Marker-assisted introgression using non-unique marker alleles I: selection on the presence of linked marker alleles

This paper investigates marker-assisted introgression of a major gene into an outbred line, where identification of the introgressed gene is incomplete because marker alleles are not unique to the base populations (the same marker allele can occur in both donor and recipient population). Those markers are used to identify the introgressed allele as well as the background genotype. The effect of using those markers, as if they were completely informative on the retention of the introgressed allele, was examined over five generations of backcrossing by using a single marker or a marker bracket for different starting frequencies of the marker alleles. Results were calculated by using both a deterministic approach, where selection is only for the desired allele, and by a stochastic approach, where selection is also on background genotype. When marker allele frequencies in donor and recipient population diverged from 1 and 0 (using a diallelic marker), the ability to retain the desired allele rapidly declined. Marker brackets performed notably better than single markers. If selection on background marker genotype was applied, the desired allele could be lost even more quickly than expected at random because the chance that the allele, which is common in the donor line, is present on the locus identifying the introgressed allele and is surrounded by alleles common in the recipient line on the background marker loci, will descend from the donor line (double recombination has taken place), is a lot smaller than the chance that this allele will stem from the recipient line (in which the allele occurs in low frequency). Marker brackets again performed better. Preselection against marker homozygotes (producing uninformative gametes) gave a slightly better retention of the introgressed allele.     

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1S6/ACT2S6 double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1S6 allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1W138/act1S6 allele pair in a two-element dTph1 transposition system by producing 10,000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.     

AB-QTL analysis reveals new alleles associated to proline accumulation and leaf wilting under drought stress conditions in barley (Hordeum vulgare L.)

ABSTRACT: BACKGROUND: Land plants have evolved several measures to maintain their life against abiotic stresses. The accumulation of proline is the most generalized response of plants under drought, heat or salt stress conditions. It is known as an osmoprotectant which also acts as an instant source of energy during drought recovery process. But, both its role and genetic inheritance are poorly understood in agriculture crops. In the present work, advanced backcross quantitative trait locus (AB-QTL) analysis was performed to elucidate genetic mechanisms controlling proline accumulation and leaf wilting in barley under drought stress conditions. RESULTS: The analysis revealed eight QTL associated to proline content (PC) and leaf wilting (WS). QTL for PC were localized on chromosome 3 H, 4 H, 5 H and 6 H. The strongest QTL effect QPC.S42.5 H was detected on chromosome 5 H where drought inducible exotic allele was associated to increase PC by 54%. QTL effects QPC.S42.3 H, QPC.S42.4 H and QPC.S42.6 H were responsible to heighten PC due to the preeminence of elite alleles over the exotic alleles which ranged from 26% to 43%. For WS, QTL have been localized on chromosome 1 H, 2 H, 3 H and 4 H. Among these, QWS.S42.1 H and QWS.S42.4 H were associated to decrease in WS due to the introgression of exotic alleles. In addition, two digenic epistatic interaction effects were detected for WS where the additive effect of exotic alleles imparted a favorable increase in the trait value. CONCLUSIONS: The present data represents a first report on whole-genome mapping of proline accumulation and leaf wilting in barley. The detected QTL are linked to new alleles from both cultivated and wild accessions which bring out an initial insight on the genetic inheritance of PC and WS. These QTL alleles are fixed in the isogenic background of Scarlett, which will allow for positional cloning of underlying genes and to develop drought resilient barley cultivars.     

Evaluation of QTL alleles from exotic sources for hybrid seed yield in the original and different genetic backgrounds of spring-type <Emphasis Type="Italic">Brassica napus</Emphasis> L.

