interspecific Hybrids of Two Sea Urchins, Strongylocentrotus nudus and S. intermedius, by RAD Sequencing

Sea urchins have long been used as research model organisms for developmental biology and evolutionary studies. Some of them are also important aquaculture species in East Asia. In this work, we report the construction of RAD-tag based high-density genetic maps by genotyping F1 interspecific hybrids derived from a crossing between a female sea urchin Strongylocentrotus nudus and a male Strongylocentrotus intermedius. With polymorphisms present in these two wild individuals, we constructed a female meiotic map containing 3,080 markers for S. nudus, and a male meiotic map for S. intermedius which contains 1,577 markers. Using the linkage maps, we were able to anchor a total of 1,591 scaffolds (495.9 Mb) accounting for 60.8% of the genome assembly of Strongylocentrotus purpuratus. A genome-wide scan resulted in the identification of one putative QTL for body size which spanned from 25.3 cM to 30.3 cM. This study showed the efficiency of RAD-Seq based high-density genetic map construction using F1 progenies for species with no prior genomic information. The genetic maps are essential for QTL mapping and are useful as framework to order and orientate contiguous scaffolds from sea urchin genome assembly. The integration of the genetic map with genome assembly would provide an unprecedented opportunity to conduct QTL analysis, comparative genomics, and population genetics studies.

Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing.

Genetic maps in barley are usually constructed from a limited number of molecular markers such as SSR (simple sequence repeat) and DarT (diversity arrays technology). These markers must be first developed before being used for genotyping. Here, we introduce a new strategy based on sequencing progeny of a doubled haploid population from Baudin × AC Metcalfe to construct a genetic map in barley. About 13,547 polymorphic SNP tags with >93% calling rate were selected to construct the genetic map. A total of 12,998 SNP tags were anchored to seven linkage groups which spanned a cumulative 967.6 cM genetic distance. The high-density genetic map can be used for QTL mapping and the assembly of WGS and BAC contigs. The genetic map was evaluated for its effectiveness and efficiency in QTL mapping and candidate gene identification. A major QTL for plant height was mapped at 105.5 cM on chromosome 3H. This QTL with LOD value of 13.01 explained 44.5% of phenotypic variation. This strategy will enable rapid and efficient establishment of high-density genetic maps in other species.

BAC-End Sequence-Based SNP Mining in Allotetraploid Cotton (Gossypium) Utilizing Resequencing Data, Phylogenetic Inferences, and Perspectives for Genetic Mapping.

A bacterial artificial chromosome library and BAC-end sequences for cultivated cotton (Gossypium hirsutum L.) have recently been developed. This report presents genome-wide single nucleotide polymorphism (SNP) mining utilizing resequencing data with BAC-end sequences as a reference by alignment of 12 G. hirsutum L. lines, one G. barbadense L. line, and one G. longicalyx Hutch and Lee line. A total of 132,262 intraspecific SNPs have been developed for G. hirsutum, whereas 223,138 and 470,631 interspecific SNPs have been developed for G. barbadense and G. longicalyx, respectively. Using a set of interspecific SNPs, 11 randomly selected and 77 SNPs that are putatively associated with the homeologous chromosome pair 12 and 26, we mapped 77 SNPs into two linkage groups representing these chromosomes, spanning a total of 236.2 cM in an interspecific F2 population (G. barbadense 3-79 × G. hirsutum TM-1). The mapping results validated the approach for reliably producing large numbers of both intraspecific and interspecific SNPs aligned to BAC-ends. This will allow for future construction of high-density integrated physical and genetic maps for cotton and other complex polyploid genomes. The methods developed will allow for future Gossypium resequencing data to be automatically genotyped for identified SNPs along the BAC-end sequence reference for anchoring sequence assemblies and comparative studies.

The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing.

Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti ‘FenDanBai’ × P. × suffruticosa ‘HongQiao’, to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 ‘SNP-only’ markers, 18 ‘InDel-only’ markers, and 56 ‘SNP&InDel’ markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony.

