Genetic linkage mapping of an annual × perennial ryegrass population

Annual (Lolium multiflorum Lam.) and perennial (L. perenne L.) ryegrass are two common forage and turfgrass species grown throughout the world. Perennial ryegrass is most commonly used for turfgrass purposes, and contamination by annual ryegrass, through physical seed mixing or gene flow, can result in a significant reduction in turfgrass quality. Seed certifying agencies in the United States currently use a test called seedling root fluorescence (SRF) to detect contamination between these species. The SRF test, however, can be inaccurate and therefore, the development of additional markers for species separation is needed. Male and female molecular-marker linkage maps of an interspecific annual × perennial ryegrass mapping population were developed to determine the map location of the SRF character and to identify additional genomic regions useful for species separation. A total of 235 AFLP markers, 81 RAPD markers, 16 comparative grass RFLPs, 106 SSR markers, 2 isozyme loci and 2 morphological characteristics, 8-h flowering, and SRF were used to construct the maps. RFLP markers from oat and barley and SSR markers from tall fescue and other grasses allowed the linkage groups to be numbered, relative to the Triticeae and the International Lolium Genome Initative reference population P150/112. The three-generation population structure allowed both male and female maps to be constructed. The male and female maps each have seven linkage groups, but differ in map length with the male map being 537 cm long and the female map 712 cm long. Regions of skewed segregation were identified in both maps with linkage groups 1, 3, and 6 of the male map showing the highest percentage of skewed markers. The (SRF) character mapped to linkage group 1 in both the male and female maps, and the 8-h flowering character was also localized to this linkage group on the female map. In addition, the Sod-1 isozyme marker, which can separate annual and perennial ryegrasses, mapped to linkage group 7. These results indicate that Lolium linkage groups 1 and 7 may provide additional markers and candidate genes for use in ryegrass species separation.Communicated by C. Möllers     

Genetic variability and QTL mapping of freezing tolerance and related traits in Medicago truncatula

Freezing is a major environmental limitation to crop productivity for a number of species including legumes. We investigated the genetic determinism of freezing tolerance in the model legume Medicago truncatula Gaertn (M. truncatula). After having observed a large variation for freezing tolerance among 15 M. truncatula accessions, the progeny of a F6 recombinant inbred line population, derived from a cross between two accessions, was acclimated to low above-freezing temperatures and assessed for: (a) number of leaves (NOL), leaf area (LA), chlorophyll content index (CCI), shoot and root dry weights (SDW and RDW) at the end of the acclimation period and (b) visual freezing damage (FD) during the freezing treatment and 2 weeks after regrowth and foliar electrolyte leakage (EL) 2 weeks after regrowth. Consistent QTL positions with additive effects for FD were found on LG1, LG4 and LG6, the latter being the most explanatory (R ² ≈ 40 %). QTL for NOL, QTL for EL, NOL and RDW, and QTL for EL and CCI colocalized with FD QTL on LG1, LG4 and LG6, respectively. Favorable alleles for these additive effects were brought by the same parent suggesting that this accession contributes to superior freezing tolerance by affecting plants’ capacity to maintain growth at low above-freezing temperatures. No epistatic effects were found between FD QTL, but for each of the studied traits, 3–6 epistatic effects were detected between loci not detected directly as QTL. These results open the way to the assessment of syntenic relationships between QTL for frost tolerance in M. truncatula and cultivated legume species.     

Association mapping, patterns of linkage disequilibrium and selection in the vicinity of the PHYTOCHROME C gene in pearl millet

Key message

Linkage analysis confirmed the association in the region of PHYC in pearl millet. The comparison of genes found in this region suggests that PHYC is the best candidate.

