Genomic architecture of ecologically divergent body shape in a pair of sympatric crater lake cichlid fishes

Determining the genetic bases of adaptations and their roles in speciation is a prominent issue in evolutionary biology. Cichlid fish species flocks are a prime example of recent rapid radiations, often associated with adaptive phenotypic divergence from a common ancestor within a short period of time. In several radiations of freshwater fishes, divergence in ecomorphological traits — including body shape, colour, lips and jaws — is thought to underlie their ecological differentiation, specialization and, ultimately, speciation. The Midas cichlid species complex (Amphilophus spp.) of Nicaragua provides one of the few known examples of sympatric speciation where species have rapidly evolved different but parallel morphologies in young crater lakes. This study identified significant QTL for body shape using SNPs generated via ddRAD sequencing and geometric morphometric analyses of a cross between two ecologically and morphologically divergent, sympatric cichlid species endemic to crater Lake Apoyo: an elongated limnetic species (Amphilophus zaliosus) and a high‐bodied benthic species (Amphilophus astorquii). A total of 453 genome‐wide informative SNPs were identified in 240 F₂ hybrids. These markers were used to construct a genetic map in which 25 linkage groups were resolved. Seventy‐two segregating SNPs were linked to 11 QTL. By annotating the two most highly supported QTL‐linked genomic regions, genes that might contribute to divergence in body shape along the benthic–limnetic axis in Midas cichlid sympatric adaptive radiations were identified. These results suggest that few genomic regions of large effect contribute to early stage divergence in Midas cichlids.     

Confirmation and fine‐mapping of clinical mastitis and somatic cell score QTL in Nordic Holstein cattle

A genome‐wide association study of 2098 progeny‐tested Nordic Holstein bulls genotyped for 36 387 SNPs on 29 autosomes was conducted to confirm and fine‐map quantitative trait loci (QTL) for mastitis traits identified earlier using linkage analysis with sparse microsatellite markers in the same population. We used linear mixed model analysis where a polygenic genetic effect was fitted as a random effect and single SNPs were successively included as fixed effects in the model. We detected 143 SNP‐by‐trait significant associations (P < 0.0001) on 20 chromosomes affecting mastitis‐related traits. Among them, 21 SNP‐by‐trait combinations exceeded the genome‐wide significant threshold. For 12 chromosomes, both the present association study and the previous linkage study detected QTL, and of these, six were in the same chromosomal locations. Strong associations of SNPs with mastitis traits were observed on bovine autosomes 6, 13, 14 and 20. Possible candidate genes for these QTL were identified. Identification of SNPs in linkage disequilibrium with QTL will enable marker‐based selection for mastitis resistance. The candidate genes identified should be further studied to detect candidate polymorphisms underlying these QTL.     

Genetic mapping of biomass yield in three interconnected Miscanthus populations

Improving biomass yield is a major goal of Miscanthus breeding. We conducted a study on one interspecific Miscanthus sinensis × Miscanthus sacchariflorus F₁ population and two intraspecific M. sinensis F₁ populations, each of which shared a common parent. A field trial was established at Urbana, IL during spring 2011, and phenotypic data were collected in 2012 and 2013 for fourteen yield traits. Six high‐density parental genetic maps, as well as a consensus genetic map integrating M. sinensis and M. sacchariflorus, were developed via the pseudotestcross strategy for noninbred parents with ≥1214 single‐nucleotide polymorphism markers generated from restriction site‐associated DNA sequencing. We confirmed for the first time a whole‐genome duplication in M. sacchariflorus relative to Sorghum bicolor, similar to that observed previously for M. sinensis. Four quantitative trait locus (QTL) analysis methods for detecting marker‐trait associations were compared: (1) individual parental map composite interval mapping analysis, (2) individual parental map stepwise analysis, (3) consensus map single‐population stepwise analysis and (4) consensus map joint‐population stepwise analysis. These four methods detected 288, 264, 133 and 109 total QTLs, which resolved into 157, 136, 106 and 86 meta‐QTLs based on QTL congruency, respectively, including a set of 59 meta‐QTLs common to all four analysis methods. Composite interval mapping and stepwise analysis co‐identified 118 meta‐QTLs across six parental maps, suggesting high reliability of stepwise regression in QTL detection. Joint‐population stepwise analysis yielded the highest resolution of QTLs compared to the other three methods across all meta‐QTLs. Strong, frequently advantageous transgressive segregation in the three populations indicated a promising future for breeding new higher‐yielding cultivars of Miscanthus.     