Development and Integration of Genome-Wide Polymorphic Microsatellite Markers onto a Reference Linkage Map for Constructing a High-Density Genetic Map of Chickpea.

The identification of informative in silico polymorphic genomic and genic microsatellite markers by comparing the genome and transcriptome sequences of crop genotypes is a rapid, cost-effective and non-laborious approach for large-scale marker validation and genotyping applications, including construction of high-density genetic maps. We designed 1494 markers, including 1016 genomic and 478 transcript-derived microsatellite markers showing in-silico fragment length polymorphism between two parental genotypes (Cicer arietinum ICC4958 and C. reticulatum PI489777) of an inter-specific reference mapping population. High amplification efficiency (87%), experimental validation success rate (81%) and polymorphic potential (55%) of these microsatellite markers suggest their effective use in various applications of chickpea genetics and breeding. Intra-specific polymorphic potential (48%) detected by microsatellite markers in 22 desi and kabuli chickpea genotypes was lower than inter-specific polymorphic potential (59%). An advanced, high-density, integrated and inter-specific chickpea genetic map (ICC4958 x PI489777) having 1697 map positions spanning 1061.16 cM with an average inter-marker distance of 0.625 cM was constructed by assigning 634 novel informative transcript-derived and genomic microsatellite markers on eight linkage groups (LGs) of our prior documented, 1063 marker-based genetic map. The constructed genome map identified 88, including four major (7–23 cM) longest high-resolution genomic regions on LGs 3, 5 and 8, where the maximum number of novel genomic and genic microsatellite markers were specifically clustered within 1 cM genetic distance. It was for the first time in chickpea that in silico FLP analysis at genome-wide level was carried out and such a large number of microsatellite markers were identified, experimentally validated and further used in genetic mapping. To best of our knowledge, in the presently constructed genetic map, we mapped highest number of new sequence-based robust microsatellite markers (634) which is an advancement over the previously documented (~300 markers) inter-specific genetic maps. This advanced high-density map will serve as a foundation for large-scale marker validation and genotyping applications, including identification and targeted mapping of trait-specific genes/QTLs (quantitative trait loci) with sub-optimal use of resources and labour in chickpea.

High-density genetic linkage map construction and QTL mapping of grain shape and size in the wheat population Yanda1817 × Beinong6.

High-density genetic linkage maps are necessary for precisely mapping quantitative trait loci (QTLs) controlling grain shape and size in wheat. By applying the Infinium iSelect 9K SNP assay, we have constructed a high-density genetic linkage map with 269 F 8 recombinant inbred lines (RILs) developed between a Chinese cornerstone wheat breeding parental line Yanda1817 and a high-yielding line Beinong6. The map contains 2431 SNPs and 128 SSR & EST-SSR markers in a total coverage of 3213.2 cM with an average interval of 1.26 cM per marker. Eighty-eight QTLs for thousand-grain weight (TGW), grain length (GL), grain width (GW) and grain thickness (GT) were detected in nine ecological environments (Beijing, Shijiazhuang and Kaifeng) during five years between 2010–2014 by inclusive composite interval mapping (ICIM) (LOD≥2.5). Among which, 17 QTLs for TGW were mapped on chromosomes 1A, 1B, 2A, 2B, 3A, 3B, 3D, 4A, 4D, 5A, 5B and 6B with phenotypic variations ranging from 2.62% to 12.08%. Four stable QTLs for TGW could be detected in five and seven environments, respectively. Thirty-two QTLs for GL were mapped on chromosomes 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 6B, 7A and 7B, with phenotypic variations ranging from 2.62% to 44.39%. QGl.cau-2A.2 can be detected in all the environments with the largest phenotypic variations, indicating that it is a major and stable QTL. For GW, 12 QTLs were identified with phenotypic variations range from 3.69% to 12.30%. We found 27 QTLs for GT with phenotypic variations ranged from 2.55% to 36.42%. In particular, QTL QGt.cau-5A.1 with phenotypic variations of 6.82–23.59% was detected in all the nine environments. Moreover, pleiotropic effects were detected for several QTL loci responsible for grain shape and size that could serve as target regions for fine mapping and marker assisted selection in wheat breeding programs.