Previously identified alleles at quantitative trait loci (QTL) for hybrid seed yield were re-evaluated in the same genetic background (in hybrid combination with the same tester) as the original QTL mapping study and also evaluated in a different genetic background (in hybrid combination with two different testers). The QTL were identified from wide crosses of exotic germplasm sources with spring-type Brassica napus L., in which alleles from the exotic germplasm sources increased hybrid seed yield. Results from the re-evaluation of six QTL, in the same genetic background and hybrid combination, indicate that several of the exotic donor QTL alleles did increase hybrid seed yield and could be successfully used for improving the original single-cross hybrid. However, results from the evaluation of seven QTL (including the same six previous QTL) in a new genetic background, in combination with two new testers, indicate that the exotic QTL alleles were often no different or produced significantly lower hybrid seed yield than the spring QTL alleles. In all studies, the QTL were also very sensitive to environmental interactions. Thus, our results indicate that although these exotic sources contain favorable QTL alleles when introgressed into one spring hybrid background, the effects are not predictive of other genetic backgrounds or hybrid combinations. Although QTL affecting hybrid seed yield have been identified, comparisons of multiple QTL alleles are needed to determine the most favorable allele at each locus. Characterization of QTL complementation across testers will be required to predict their effects in multiple hybrid combinations.     

Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker

Grain protein content (GPC) is important for human nutrition and has a strong influence on pasta and bread quality. A quantitative trait locus, derived from a Triticum turgidum ssp. dicoccoides accession (DIC), with an average increase in GPC of 14 g kg(-1) was mapped on chromosome 6BS. Using the wheat-rice colinearity, a high-density map of the wheat region was developed and the quantitative trait locus was mapped as a simple Mendelian locus designated Gpc-B1. A physical map of approx. 250 kb of the Gpc-B1 region was developed using a tetraploid wheat bacterial artificial chromosome library. The constructed physical map included the two Gpc-B1 flanking markers and one potential candidate gene from the colinear rice region completely linked to Gpc-B1. The relationship between physical and genetic distances and the feasibility of isolating genes by positional cloning in wheat are discussed. A high-throughput codominant marker, Xuhw89, was developed. A 4-bp deletion present in the DIC allele was absent in a collection of 117 cultivated tetraploid and hexaploid wheat germplasm, suggesting that this marker will be useful to incorporate the high GPC allele from the DIC accession studied here into commercial wheat varieties.     

Marker-assisted introgression using non-unique marker alleles II: selection on probability of presence of the introgressed allele

If marker alleles that identify a gene for introgression are not completely unique to the different base populations, the trait allele can be lost quickly during the process of backcrossing. This study considers ways to deal with incompletely informative markers in order to retain the desired allele. Selection was based on the probability of the presence of the desired (introgressed) trait allele, which was calculated for each marker genotype, using a single marker or a diallelic or triallelic marker bracket. The percentage of individuals retaining the introgressed allele was calculated over five generations of backcrossing, for selected fractions between 0 and 1, for marker alleles that could occur in both base populations. The best results were obtained with a rather large selected fraction, when all individuals, heterozygous and homozygous for the most desirable allele at the marker loci, were selected. Additional selection against marker homozygotes (which might have the highest probability of carrying the desired-trait allele, but produce uninformative gametes) altered the optimum selected fraction, making the selected fraction more consistently inversely related to a better retention of the desired-trait allele. A marker bracket was found to give a better retention of the desired-trait allele than a single marker and triallelic markers were better than diallelic markers, giving a retention of almost 50%. The earlier that preselection of parents (on informativeness) took place the better the overall result; preselection should occur preferably in the base populations. Preselection could make marker alleles unique to alternative base populations and markers would effectively become fully informative. Selection in the base populations might not be possible or not desirable, for example, because of the available number of individuals. This is unlikely to be a problem when parents are paired up to exclude any common marker alleles.     

Efficient use of DNA molecular markers to construct industrial yeast strains

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure.     

Effect of an introgression from Aegilops cylindrica host on manifestation of productivity traits in winter common wheat F2 plants

The effect of introgression of a chromosome 1D segment from Aegilops cylindrica to winter common wheat on productivity traits in F2 plants was studied using storage protein loci as genetic markers. An allele of the gliadin-coding Gli-D1 locus served as a marker of the introgression. Using of two- and three-locus interaction models, it was shown that the introgression tagged with Gli-D1 affected the manifestation of productivity traits (productive tillering, grain weight per plant and grain number per plant) through interaction with other marker storage protein loci: Glu-B1, Glu-D1, and Gli-B2.     