Spurious linkage between markers in QTL mapping

Selective genotyping of extreme progeny is a powerful method to increase the information content per individual when looking for quantitative trait loci (QTLs) using molecular markers for which a map is known. However, if marker information from the selected individuals is used to construct the map of the markers, this can lead to distorted segregation of the markers that in turn can lead to the estimation of a spurious linkage between independently inherited markers. The mistaken estimation of linkage between independently inherited markers will occur when there are two (or more) independently inherited QTLs linked to two (or more) markers and the same individuals are used to estimate the map of the markers and to do the QTL estimation. The incorrect linkage occurs because in selecting individuals from the tails of the phenotypic distribution we will also be selecting certain combinations of the markers instead of obtaining a random sample of the true distribution of the marker genotypes. Analytical results are outlined and the analyses of a simulated data set illustrate the problems that could arise when data from individuals chosen by selective genotyping are incorrectly employed to construct a marker map. A strategy is proposed to remedy this problem.     

Development of public immortal mapping populations,molecular markers and linkage maps for rapid cycling <Emphasis Type="Italic">Brassica rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

Publicly available genomic tools help researchers integrate information and make new discoveries. In this paper, we describe the development of immortal mapping populations of rapid cycling, self-compatible lines, molecular markers, and linkage maps for Brassica rapa and B. oleracea and make the data and germplasm available to the Brassica research community. The B. rapa population consists of 160 recombinant inbred (RI) lines derived from the cross of highly inbred lines of rapid cycling and yellow sarson B. rapa. The B. oleracea population consists of 155 double haploid (DH) lines derived from an F1 cross between two DH lines, rapid cycling and broccoli. A total of 120 RFLP probes, 146 SSR markers, and one phenotypic trait (flower color) were used to construct genetic linkage maps for both species. The B. rapa map consists of 224 molecular markers distributed along 10 linkage groups (A1–A10) with a total distance of 1125.3 cM and a marker density of 5.7 cM/marker. The B. oleracea genetic map consists of 279 molecular markers and one phenotypic marker distributed along nine linkage groups (C1–C9) with a total distance of 891.4 cM and a marker density of 3.2 cM/marker. A syntenic analysis with Arabidopsis thaliana identified collinear genomic blocks that are in agreement with previous studies, reinforcing the idea of conserved chromosomal regions across the Brassicaceae.     

Population structure and genetic basis of the agronomic traits of upland cotton in China revealed by a genome‐wide association study using high‐density SNPs

Gossypium hirsutum L. represents the largest source of textile fibre, and China is one of the largest cotton‐producing and cotton‐consuming countries in the world. To investigate the genetic architecture of the agronomic traits of upland cotton in China, a diverse and nationwide population containing 503 G. hirsutum accessions was collected for a genome‐wide association study (GWAS) on 16 agronomic traits. The accessions were planted in four places from 2012 to 2013 for phenotyping. The CottonSNP63K array and a published high‐density map based on this array were used for genotyping. The 503 G. hirsutum accessions were divided into three subpopulations based on 11 975 quantified polymorphic single‐nucleotide polymorphisms (SNPs). By comparing the genetic structure and phenotypic variation among three genetic subpopulations, seven geographic distributions and four breeding periods, we found that geographic distribution and breeding period were not the determinants of genetic structure. In addition, no obvious phenotypic differentiations were found among the three subpopulations, even though they had different genetic backgrounds. A total of 324 SNPs and 160 candidate quantitative trait loci (QTL) regions were identified as significantly associated with the 16 agronomic traits. A network was established for multieffects in QTLs and interassociations among traits. Thirty‐eight associated regions had pleiotropic effects controlling more than one trait. One candidate gene, Gh_D08G2376, was speculated to control the lint percentage (LP). This GWAS is the first report using high‐resolution SNPs in upland cotton in China to comprehensively investigate agronomic traits, and it provides a fundamental resource for cotton genetic research and breeding.     

The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.)