Development of chromosome-specific markers with high polymorphism for allotetraploid cotton based on genome-wide characterization of simple sequence repeats in diploid cottons (Gossypium arboreum L. and Gossypium raimondii Ulbrich).

BackgroundTetraploid cotton contains two sets of homologous chromosomes, the At- and Dt-subgenomes. Consequently, many markers in cotton were mapped to multiple positions during linkage genetic map construction, posing a challenge to anchoring linkage groups and mapping economically-important genes to particular chromosomes. Chromosome-specific markers could solve this problem. Recently, the genomes of two diploid species were sequenced whose progenitors were putative contributors of the At- and Dt-subgenomes to tetraploid cotton. These sequences provide a powerful tool for developing chromosome-specific markers given the high level of synteny among tetraploid and diploid cotton genomes. In this study, simple sequence repeats (SSRs) on each chromosome in the two diploid genomes were characterized. Chromosome-specific SSRs were developed by comparative analysis and proved to distinguish chromosomes.ResultsA total of 200,744 and 142,409 SSRs were detected on the 13 chromosomes of Gossypium arboreum L. and Gossypium raimondii Ulbrich, respectively. Chromosome-specific SSRs were obtained by comparing SSR flanking sequences from each chromosome with those from the other 25 chromosomes. The average was 7,996 per chromosome. To confirm their chromosome specificity, these SSRs were used to distinguish two homologous chromosomes in tetraploid cotton through linkage group construction. The chromosome-specific SSRs and previously-reported chromosome markers were grouped together, and no marker mapped to another homologous chromosome, proving that the chromosome-specific SSRs were unique and could distinguish homologous chromosomes in tetraploid cotton. Because longer dinucleotide AT-rich repeats were the most polymorphic in previous reports, the SSRs on each chromosome were sorted by motif type and repeat length for convenient selection. The primer sequences of all chromosome-specific SSRs were also made publicly available.ConclusionChromosome-specific SSRs are efficient tools for chromosome identification by anchoring linkage groups to particular chromosomes during genetic mapping and are especially useful in mapping of qualitative-trait genes or quantitative trait loci with just a few markers. The SSRs reported here will facilitate a number of genetic and genomic studies in cotton, including construction of high-density genetic maps, positional gene cloning, fingerprinting, and genetic diversity and comparative evolutionary analyses among Gossypium species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1265-2) contains supplementary material, which is available to authorized users.

Construction of a SNP-based high-density genetic map for pummelo using RAD sequencing

Pummelo (Citrus grandis) is one of the most important gene pools for citrus breeding programmes. A high-density linkage map is a valuable tool for functional genomics and genetic breeding studies. A newly developed genome sequence-based marker technology, restriction site-associated DNA (RAD) sequencing, has proven to be powerful for the rapid discovery and genotyping of genome-wide SNP markers and for the high-density genetic map construction. We present the construction of a high-density genetic map of pummelo using RAD sequencing. An F1 population of 124 individuals and its parents (‘Pingshan’ pummelo and ‘Guanxi’ pummelo) were applied to the map construction. One thousand five hundred forty three high-quality single nucleotide polymorphism (SNP) markers were developed and mapped to nine linkage groups. In addition, 20 simple sequence repeat (SSR) markers were included and showed general consistency with the SNP markers. These 1563 markers constituted a total genetic length of 976.58 cM and an average of 0.62 cM between adjacent loci. The number of markers within each linkage group (LG) ranged from 81 (for LG4) to 285 (for LG2). A comparison of the genetic maps to the published sweet orange (Citrus sinensis) genome revealed both conservation and variations. The alignment of the LGs from this map was also shown in comparison with a previously genetic linkage map from pummelo. This study showed that the RAD sequencing allowed the rapid discovery of a large number of SNPs in the pummelo. The SNP-based high-density genetic map for pummelo was successfully generated by using these designed SNP markers. The completed genetic map is a valuable resource for further pummelo genetic studies and provides useful information for gene positional cloning, MAS breeding and C. grandis genome assembly.