Abstract

Major efforts are currently underway to dissect the phenotype–genotype relationship in plants and animals using existing populations. This method exploits historical recombinations accumulated in these populations. However, linkage disequilibrium sometimes extends over a relatively long distance, particularly in genomic regions containing polymorphisms that have been targets for selection. In this case, many genes in the region could be statistically associated with the trait shaped by the selected polymorphism. Statistical analyses could help in identifying the best candidate genes into such a region where an association is found. In a previous study, we proposed that a fragment of the PHYTOCHROME C gene (PHYC) is associated with flowering time and morphological variations in pearl millet. In the present study, we first performed linkage analyses using three pearl millet F₂ families to confirm the presence of a QTL in the vicinity of PHYC. We then analyzed a wider genomic region of ~100 kb around PHYC to pinpoint the gene that best explains the association with the trait in this region. A panel of 90 pearl millet inbred lines was used to assess the association. We used a Markov chain Monte Carlo approach to compare 75 markers distributed along this 100-kb region. We found the best candidate markers on the PHYC gene. Signatures of selection in this region were assessed in an independent data set and pointed to the same gene. These results foster confidence in the likely role of PHYC in phenotypic variation and encourage the development of functional studies.     

Structural and functional mapping of α-fetoprotein

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.     

The β subunit locus of the human fibronectin receptor: DNA restriction fragment length polymorphism and linkage mapping studies

Summary The beta subunit of the human fibronectin receptor (FNRB) is a transmembrane protein belonging to the VLA (very late antigens of activation) family. Using pGEM-32, a 2.5-kb partial cDNA clone corresponding to the 3 portion of the human FNRB locus, multiple restriction fragment length polymorphisms (RFLPs) were revealed on DNAs from unrelated Caucasians. RFLPs detected by five enzymes, BanII, HinfI, KpnI, BglII, and SacI, are of the simple two-allele form, and pairwise linkage analyses of these RFLPs with numerous known DNA markers from the chromosome-10 pericentromeric region not only confirmed the chromosome-10 assignment of the functional FNRB gene but also supported its localization at p11.2 suggested by in situ hybridization. An infrequent MspI RFLP was detected by pB/R2, a 4.6-kb genomic clone from the FNRB locus. Another type of DNA polymorphism was also revealed by the cDNA clone and it was visualized on the Southern blot analyses as the presence or absence of an extra band (or a set of extra bands). It seems to stem from a stretch of DNA sequence present in some individuals at one single locus but absent in others, and is of non-chromosome-10 origin based on linkage analyses with known chromosome 10 markers. This presence/absence type of polymorphism could be revealed by all of the 25 restriction enzymes tested and is similar in nature to that previously reported with one of the human dihydrofolate reductase pseudogenes, DHFRP1. Dissection of the pGEM-32 clone demonstrated that the region revealing the non-chromosome-10 sequences is within a fragment about 1.7 kb in length extending from about 600 nucleotides preceding the stop codon down to the end of the cloned FNRB 3 untranslated region. Due to its high polymorphism information content (PIC) value (0.71 for haplotypes of BanII, HinfI, and KpnI RFLPs) and proximity to the centromere, FNRB will prove to be a highly useful marker for genetic linkage studies of multiple endocrine neoplasia type 2A (MEN2A) as well as for chromosome-10 linkage studies in general.     

Box–Cox transformation for QTL mapping

The maximum likelihood method of QTL mapping assumes that the phenotypic values of a quantitative trait follow a normal distribution. If the assumption is violated, some forms of transformation should be taken to make the assumption approximately true. The Box–Cox transformation is a general transformation method which can be applied to many different types of data. The flexibility of the Box–Cox transformation is due to a variable, called transformation factor, appearing in the Box–Cox formula. We developed a maximum likelihood method that treats the transformation factor as an unknown parameter, which is estimated from the data simultaneously along with the QTL parameters. The method makes an objective choice of data transformation and thus can be applied to QTL analysis for many different types of data. Simulation studies show that (1) Box–Cox transformation can substantially increase the power of QTL detection; (2) Box–Cox transformation can replace some specialized transformation methods that are commonly used in QTL mapping; and (3) applying the Box–Cox transformation to data already normally distributed does not harm the result.     

Characterization and study of a κ-casein-like chymosin-sensitive linkage

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of κ-caseins. In all these proteins, this linkage is observed within an exposed β-strand-like structure, as also predicted for κ-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the κ-casein/chymosin complex.     

Does probabilistic modelling of linkage disequilibrium evolution improve the accuracy of QTL location in animal pedigree?