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2–5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5–14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Narrowing down the single homoeologous FaPFRU locus controlling flowering in cultivated octoploid strawberry using a selective mapping strategy

Extending the period of fruit production is a way to substantially increase crop yield in many fruit or ornamental species. In the cultivated octoploid strawberry (Fragaria × ananassa), the most consumed small fruit worldwide, fruit production season can be extended by selecting the perpetual flowering (PF) cultivars. This trait is of considerable interest to growers and to the food industry. Four homoeologous loci controlling a single trait can be expected in such a complex octoploid species. However, we recently showed that the PF trait is under the control of the single dominant FaPFRU locus (J. Exp. Bot., 2013, 64 , 1837), making it potentially amenable to marker‐assisted selection (MAS). Here, we report the successful use of a strategy, based on a selective mapping using a reduced sample of individuals, to identify nine markers in close linkage to the FaPFRU allelic variant. Thus, this strategy can be used to fine map the target homoeologous loci in other complex polyploid crop species. Recombinant analysis further enabled us to reduce the locus to a region flanked by two markers, Bx083_206 and Bx215_131, corresponding to a 1.1 Mb region in the diploid F. vesca reference genome. This region comprises 234 genes, including 15 flowering associated genes. Among these, the FLOWERING LOCUS T (FT) is known to be a key activator of flowering. The close association between the PF trait and the FaPFRU flanking markers was validated using an additional segregating population and genetic resources. This study lays the foundation for effective and rapid breeding of PF strawberry cultivars by MAS.     

A genome‐wide association study of 23 agronomic traits in Chinese wheat landraces

Uncovering the genetic basis of agronomic traits in wheat landraces is important for ensuring global food security via the development of improved varieties. Here, 723 wheat landraces from 10 Chinese agro‐ecological zones were evaluated for 23 agronomic traits in six environments. All accessions could be clustered into five subgroups based on phenotypic data via discriminant function analysis, which was highly consistent with genotypic classification. A genome‐wide association study was conducted for these traits using 52 303 DArT‐seq markers to identify marker‐trait associations and candidate genes. Using both the general linear model and the mixed linear model, 149 significant markers were identified for 21 agronomic traits based on best linear unbiased prediction values. Considering the linkage disequilibrium decay distance in this study, significant markers within 10 cM were combined as a quantitative trait locus (QTL), with a total of 29 QTL identified for 15 traits. Of these, five QTL for heading date, flag leaf width, peduncle length, and thousand kernel weight had been reported previously. Twenty‐five candidate genes associated with significant markers were identified. These included the known vernalization genes VRN‐B1 and vrn‐B3 and the photoperiod response genes Ppd and PRR. Overall, this study should be helpful in elucidating the underlying genetic mechanisms of complex agronomic traits and performing marker‐assisted selection in wheat.     

A microsatellite-based genetic linkage map of the cichlid fish, Astatotilapia burtoni (Teleostei): a comparison of genomic architectures among rapidly speciating cichlids

Cichlid fishes compose an astonishingly large number of species and formed species flocks in record-breaking time. To facilitate efficient genome scans and comparisons of cichlid genomes, we constructed a medium-density genetic linkage map of microsatellite markers of Astatotilapia burtoni. The mapping cross was derived from two inbred laboratory lines to obtain F₂ progeny by intercrossing. The map revealed 25 linkage groups spanning 1249.3 cM of the genome (size ~950 Mb) with an average marker spacing of 6.12 cM. The seven Hox clusters, ParaHox C1, and two paralogs of Pdgfrβ were mapped to different linkage groups, thus supporting the hypothesis of a teleost-specific genome duplication. The A. burtoni linkage map was compared to the other two available maps for cichlids using shared markers that showed conservation and synteny among East African cichlid genomes. Interesting candidate genes for cichlid speciation were mapped using SNP markers.     

Genetic mapping in hybrid zones

Recent advances in genetic mapping methodologies make it feasible to localize quantitative trait loci (QTL) that contribute to adaptation and speciation. However, it has not been possible to employ these methods in many wild species because of difficulties associated with creating and propagating recombinant populations of sufficient size for QTL mapping. Natural hybrid zones contain recombinant individuals resulting from many generations of hybridization and thus offer a potential solution to these problems. For studies of speciation, hybrid zones offer the possibility of mapping QTL simultaneously with assessments of their effects on assortative mating, hybrid fitness, and interspecific gene flow. Here, we explore the problems and prospects associated with genetic map building and QTL analyses in natural hybrid zones by analyzing correlations among markers of known genomic location in four hybrid zones between the wild sunflower species Helianthus annuus and Helianthus petiolaris. Results indicate that mapping in hybrid zones presents many challenges. These include overlap in the strength of marker correlations between linked and unlinked markers, unevenness in marker frequencies along linkages, and heterogeneity in the relationship between marker distances and correlations. All make it difficult to accurately group and order markers or to estimate the distances between them. These problems can be ameliorated by sampling strategies that maximize the difference in linkage disequilibria between linked and unlinked markers and that minimize differences in frequencies among markers or QTL. In addition, studies that employ a previously determined molecular marker map for gene localization have a greater likelihood of success than those that rely on the hybrid zone data for both map construction and QTL analyses.     