A high-density genetic map of cucumber derived from Specific Length Amplified Fragment sequencing (SLAF-seq).

High-density genetic map provides an essential framework for accurate and efficient genome assembly and QTL fine mapping. Construction of high-density genetic maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. In this research, a high-density genetic map of cucumber (Cucumis sativus L.) was successfully constructed across an F2 population by a recently developed Specific Length Amplified Fragment sequencing (SLAF-seq) method. In total, 18.69 GB of data containing 93,460,000 paired-end reads were obtained after preprocessing. The average sequencing depth was 44.92 in the D8 (female parent), 42.16 in the Jin5-508 (male parent), and 5.01 in each progeny. 79,092 high-quality SLAFs were detected, of which 6784 SLAFs were polymorphic, and 1892 of the polymorphic markers met the requirements for constructing genetic map. The genetic map spanned 845.87 cm with an average genetic distance of 0.45 cm. It is a reliable linkage map for fine mapping and molecular breeding of cucumber for its high marker density and well-ordered markers.

High-density genetic map construction and QTLs analysis of grain yield-related traits in sesame (Sesamum indicum L.) based on RAD-Seq techonology.

BackgroundSesame (Sesamum indicum L., 2n = 26) is an important oilseed crop with an estimated genome size of 369 Mb. The genetic basis, including the number and locations of quantitative trait loci (QTLs) of sesame grain yield and quality remain poorly understood, due in part to the lack of reliable markers and genetic maps. Here we report on the construction of a hitherto most high-density genetic map of sesame using the restriction-site associated DNA sequencing (RAD-seq) combined with 89 PCR markers, and the identification of grain yield-related QTLs using a recombinant inbred line (RIL) population.ResultIn total, 3,769 single-nucleotide polymorphism (SNP) markers were identified from RAD-seq, and 89 polymorphic PCR markers were identified including 44 expressed sequence tag-simple sequence repeats (EST-SSRs), 10 genomic-SSRs and 35 Insertion-Deletion markers (InDels). The final map included 1,230 markers distributed on 14 linkage groups (LGs) and was 844.46 cM in length with an average of 0.69 cM between adjacent markers. Using this map and RIL population, we detected 13 QTLs on 7 LGs and 17 QTLs on 10 LGs for seven grain yield-related traits by the multiple interval mapping (MIM) and the mixed linear composite interval mapping (MCIM), respectively. Three major QTLs had been identified using MIM with R2 > 10.0% or MCIM with ha2 > 5.0%. Two co-localized QTL groups were identified that partially explained the correlations among five yield-related traits.ConclusionThree thousand eight hundred and four pairs of new DNA markers including SNPs and InDels were developed by RAD-seq, and a so far most high-density genetic map was constructed based on these markers in combination with SSR markers. Several grain yield-related QTLs had been identified using this population and genetic map. We report here the first QTL mapping of yield-related traits with a high-density genetic map using a RIL population in sesame. Results of this study solidified the basis for studying important agricultural traits and implementing marker-assisted selection (MAS) toward genetic improvement in sesame.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0274-7) contains supplementary material, which is available to authorized users.

High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers.