Background

Since 2001, the use of more and more dense maps has made researchers aware that combining linkage and linkage disequilibrium enhances the feasibility of fine-mapping genes of interest. So, various method types have been derived to include concepts of population genetics in the analyses. One major drawback of many of these methods is their computational cost, which is very significant when many markers are considered. Recent advances in technology, such as SNP genotyping, have made it possible to deal with huge amount of data. Thus the challenge that remains is to find accurate and efficient methods that are not too time consuming. The study reported here specifically focuses on the half-sib family animal design. Our objective was to determine whether modelling of linkage disequilibrium evolution improved the mapping accuracy of a quantitative trait locus of agricultural interest in these populations. We compared two methods of fine-mapping. The first one was an association analysis. In this method, we did not model linkage disequilibrium evolution. Therefore, the modelling of the evolution of linkage disequilibrium was a deterministic process; it was complete at time 0 and remained complete during the following generations. In the second method, the modelling of the evolution of population allele frequencies was derived from a Wright-Fisher model. We simulated a wide range of scenarios adapted to animal populations and compared these two methods for each scenario.

Results

Our results indicated that the improvement produced by probabilistic modelling of linkage disequilibrium evolution was not significant. Both methods led to similar results concerning the location accuracy of quantitative trait loci which appeared to be mainly improved by using four flanking markers instead of two.

Conclusions

Therefore, in animal half-sib designs, modelling linkage disequilibrium evolution using a Wright-Fisher model does not significantly improve the accuracy of the QTL location when compared to a simpler method assuming complete and constant linkage between the QTL and the marker alleles. Finally, given the high marker density available nowadays, the simpler method should be preferred as it gives accurate results in a reasonable computing time.     

QTLs mapping for fruit size and shape in chromosomes 2 and 4 in pepper and a comparison of the pepper QTL map with that of tomato

Quantitative trait locus (QTL) mapping for fruit weight and shape in pepper (Capsicum spp.) was performed using C. chinense and C. frutescens introgression lines of chromosomes 2 and 4. In chromosome 2, a single major fruit-weight QTL, fw2.1, was detected in both populations that explained 62% of the trait variation. This QTL, as well as a fruit-shape QTL, fs2.1, which had a more minor effect, were localized to the tomato fruit-shape gene ovate. The cloned tomato fruit-weight QTL, fw2.2, did not play a major role in controlling fruit size variations in pepper. In chromosome 4, two fruit-weight QTLs, fw4.1 and fw4.2, were detected in the same genomic regions in both mapping populations. In addition, a single fruit-shape QTL was detected in each of the mapping populations that co-localized with one of the fruit-weight QTLs, suggesting pleiotropy or close linkage of the genes controlling size and shape. fw2.1 and fw4.2 represent major fruit-weight QTLs that are conserved in the three Capsicum species analyzed to date for fruit-size variations. Co-localization of the pepper QTLs with QTLs identified for similar traits in tomato suggests that the pepper and tomato QTLs are orthologous. Compared to fruit-shape QTLs, fruit-weight QTLs were more often conserved between pepper and tomato. This implies that different modes of selection were employed for these traits during domestication of the two Solanaceae species.S. Zygier and A. Ben Chaim contributed equally to this work.     

Simultaneous mapping of epistatic QTL in DU6i × DBA/2 mice

We have mapped epistatic quantitative trait loci (QTL) in an F₂ cross between DU6i × DBA/2 mice. By including these epistatic QTL and their interaction parameters in the genetic model, we were able to increase the genetic variance explained substantially (8.8%–128.3%) for several growth and body composition traits. We used an analysis method based on a simultaneous search for epistatic QTL pairs without assuming that the QTL had any effect individually. We were able to detect several QTL that could not be detected in a search for marginal QTL effects because the epistasis cancelled out the individual effects of the QTL. In total, 23 genomic regions were found to contain QTL affecting one or several of the traits and eight of these QTL did not have significant individual effects. We identified 44 QTL pairs with significant effects on the traits, and, for 28 of the pairs, an epistatic QTL model fit the data significantly better than a model without interactions. The epistatic pairs were classified by the significance of the epistatic parameters in the genetic model, and visual inspection of the two-locus genotype means identified six types of related genotype–phenotype patterns among the pairs. Five of these patterns resembled previously published patterns of QTL interactions.     

