Development of an RFLP map in diploid alfalfa

Summary We have developed a restriction fragment length polymorphism (RFLP) linkage map in diploid alfalfa (Medicago sativa L.) to be used as a tool in alfalfa improvement programs. An F₂ mapping population of 86 individuals was produced from a cross between a plant of the W2xiso population (M. sativa ssp. sativa) and a plant from USDA PI440501 (M. sativa ssp. coerulea). The current map contains 108 cDNA markers covering 467.5 centimorgans. The short length of the map is probably due to low recombination in this cross. Marker order may be maintained in other populations even though the distance between clones may change. About 50% of the mapped loci showed segregation distortion, mostly toward excess heterozygotes. This is circumstantial evidence supporting the maximum heterozygote theory which states that relative vigor is dependent on maximizing the number of loci with multiple alleles. The application of the map to tetraploid populations is discussed.     

Prevalence of segregation distortion in diploid alfalfa and its implications for genetics and breeding applications

Segregation distortion (SD) is often observed in plant populations; its presence can affect mapping and breeding applications. To investigate the prevalence of SD in diploid alfalfa (Medicago sativa L.), we developed two unrelated segregating F₁ populations and one F₂ population. We genotyped all populations with SSR markers and assessed SD at each locus in each population. The three maps were syntenic and largely colinear with the Medicago truncatula genome sequence. We found genotypic SD for 24 and 34% of markers in the F₁ populations and 68% of markers in the F₂ population; distorted markers were identified on every linkage group. The smaller percentage of genotypic SD in the F₁ populations could be because they were non-inbred and/or due to non-fully informative markers. For the F₂ population, 60 of 90 mapped markers were distorted, and they clustered into eight segregation distortion regions (SDR). Most SDR identified in the F₁ populations were also identified in the F₂ population. Genotypic SD was primarily due to zygotic rather than allelic distortion, suggesting zygotic not gametic selection is the main cause of SD. On the F₂ linkage map, distorted markers in all SDR except two showed heterozygote excess. The severe SD in the F₂ population likely biased genetic distances among markers and possibly also marker ordering and could affect QTL mapping of agronomic traits. To reduce the effects of SD and non-fully informative markers, we suggest constructing linkage maps and conducting QTL mapping in advanced generation populations.     

Chromosomal regions associated with segregation distortion in maize

Segregation distortion skews the genotypic frequencies from their Mendelian expectations. Our objectives in this study were to assess the frequency of occurrence of segregation distortion in maize, identify chromosomal regions consistently associated with segregation distortion, and examine the effects of gametophytic factors on linkage mapping. We constructed a simple sequence repeat (SSR) linkage map for a LH200/LH216 F₂Syn3 (i.e., random-mated three times) population, and compared the segregation distortion in this map with the segregation distortion in three published linkage maps. Among 1,820 codominant markers across the four mapping populations, 301 (17%) showed segregation distortion (P < 0.05). The frequency of markers showing segregation distortion ranged from 19% in the Tx303/CO159 mapping population to 36% in the B73/Mo17 mapping population. A positive relationship was found between the number of meioses and the frequency of segregation distortion detected in a population. On a given chromosome, nearly all of the markers showing segregation distortion favored the allele from the same parent. A total of 18 chromosomal regions on the ten maize chromosomes were associated with segregation distortion. The consistent location of these chromosomal regions in four populations suggested the presence of segregation distortion regions (SDRs). Three known gametophytic factors are possible genetic causes of these SDRs. As shown in previous research, segregation distortion does not affect the estimate of map distance when only one gametophytic factor is present in an SDR.     

An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.)

A genetic map constructed from a population segregating for a trait of interest is required for QTL identification. The goal of this study was to construct a molecular map of tetraploid alfalfa (Medicago sativa.) using simple sequence repeat (SSR) markers derived primarily from expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) inserts of M. truncatula. This map will be used for the identification of drought tolerance QTL in alfalfa. Two first generation backcross populations were constructed from a cross between a water-use efficient, M. sativa subsp. falcata genotype and a low water-use efficient M. sativa subsp. sativa genotype. The two parents and their F₁ were screened with 1680 primer pairs designed to amplify SSRs, and 605 single dose alleles (SDAs) were amplified. In the F₁, 351 SDAs from 256 loci were mapped to 41 linkage groups. SDAs not inherited by the F₁, but transmitted through the recurrent parents and segregating in the backcross populations, were mapped to 43 linkage groups, and 44 of these loci were incorporated into the composite maps. Homologous linkage groups were joined to form eight composite linkage groups representing the eight chromosomes of M. sativa. The composite maps consist of eight composite linkage groups with 243 SDAs from M. truncatula EST sequences, 38 SDAs from M. truncatula BAC clone sequences, and five SDAs from alfalfa genomic SSRs. The total composite map length is 624 cM, with average marker density per composite linkage group ranging from 1.5 to 4.4 cM, and an overall average density of 2.2 cM. Segregation distortion was 10%, and distorted loci tended to cluster on individual homologues of several linkage groups. Electronic Supplementary Material Supplementary material is available for this article at     

A molecular marker linkage map of tetraploid alfalfa (Medicago sativa L.)