Pear (Pyrus spp) is an important fruit crop, grown in all temperate regions of the world, with global production ranked after grape and apples among deciduous tree crops. A high-density linkage map is a valuable tool for fine mapping quantitative trait loci (QTL) and map-based gene cloning. In this study, we firstly constructed a high-density linkage map of pear using SNPs integrated with SSRs, developed by the rapid and robust technology of restriction-associated DNA sequencing (RADseq). The linkage map consists of 3143 SNP markers and 98 SSRs, 3241 markers in total, spanning 2243.4 cM, with an average marker distance of 0.70 cM. Anchoring SSRs were able to anchor seventeen linkage groups to their corresponding chromosomes. Based on this high-density integrated pear linkage map and two years of fruit phenotyping, a total of 32 potential QTLs for 11 traits, including length of pedicel (LFP), single fruit weight (SFW), soluble solid content (SSC), transverse diameter (TD), vertical diameter (VD), calyx status (CS), flesh colour (FC), juice content (JC), number of seeds (NS), skin colour (SC), and skin smooth (SS), were identified and positioned on the genetic map. Among them, some important fruit-related traits have for the first time been identified, such as calyx status, length of pedicel, and flesh colour, and reliable localization of QTLs were verified repeatable. This high-density linkage map of pear is a worthy reference for mapping important fruit traits, QTL identification, and comparison and combination of different genetic maps.

Construction of a SNP-based genetic linkage map in cultivated peanut based on large scale marker development using next-generation double-digest restriction-site-associated DNA sequencing (ddRADseq).

BackgroundCultivated peanut, or groundnut (Arachis hypogaea L.), is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). In recent years, many efforts have been made to construct linkage maps in cultivated peanut, but almost all of these maps were constructed using low-throughput molecular markers, and most show a low density, directly influencing the value of their applications. With advances in next-generation sequencing (NGS) technology, the construction of high-density genetic maps has become more achievable in a cost-effective and rapid manner. The objective of this study was to establish a high-density single nucleotide polymorphism (SNP)-based genetic map for cultivated peanut by analyzing next-generation double-digest restriction-site-associated DNA sequencing (ddRADseq) reads.ResultsWe constructed reduced representation libraries (RRLs) for two A. hypogaea lines and 166 of their recombinant inbred line (RIL) progenies using the ddRADseq technique. Approximately 175 gigabases of data containing 952,679,665 paired-end reads were obtained following Solexa sequencing. Mining this dataset, 53,257 SNPs were detected between the parents, of which 14,663 SNPs were also detected in the population, and 1,765 of the obtained polymorphic markers met the requirements for use in the construction of a genetic map. Among 50 randomly selected in silico SNPs, 47 were able to be successfully validated. One linkage map was constructed, which was comprised of 1,685 marker loci, including 1,621 SNPs and 64 simple sequence repeat (SSR) markers. The map displayed a distribution of the markers into 20 linkage groups (LGs A01–A10 and B01–B10), spanning a distance of 1,446.7 cM. The alignment of the LGs from this map was shown in comparison with a previously integrated consensus map from peanut.ConclusionsThis study showed that the ddRAD library combined with NGS allowed the rapid discovery of a large number of SNPs in the cultivated peanut. The first high density SNP-based linkage map for A. hypogaea was generated that can serve as a reference map for cultivated Arachis species and will be useful in genetic mapping. Our results contribute to the available molecular marker resources and to the assembly of a reference genome sequence for the peanut.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-351) contains supplementary material, which is available to authorized users.

Current status and perspective of RAD-seq in genomic research

The restriction-site associated DNA sequencing (RAD-seq) is a high-throughput sequencing technique developed from the next-generation sequencing (NGS). This method can reduce the representation of the complex genome while mapping thousands of polymorphic markers with or without a reference genome. It has been extensively used for high-density genetic map construction, fine mapping of important genes, genome sequence assembly, population genomic research, as well as phylogenetic research and so on. Here, we introduce the technological principle and development of RAD-seq combined with the sequencing applications in various species. Due to its uniqueness, RAD-seq will have a wide application in genetic analysis of complex genomic research in the future.

Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes

BackgroundGenetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.FindingsShiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.ConclusionsWe used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.

Correction: Construction of High-Density Genetic Linkage Maps and Mapping of Growth-Related Quantitative Trail Loci in the Japanese Flounder (Paralichthys olivaceus)

Correction: Construction of High-Density Genetic Linkage Maps and Mapping of Growth-Related Quantitative Trail Loci in the Japanese Flounder (Paralichthys olivaceus)

Construction of high-density genetic linkage maps and mapping of growth-related quantitative trail loci in the Japanese flounder (Paralichthys olivaceus).