Genetic mapping of a major codominant QTL associated with β-carotene accumulation in watermelon

The common flesh color of commercially grown watermelon is red due to the accumulation of lycopene. However, natural variation in carotenoid composition that exists among heirloom and exotic accessions results in a wide spectrum of flesh colors. We previously identified a unique orange flesh watermelon accession (NY0016) that accumulates mainly β-carotene and no lycopene. We hypothesized this unique accession could serve as a viable source for increasing provitamin A content in watermelon. Here we characterize the mode of inheritance and genetic architecture of this trait. Analysis of testcrosses of NY0016 with yellow and red fruited lines indicated a codominant mode of action as F₁ fruits exhibited a combination of carotenoid profiles from both parents. We combined visual color phenotyping with genotyping-by-sequencing of an F_2:3 population from a cross of NY0016 by a yellow fruited line, to map a major locus on chromosome 1, associated with β-carotene accumulation in watermelon fruit. The QTL interval is approximately 20 cM on the genetic map and 2.4 Mb on the watermelon genome. Trait-linked marker was developed and used for validation of the QTL effect in segregating populations across different genetic backgrounds. This study is a step toward identification of a major gene involved in carotenoid biosynthesis and accumulation in watermelon. The codominant inheritance of β-carotene provides opportunities to develop, through marker-assisted breeding, β-carotene-enriched red watermelon hybrids.     

Linkage disequilibrium analyses and restriction mapping of four RFLPs at the proα2(I) collagen locus: lack of correlation between linkage disequilibrium and physical distance

Summary Restriction fragment length polymorphisms (RRLPs) located at short distances may demonstrate linkage disequilibrium. Under the assumption that the distances between the loci of the RFLPs are inversely related to the linkage disequilibria, gene order may be deduced. However, if the assumption is invalid, the results may be incorrect. We have studied four different DNA polymorphisms at the COLIA2 locus in 180 unrelated Norwegian individuals. Observed frequencies (presence/absence) for the different polymorphic sites were as follows: site A (EcoRI) 0.30/0.70, site B (MspI) 0.83/0.16, site C (StuI) 0.86/0.14, and site D (RsaI) 0.66/0.34. Of 16 possible haplotypes 12 were demonstrated, and 2 additional were deduced to be present. Restriction mapping of the four polymorphic sites gave the following order of the sites from the 5 to the 3 of the gene: A-D-B-C. Linkage disequilibrium was not found between the sites A and D; strong disequilibrium was found between sites A and C, and B and C; and less strong, between A and B, B and D, and C and D. Analysis of linkage disequilibrium coefficients between all pairs of loci demonstrated that there is no consistent relationship between linkage disequilibrium and physical distance (=-0.07). These results suggest that for a small region of the genome, factors such as deviating mutation rate and gene conversion may add significantly to rearrangements by recombination. Thus, a deduced gene order from linkage disequilibrium data has to be regarded with great caution.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

Prepulse inhibition （PPI） of tactile startle response in recombinant congenic strains of mice： QTL mapping and comparison with acoustic PPI

Prepulse inhibition (PPI) of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems.We analyzed the tactile startle response (TSR) and PPI of TSR (tPPD,using light as a prepulse stimulus,in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them.Parental strains were significantly different for TSR,but were comparable for tPPI.Among the congenic strains,variation for TSR was significant in both genetic backgrounds,but that of tPPI was significant only for the C57BL/6J background.Provisional mapping for loci modulating TSR and tPPI was carded out.Using mapping data from our previous study on acoustic startle responses (ASR) and PPI of ASR (aPPI),no common markers for aPPI and tPPI were identified.However,some markers were significantly associated with both ASR and TSIL at least in one genetic background.These results indicate cross-modal genetic regulation for the startle response but not for PPI,in these mouse strains.     

Syntenic mapping and chromosomal localization of bovine α and β interferon genes

The previous assignment of bovine -(IFNA) and -(IFNB) interferon gene families to syntenic group U18 was confirmed with additional cDNA probes and a bovine-rodent hybrid somatic cell panel representing all 29 bovine autosomal syntenic groups. Fluorescent in situ hybridization (FISH) localized these genes to bovine Chromosome (Chr) 8 band 15 and demonstrates that with biotinylated plasmids, as few as five tandemly arrayed sequences can be detected by conventional fluorescent microscopy. This technique can be applied to physical mapping of other multicopy genes in domestic animals.     