Development of candidate introgression lines using an exotic barley accession (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> ssp.<Emphasis Type="Italic"> spontaneum</Emphasis>) as donor

In the present paper, we report on the selection of two sets of candidate introgression lines (pre-ILs) in spring barley. Two BC₂DH populations, S42 and T42, were generated by introgressing an accession of Hordeum vulgare ssp. spontaneum (ISR42-8, from Israel) into two different spring barley cultivars, Scarlett (S) and Thuringia (T). From these BC₂DH populations two sets with 49 (S42) and 43 (T42) pre-ILs were selected, and their genomic architecture as revealed by SSR marker analysis was characterised. The selected pre-ILs cover at least 98.1% (S42) and 93.0% (T42) of the exotic genome in overlapping introgressions and contain on average 2 (S42) and 1.5 (T42) additional non-target introgressions. In order to illustrate a potential application and validation of these pre-ILs, the phenotypic effect of the exotic introgression at the locus of the major photoperiod response gene Ppd-H1 was analysed. Pre-ILs carrying the introgression at the Ppd-H1 locus flowered significantly earlier than the elite parents, and the introgression maintained its effect across the two genetic backgrounds and across four tested environments. The selected pre-ILs represent a first promising step towards the assessment and utilization of genetic variation present in exotic barley. They may promote the breeding progress, serve for the verification of QTL effects and provide a valuable resource for the unravelling of gene function, e.g. by expression profiling or map-based cloning.     

Simulating genealogies of selected alleles in a population of variable size

An importance-sampling method is presented that allows the simulation of the history of a selected allele in a population of variable size. A sample path describing the number of copies of an allele that arose as a single mutant is generated by simulating backwards from the current frequency until the allele is lost. The mathematical expectation of a quantity or statistic is then estimated by taking averages over replicate simulations, weighting each replicate by the ratio of its probabilities under the Markov chains for the forward and backwards processes. This method was used to find the average age of a selected allele in an exponentially growing population. In terms of the effect on average allele age, selection in favour of an allele is not equivalent to exponential growth. To generate gene genealogies of a sample of copies of a selected allele, the neutral coalescent model is simulated for the subpopulation containing only the selected allele. From the resulting intra-allelic genealogy, it is possible to calculate the likelihood of the selection intensity as a function of the observed level of variability at marker loci closely linked to the selected allele. This method was used to estimate the intensity of selection affecting the delta 32 allele at the CCR5 locus in Europeans and a mutant at the MLH1 locus associated with colorectal cancer in the Finnish population.     

Assessment of parent-of-origin effects in linkage analysis of quantitative traits

Methods are presented for incorporation of parent-of-origin effects into linkage analysis of quantitative traits. The estimated proportion of marker alleles shared identical by descent is first partitioned into a component derived from the mother and a component derived from the father. These parent-specific estimates of allele sharing are used in variance-components or Haseman-Elston methods of linkage analysis so that the effect of the quantitative-trait locus carried on the maternally derived chromosome is potentially different from the effect of the locus on the paternally derived chromosome. Statistics for linkage between trait and marker loci derived from either or both parents are then calculated, as are statistics for testing whether the effect of the maternally derived locus is equal to that of the paternally derived locus. Analyses of data simulated for 956 siblings from 263 nuclear families who had participated in a linkage study revealed that type I error rates for these statistics were generally similar to nominal values. Power to detect an imprinted locus was substantially increased when analyzed with a model allowing for parent-of-origin effects, compared with analyses that assumed equal effects; for example, for an imprinted locus accounting for 30% of the phenotypic variance, the expected LOD score was 4.5 when parent-of-origin effects were incorporated into the analysis, compared with 3.1 when these effects were ignored. The ability to include parent-of-origin effects within linkage analysis of quantitative traits will facilitate genetic dissection of complex traits.     

Current status and future possibilities of molecular genetics techniques in <Emphasis Type="Italic">Brassica napus</Emphasis>

As PCR methods have improved over the last 15 years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.     

Linkage analysis of "necessary" disease loci versus "susceptibility" loci.