High-density genetic linkage maps were constructed for the Japanese flounder (Paralichthys olivaceus). A total of 1624 microsatellite markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 resulted in the mapping of 1487 markers to 24 linkage groups, a result which was consistent with the 24 chromosomes seen in chromosome spreads. The female map was composed of 1257 markers, covering a total of 1663.8 cM with an average interval 1.35 cM between markers. The male map consisted of 1224 markers, spanning 1726.5 cM, with an average interval of 1.44 cM. The genome length in the Japanese flounder was estimated to be 1730.3 cM for the females and 1798.0 cM for the males, a coverage of 96.2% for the female and 96.0% for the male map. The mean recombination at common intervals throughout the genome revealed a slight difference between sexes, i.e. 1.07 times higher in the male than female. High-density genetic linkage maps are very useful for marker-assisted selection (MAS) programs for economically valuable traits in this species and for further evolutionary studies in flatfish and vertebrate species. Furthermore, four quantiative trait loci (QTL) associated with growth traits were mapped on the genetic map. One QTL was identified for body weight on LG 14 f, which explained 14.85% of the total variation of the body weight. Three QTL were identified for body width on LG14f and LG14m, accounting for 16.75%, 13.62% and 13.65% of the total variation in body width, respectively. The additive effects were evident as negative values. There were four QTL for growth traits clustered on LG14, which should prove to be very useful for improving growth traits using molecular MAS.

Molecular markers and their application in genetic diversity of penaeid shrimp

Penaeidae is a family of shrimp, and it contains many species of economic importance, such as the tiger prawn (Penaeus monodon), white leg shrimp, Atlantic white shrimp and Indian prawn. Identification and population genotype structure of penaeid shrimp have been enhanced by molecular markers that can be classified into three types, namely allozyme, mitochondrial and nuclear markers. The widely used mitochondrial DNA markers are 12S rDNA, 16S rDNA, cytochrome b and control region. Random amplification of polymorphic DNA, amplified fragment length polymorphism, restriction fragment length polymorphism, single-stranded conformational polymorphism and microsatellites are the most commonly used nuclear markers for DNA fingerprinting. Molecular markers play a crucial role in penaeid shrimp to evaluate phenotypic and genetic variation, assess demographic bottleneck, study natural population structure, compare wild and hatchery populations, preserve genetic biodiversity, construct chromosome maps and detect whether genetic tag propagation–assisted rehabilitation programs are effective. Increase in the number of molecular markers, construction of high-density genetic maps and implementation of genomic resources (including genome sequencing) are expected to provide tools for the genetic improvement in these aquaculture species through marker-assisted selection. Molecular markers are versatile tools for the identification of populations with genetic crisis by comparing genetic diversities, which helps to establish management units within these threatened species.

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-009-1059-5) contains supplementary material, which is available to authorized users.

Barley Genomics: An Overview

Barley (Hordeum vulgare), first domesticated in the Near East, is a well-studied crop in terms of genetics, genomics, and breeding and qualifies as a model plant for Triticeae research. Recent advances made in barley genomics mainly include the following: (i) rapid accumulation of EST sequence data, (ii) growing number of studies on transcriptome, proteome, and metabolome, (iii) new modeling techniques, (iv) availability of genome-wide knockout collections as well as efficient transformation techniques, and (v) the recently started genome sequencing effort. These developments pave the way for a comprehensive functional analysis and understanding of gene expression networks linked to agronomically important traits. Here, we selectively review important technological developments in barley genomics and related fields and discuss the relevance for understanding genotype-phenotype relationships by using approaches such as genetical genomics and association studies. High-throughput genotyping platforms that have recently become available will allow the construction of high-density genetic maps that will further promote marker-assisted selection as well as physical map construction. Systems biology approaches will further enhance our knowledge and largely increase our abilities to design refined breeding strategies on the basis of detailed molecular physiological knowledge.