An updated ‘Essex’ by ‘Forrest’ linkage map and first composite interval map of QTL underlying six soybean traits

DNA marker maps based on single populations are the basis for gene, loci and genomic analyses. Individual maps can be integrated to produce composite maps with higher marker densities if shared marker orders are consistent. However, estimates of marker order in composite maps must include sets of markers that were not polymorphic in multiple populations. Often some of the pooled markers were not codominant, or were not correctly scored. The soybean composite map was composed of data from five separate populations based on northern US germplasm but does not yet include ‘Essex’ by ‘Forrest’ recombinant inbred line (RIL) population (E × F) or any southern US soybean cultivars. The objectives were, to update the E × F map with codominant markers, to compare marker orders among this map, the Forrest physical map and the composite soybean map and to compare QTL identified by composite interval maps to the earlier interval maps. Two hundred and thirty seven markers were used to construct the core of the E × F map. The majority of marker orders were consistent between the maps. However, 19 putative marker inversions were detected on 12 of 20 linkage groups (LG). Eleven marker distance compressions were also found. The number of inverted markers ranged from 1 to 2 per LG. Thus, marker order inversions may be common in southern compared to northern US germplasm. A total of 61 QTL among 37 measures of six traits were detected by composite interval maps, interval maps and single point analysis. Seventeen of the QTL found in composite intervals had previously been detected among the 29 QTL found in simple interval maps. The genomic locations of the known QTL were more closely delimited. A genome sequencing project to compare Southern and Northern US soybean cultivars would catalog and delimit inverted regions and the associated QTL. Gene introgression in cultivar development programs would be accelerated.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Mapping and confirmation of a new sudden death syndrome resistance QTL on linkage group D2 from the soybean genotypes PI 567374 and ‘Ripley’

The use of resistant cultivars is the most effective method for controlling sudden death syndrome (SDS), caused by Fusarium solani f. sp. glycines (FSG) (syn. Fusarium virguliforme Akoi, O’Donnell, Homma and Lattanzi), in soybean [Glycine max (L.) Merr.]. Previous research has led to the identification of soybean genotypes with partial resistance to SDS and quantitative trait loci (QTL) controlling this resistance. The objective of our study was to map QTL conferring SDS resistance in populations developed from the crosses Ripley × Spencer (R×S-1) and PI 567374 × Omaha (P×O-1). Both Ripley and PI 567374 have partial resistance to SDS and Spencer and Omaha are susceptible. The R×S-1 population was evaluated for SDS resistance in three field environments and the P×O-1 population was greenhouse evaluated. Three SDS resistance QTL were mapped in the R×S-1 population and two in the P×O-1 population. One resistance QTL was mapped to the same location on linkage group (LG) D2 in both backgrounds. This QTL was then tested in a population of F₂ plants developed through one backcross (BC1F₂) in the PI 567374 source and in a population of F₈ plants derived from a heterozygous F₅ plant in the Ripley source. The LG D2 QTL was also significant in confirmation populations in both resistant backgrounds. Since none of the SDS resistance QTL identified in the R×S-1 or P×O-1 populations mapped to previously reported SDS resistance regions, these new QTL should be useful sources of SDS resistance for soybean breeders.     

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment com-bined with high chloroform Carnory’s fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromo-some 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed.Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

Physical mapping shows close linkage between the α-galactosidase A gene (GLA) and the DXS178 locus

X-linked agammaglobulinaemia (XLA) is an inherited disorder characterised by a lack of circulating B-cells and antibodies. While the gene involved in XLA has not yet been identified, the locus for the disorder is tightly linked to the polymorphic marker DXS178, which maps to Xq22. Fabry disease is an X-linked recessive disorder caused by a deficiency in the lysosomal enzyme -galactosidase A. The gene encoding this enzyme has been characterized and also maps to Xq22. Using pulsed field gel electrophoresis we have constructed a long-range restriction map that shows that the -galactosidase A gene (GLA) and DXS178 lie no more than 140 kb apart on a stretch of DNA containing a number of putative CpG islands. We have also isolated yeast artifical chromosome (YAC) clones that confirm this physical linkage. The localisation of DXS178 near the -galactosidase A gene will facilitate carrier detection in Fabry families using restriction fragment length polymorphism (RFLP) analysis. The identification of a number of CpG islands near DXS178 also provides candidate locations for the gene responsible for XLA.