The association of some diseases with specific alleles of certain genetic markers has been difficult to explain. Several explanations have been proposed for the phenomenon of association, e.g. the existence of multiple, interacting genes (epistasis) or a disease locus in linkage disequilibrium with the marker locus. One might suppose that when marker data from families with associated diseases are analyzed for linkage, the existence of the association would assure that linkage will be found, and found at a tight recombination fraction. In fact, however, linkage analyses of some diseases associated with HLA, as well as diseases associated with alleles at other loci located throughout the genome, show significant evidence against linkage, and others show loose linkage, to the puzzlement of many researchers. In part, the puzzlement arises because linkage analysis is ideal for looking for loci that are necessary, even if not sufficient, for disease expression but may be much less useful for finding loci that are neither necessary nor sufficient for disease expression (so-called susceptibility loci). This work explores what happens when one looks for linkage to susceptibility loci. A susceptibility locus in this case means that the allele increases risk but is neither necessary nor sufficient for disease expression. It might be either an allele at the marker locus itself that is increasing susceptibility or an allele at a locus in linkage disequilibrium with the marker. This work uses computer simulation to examine how linkage analyses behave when confronted with data from such a model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marker-Assisted Introgression in Backcross Breeding Programs

The efficiency of marker-assisted introgression in backcross populations derived from inbred lines was investigated by simulation. Background genotypes were simulated assuming that a genetic model of many genes of small effects in coupling phase explains the observed breed difference and variance in backcross populations. Markers were efficient in introgression backcross programs for simultaneously introgressing an allele and selecting for the desired genomic background. Using a marker spacing of 10-20 cM gave an advantage of one to two backcross generations selection relative to random or phenotypic selection. When the position of the gene to be introgressed is uncertain, for example because its position was estimated from a trait gene mapping experiment, a chromosome segment should be introgressed that is likely to include the allele of interest. Even for relatively precisely mapped quantitative trait loci, flanking markers or marker haplotypes should cover ~10-20 cM around the estimated position of the gene, to ensure that the allele frequency does not decline in later backcross generations.     

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain. To do this analysis we chose to examine the relative Ig H chain V region gene usage of pre-B cell lines derived from (C57BL/6 x BALB/c)F1 fetal liver cells by Southern blot analysis. We found a high frequency of Vh-gene rearrangements (77% of the alleles had VDJ rearrangements) and these rearrangements occurred to Vh-genes throughout the Vh locus and were not confined to the D-proximal Vh-genes as has been previously observed with lines from other mouse strains. The Vh-gene usage pattern is similar on both alleles indicating that at least one of the determinants of which Vh-gene is used is trans-acting and acts similarly on each allele. Furthermore, one allele, Ighb (donated by the C57BL/6 parent), rearranged Vh-genes more frequently than the other allele, Igha (donated by the BALB/c parent) suggesting that one of the determinants of Vh-gene rearrangement may be acting in an allele-specific manner.     

Disequilibrium mapping of a quantitative-trait locus in an expanding population.

Linkage disequilibrium (LD) mapping can be successful if there is strong nonrandom association between marker alleles and an allele affecting a trait of interest. The principles of LD mapping of dichotomous traits are well understood, but less is known about LD mapping of a quantitative-trait locus (QTL). It is shown in this report that selective genotyping can increase the power to detect and map a rare allele of large effect at a QTL. Two statistical tests of the association between an allele and a quantitative character are proposed. These tests are approximately independent, so information from them can be combined. Analytic theory is developed to show that these two tests are effective in detecting the presence of a low-frequency allele with a relatively large effect on the character when the QTL is either already a candidate locus or closely linked to a marker locus that is in strong LD with the QTL. The latter situation is expected in a rapidly growing population in which the allele of large effect was present initially in one copy. Therefore, the proposed tests are useful under the same conditions as those for successful LD mapping of a dichotomous trait or disease. Simulations show that, for detection of the presence of a QTL, these tests are more powerful than a simple t-test. The tests also provide a basis for defining a measure of association, gamma, between a low-frequency allele at a putative QTL and a low-frequency allele at a marker locus.     