A genetic linkage map was constructed for an F₁ genotype of auto-tetraploid alfalfa (Medicago sativa L.) using two backcross populations of 101 individuals each and 82 single-dose restriction fragments segregating in each population. The percentages of marker loci deviating from Mendelian ratios were considerably less than reported for inbred diploid mapping populations (4–9% compared to 18–54%), probably due to the greater buffering capacity of autotetraploids against the effects of deleterious recessive alleles. Four homologous coupling-phase cosegregation groups were detected for seven of the eight linkage groups of diploid alfalfa and aligned using probes in common. No cosegregation groups were found for linkage group 7 due to the lack of polymorphisms in this cross. A composite map was generated by integrating the four homologous cosegregation groups and consisted of 88 loci on seven linkage groups covering 443 cM. The locus map-orders and distances were in general agreement with those found in diploid alfalfa. The mapping population segregates for winterhardiness, fall dormancy, and freezing tolerance; and the map will be used to locate genomic regions affecting these traits. Received: 9 December 1998 / Accepted: 22 June 1999     

A first linkage map of globe artichoke (Cynara cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP and microsatellite markers

We present the first genetic maps of globe artichoke (Cynara cardunculus var. scolymus L. 2n=2x=34), constructed with a two-way pseudo-testcross strategy. A F₁ mapping population of 94 individuals was generated between a late-maturing, non-spiny type and an early-maturing spiny type. The 30 AFLP, 13 M-AFLP and 9 S-SAP primer combinations chosen identified, respectively, 352, 38 and 41 polymorphic markers. Of 32 microsatellite primer pairs tested, 12 identified heterozygous loci in one or other parent, and 7 were fully informative as they segregated in both parents. The female parent map comprised 204 loci, spread over 18 linkage groups and spanned 1330.5 cM with a mean marker density of 6.5 cM. The equivalent figures for the male parent map were 180 loci, 17 linkage groups, 1239.4 and 6.9 cM. About 3% of the AFLP and AFLP-derived markers displayed segregation distortion with a P value below 0.01, and were not used for map construction. All the SSR loci were included in the linkage analysis, although one locus did show some segregation distortion. The presence of 78 markers in common to both maps allowed the alignment of 16 linkage groups. The maps generated provide a firm basis for the mapping of agriculturally relevant traits, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.     

Analysis of Segregation Distortion of Molecular Markers in F2 Population of Rice

A genetic linkage map comprising 148 SSR markers loci was constructed using an F₂ population consisting of 90 lines derived from a sub-specific cross between a japonica variety Nipponbare and an indica variety Guangluai-4. The F₂ population showed high significantly distorted segregations. Among these SSR markers, 49 markers (33.11%) showed the genetics distortion(P<0.05). Of them, 36 markers deviated toward male parent indica GuangLuAi-4 and 13 markers toward heterozygote, but none toward the female parent Nipponbare. It was found that the segregation distortion might be caused by gametophyte and zygote. Since most gametophyte loci and sterility loci were mapped in segregation distortion regions, it indicated that the segregation distortion may be caused by these gametophyte loci and sterility loci. Finally, this research also analyzed the skewed segregation of some markers, which had not been mapped on chromosome.     

A genetic linkage map of Guinea yam ( Dioscorea rotundata Poir.) based on AFLP markers

A genetic linkage map of the tetraploid white yam (Dioscorea rotundata Poir.) was constructed based on 341 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F₁ cross. The F₁ mapping population was produced by crossing a landrace cultivar TDr 93-1 as female parent to a breeding line TDr 87/00211 as the male parent. The marker segregation data were split into maternal and paternal data sets, and separate genetic linkage maps were constructed since the mapping population was an F₁ cross between two presumed heterozygous parents. The markers segregated like a diploid cross-pollinator population suggesting that the D. rotundata genome is an allo-tetraploid (2n = 4x = 40). The maternal map comprised 155 markers mapped on 12 linkage groups with a total map length of 891 cM. Three linkage groups consisted of maternal parent markers only. The paternal map consisted of 157 markers mapped on 13 linkage groups with a total map length of 852 cM. Three and one quantitative trait loci (QTLs) with effects on resistance to Yam Mosaic Virus (YMV) were identified on the maternal and paternal linkage maps, respectively. Prospects for detecting more QTLs and using marker-assisted selection in white yam breeding appear good, but this is subject to the identification of additional molecular markers to cover more of the genome.     

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

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

Modeling segregation distortion for viability selection I. Reconstruction of linkage maps with distorted markers

Molecular markers have been widely used to map quantitative trait loci (QTL). The QTL mapping partly relies on accurate linkage maps. The non-Mendelian segregation of markers, which affects not only the estimation of genetic distance between two markers but also the order of markers on a same linkage group, is usually observed in QTL analysis. However, these distorted markers are often ignored in the real data analysis of QTL mapping so that some important information may be lost. In this paper, we developed a multipoint approach via Hidden Markov chain model to reconstruct the linkage maps given a specified gene order while simultaneously making use of distorted, dominant and missing markers in an F₂ population. The new method was compared with the methods in the MapManager and Mapmaker programs, respectively, and verified by a series of Monte Carlo simulation experiments along with a working example. Results showed that the adjusted linkage maps can be used for further QTL or segregation distortion locus (SDL) analysis unless there are strong evidences to prove that all markers show normal Mendelian segregation.