Molecular mapping in oil radish (Raphanus sativus L.) and QTL analysis of resistance against beet cyst nematode (Heterodera schachtii)

The beet cyst nematode (Heterodera schachtii Schmidt) can be controlled biologically in highly infected soils of sugar beet rotations using resistant varieties of oil radish (Raphanus sativus L. ssp. oleiferus DC.) as a green crop. Resistant plants stimulate infective juveniles to invade roots, but prevent them after their penetration to complete the life cycle. The resistance trait has been transferred successfully to susceptible rapeseed by the addition of a complete radish chromosome. The aim of the study was to construct a genetic map for radish and to develop resistance-associated markers. The map with 545 RAPD, dpRAPD, AFLP and SSR markers had a length of 1,517 cM, a mean distance of 2.8 cM and consisted of nine linkage groups having sizes between 120 and 232 cM. Chromosome-specific markers for the resistance-bearing chromosome d and the other eight radish chromosomes, developed previously from a series of rapeseed-radish addition lines, were enclosed as anchor markers. Each of the extra chromosomes in the addition lines could be unambiguously assigned to one of the radish linkage groups. The QTL analysis of nematode resistance was realized in the intraspecific F₂ mapping population derived from a cross between varieties ‘Pegletta’ (nematode resistant) x ‘Siletta Nova’ (susceptible). A dominant major QTL Hs1 ^Rph explaining 46.4% of the phenotypic variability was detected in a proximal position of chromosome d. Radish chromosome-specific anchor markers with known map positions were made available for future recombination experiments to incorporate segments carrying desired genes as Hs1 ^Rph from radish into rapeseed by means of chromosome addition lines.     

Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus

Genomic rearrangements arising during polyploidization are an important source of genetic and phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among homoeologous chromosomes, due to interhomoeologue pairing, and deletions without compensating homoeologous duplications are observed in both natural B. napus and synthetic B. napus. Rearrangements of large or small chromosome segments induce gene copy number variation (CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring is difficult to deal with in linkage mapping studies. Here, we demonstrate how high‐density genetic mapping with codominant, physically anchored SNP markers can detect segmental homoeologous exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements detected in genetic mapping data by whole‐genome resequencing of parental lines along with cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC‐FISH) coupled with PCR using primers specific to the rearranged region. Using a well‐known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in narrowing the QTL to a small defined interval that enables delineation of key candidate genes.     

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.     

Characterization,development and mapping of Unigene-derived microsatellite markers in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Molecular variation within known genes controlling specific functions provide candidate gene-based markers which are tightly linked with the trait of interest. Unigene-derived microsatellite markers, with their unique identity and positions, offer the advantage of unraveling variation in the expressed component of the genome. We characterized ≥12-bp-long microsatellite loci from 13,899 unique sequences of sorghum [Sorghum bicolor (L.) Moench] available in the NCBI unigene database for their abundance and possible use in sorghum breeding. Analysis of 12,464 unigenes (≥200-bp) using MISA software identified 14,082 simple sequence repeats (SSRs) in 7,370 unigenes, from which 1,519 unigene SSR markers were developed. The average frequency of SSR was 1 per1.6 kb and 1.0 per 1.1 unigene; hexamers followed by trimers were found in abundance, of which 33.3% AT-rich and CCG repeats were the most abundant. Of the 302 unigene SSRs tested, 60 (19.8%) were polymorphic between the two parents, M35-1 and B35 of a recombinant inbred line (RIL) mapping population. A mapping population consisting of 500 RILs was developed using the above two parents, and a subset of random 245 RILs was used for genotyping with polymorphic SSRs. We developed a linkage map containing 231 markers, of which 228 (174 genomic and 54 genic) were microsatellites and three were morphological markers. Markers were distributed over 21 linkage groups, and spanned a genetic distance of 1235.5 cM. This map includes 81 new SSRs, of which 35 (21 unigene and 14 genomic) were developed in the present study and 46 from other studies. The order of the SSR markers mapped in the present study was confirmed physically by BLAST search against the whole-genome shotgun sequence of sorghum. Many unigene sequences used for marker development in this study include genes coding for important regulatory proteins and functional proteins that are involved in stress-related metabolism. The unigene SSR markers used together with other SSR markers to construct the sorghum genetic map will have applications in studies on comparative mapping, functional diversity analysis and association mapping, and for quantitative trait loci detection for drought and other agronomically important traits in sorghum.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity

Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F₂ mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F₂ plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker‐assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.