On the use of the transmission disequilibrium test to detect pseudo‐autosomal variants affecting traits with sex‐limited expression

We herein describe the realization of a genome‐wide association study for scrotal hernia and cryptorchidism in Norwegian and Belgian commercial pig populations. We have used the transmission disequilibrium test to avoid spurious associations due to population stratification. By doing so, we obtained genome‐wide significant signals for both diseases with SNPs located in the pseudo‐autosomal region in the vicinity of the pseudo‐autosomal boundary. By further analyzing these signals, we demonstrate that the observed transmission disequilibria are artifactual. We determine that transmission bias at pseudo‐autosomal markers will occur (i) when analyzing traits with sex‐limited expression and (ii) when the allelic frequencies at the marker locus differ between X and Y chromosomes. We show that the bias is due to the fact that (i) sires will preferentially transmit the allele enriched on the Y (respectively X) chromosome to affected sons (respectively daughters) and (ii) dams will appear to preferentially transmit the allele enriched on the Y (respectively X) to affected sons (respectively daughters), as offspring inheriting the other allele are more likely to be non‐informative. We define the conditions to mitigate these issues, namely by (i) extracting information from maternal meiosis only and (ii) ignoring trios for which sire and dam have the same heterozygous genotype. We show that by applying these rules to scrotal hernia and cryptorchidism, the pseudo‐autosomal signals disappear, confirming their spurious nature.     

Robust indices of Hardy-Weinberg disequilibrium for QTL fine mapping

Hardy-Weinberg disequilibrium (HWD) measures have been proposed using dense markers to fine map a quantitative trait locus (QTL) to regions < approximately 1 cM. Earlier HWD measures may introduce bias in the fine mapping because they are dependent on marker allele frequencies across loci. Hence, HWD indices that do not depend on marker allele frequencies are desired for fine mapping. Based on our earlier work, here we present four new HWD indices that do not depend on marker allele frequencies. Two are for use when marker allele frequencies in a study population are known, and two are for use when marker allele frequencies in a study population are not known and are only known in the extreme samples. The new measures are a function of the genetic distance between the marker locus and a QTL. Through simulations, we investigated and compared the fine mapping performance of the new HWD measures with that of the earlier ones. Our results show that when marker allele frequencies vary across loci, the new measures presented here are more robust and powerful.     

In an elite cross of maize a major quantitative trait locus controls one-fourth of the genetic variation for grain yield

Quantitative trait loci (QTLs) for grain yield, dry matter content and test weight were identified in an F₂ segregating population derived from a single cross between two elite maize lines (B73 and A7) and testcrossed to two genetically divergent in breds. Most of the QTLs inferred were consistent across locations, indicating that the expression of the genes influencing the traits under investigation was largely independent of the environment. By using two different tester lines we found that QTLs exhibited by one tester may not necessarily be detected with the second one. Only loci with larger effects were consistent across testers, suggesting that interaction with tester alleles may contribute to the identification of QTLs in a specific fashion. Analysis across both testers revealed four significant QTLs for grain yield that explained more than 35% of the phenotypic variation and showed an overall phenotypic effect of more than 2t/ha. The major QTL for grain yield, located in the proximity of the Nucleolus Organiser Region, accounted for 24.5% of the phenotypic variation for grain yield and showed an average effect of allele substitution of approximately 1 t/ha. Marker-assisted introgression of the superior A7 allele at this locus in the B73 genetic background will not differ from qualitative trait introgression and will eventually lead to new lines having superior testcross performance.     

Genomic contributions in livestock gene introgression programmes

The composition of the genome after introgression of a marker gene from a donor to a recipient breed was studied using analytical and simulation methods. Theoretical predictions of proportional genomic contributions, including donor linkage drag, from ancestors used at each generation of crossing after an introgression programme agreed closely with simulated results. The obligate drag, the donor genome surrounding the target locus that cannot be removed by subsequent selection, was also studied. It was shown that the number of backcross generations and the length of the chromosome affected proportional genomic contributions to the carrier chromosomes. Population structure had no significant effect on ancestral contributions and linkage drag but it did have an effect on the obligate drag whereby larger offspring groups resulted in smaller obligate drag. The implications for an introgression programme of the number of backcross generations, the population structure and the carrier chromosome length are discussed. The equations derived describing contributions to the genome from individuals from a given generation provide a framework to predict the genomic composition of a population after the introgression of a favourable donor allele. These ancestral contributions can be assigned a value and therefore allow the prediction of genetic lag.