The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.     

RFLP analysis of the size of chromosomal segments retained around the Tm-2 locus of tomato during backcross breeding

Summary Genes introduced into cultivated plants by backcross breeding programs are flanked by introgressed segments of DNA derived from the donor parent. This phenomenon is known as linkage drag and is frequently thought to affect traits other than the one originally targeted. The Tm-2 gene of Lycopersicon peruvianum, which confers resistance to tobacco mosaic virus, was introduced into several different tomato cultivars (L. esculentum) by repeated backcrossing. We have measured the sizes of the introgressed segments flanking the Tm-2 locus in several of these cultivars using a high density map of restriction fragment length polymorphic (RFLP) markers. The smallest introgressed segment is estimated to be 4 cM in length, while the longest is over 51 cM in length and contains the entire short arm of chromosome 9. Additionally, RFLP analysis was performed on remnant seed from different intermediate generations corresponding to two different backcross breeding programs for TMV resistance. The results reveal that plants containing desirable recombination near the resistance gene were rarely selected during backcrossing and, as a result, the backcross breeding method was largely ineffective in reducing the size of linked DNA around the resistance gene. We propose that, by monitoring recombination around genes of interest with linked RFLP markers, one can quickly and efficiently reduce the amount of linkage drag associated with introgression. Using such a procedure, it is estimated that an introgressed segment can be obtained in two generations that is as small as that which would otherwise require 100 backcross generations without RFLP selection.     

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.     

Selection strategies for marker-assisted backcrossing with high-throughput marker systems

Application of marker-assisted backcrossing for gene introgression is still limited by the high costs of marker analysis. High-throughput (HT) assays promise to reduce these costs, but new selection strategies are required for their efficient implementation in breeding programs. The objectives of our study were to investigate the properties of HT marker systems compared to single-marker (SM) assays, and to develop optimal selection strategies for marker-assisted backcrossing with HT assays. We employed computer simulations with a genetic model consisting of 10 chromosomes of 160 cM length to investigate the introgression of a dominant target gene. We found that a major advantage of HT marker systems is that they can provide linkage maps with equally spaced markers, whereas the possibility to provide linkage maps with high marker densities smaller than 10 cM is only of secondary use in marker-assisted backcrossing. A three-stage selection strategy that combines selection for recombinants at markers flanking the target gene with SM assays and genome-wide background selection with HT markers in the first backcross generation was more efficient than genome-wide background selection with HT markers alone. Selection strategies that combine SM and HT assays were more efficient than genome-wide background selection with HT assays alone. This result was obtained for a broad range of cost ratios of HT and SM assays. A further considerable reduction of the costs could be achieved if the population size in the first backcross generation was twice the population size in generations BC₂ and BC₃ of a three-generation backcrossing program. We conclude that selection strategies combining SM and HT assays have the potential to greatly increase the efficiency and flexibility of marker-assisted backcrossing.     

Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.     

Close linkage of the olfactory marker protein gene to the mouse deafness mutation shaker-1.

One thousand sixty-six progeny have been generated from a backcross segregating for the mouse deafness mutation, shaker-1 (sh-1). One thousand fifty-two mice were analyzed for a protein polymorphism segregating for the distal flanking marker, beta-globin (Hbb), and 13 recombinants between Hbb and sh-1 were identified. One thousand eight mice were analyzed for a restriction fragment length polymorphism segregating for the proximal flanking marker, tyrosinase (c), and 54 recombinants between c and sh-1 were identified, completing a panel of 67 recombinant mice from the backcross in the vicinity of the sh-1 mutation. This panel allows the identification of markers closely linked to the sh-1 mutation that may act as start points for a chromosomal walk to the gene. One such marker, the olfactory marker protein gene (Omp), is recombinant with sh-1 in only one mouse from the recombinant panel. Thus, the Omp gene lies 0.1 cM from sh-1, on average, a distance of 200 kb. Haplotype analysis indicates that Omp lies proximal to sh-1.     

Using Markers in Gene Introgression Breeding Programs

We investigate the use of markers to hasten the recovery of the recipient genome during an introgression breeding program. The effects of time and intensity of selection, population size, number and position of selected markers are studied for chromosomes either carrying or not carrying the introgressed gene. We show that marker assisted selection may lead to a gain in time of about two generations, an efficiency below previous theoretical predictions. Markers are most useful when their map position is known. In the early generations, it is shown that increasing the number of markers over three per non-carrier chromosome is not efficient, that the segment surrounding the introgressed gene is better controlled by rather distant markers unless high selection intensity can be applied, and that selection on this segment first can reduce the selection intensity available for selection on non-carrier chromosomes. These results are used to propose an optimal strategy for selection on the whole genome, making the most of available material and conditions (e.g., population size and fertility, genetic map).     

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.     

Use of near-isogenic lines derived by backcrossing or selfing to map qualitative traits

Near-isogenic lines (NILs) are a valuable resource for detecting linkages between qualitative trait loci and molecular markers. Molecular marker studies are expensive and methods that require genotyping fewer individuals, such as the NIL-analysis method, are desirable. We present a theory for using sets of NILs to detect linkages between molecular markers and introgressed loci. The probability that a marker a specific distance from the introgressed gene will have a donor parent allele in a near-isogenic line is a function of the distance between the marker and the gene, and the number of back-crosses and/or selfs used in deriving the NIL. The binomial probability formula is used to calculate the probability of having a donor parent allele at a given marker when sets of NILs are used. The formulae given allow calculation of the probability that a marker is linked to the introgressed gene, as well as the probability that a gene will be successfully detected when using given numbers of NILs, backcrosses, and molecular markers.     

标记辅助导入中不同前景和背景选择方法的比较

标记辅助导入是分子遗传信息应用于动物育种的一个重要方面,其目的是在标记信息的辅助下将一个品种（供体）中的一个或多个优良基因导入另一个品种（受体）,同时还要尽可能地保持受体群体原有的遗传背景。标记辅助导入的过程包括3个阶段,第一阶段是杂交,即供体与受体杂交产生F1代个体,第二阶段是回交,即F1个体以及后续各个世代的后代个体重复地与受体回交,以使受体的遗传背景得到恢复,第三阶段是横交,即重复回交后得到的个体彼此问交配,以便获得供体基因的纯合个体,使该基因在群体中固定。在回交和横交阶段,都要对参与交配的个体进行选择。在选择中,要分别进行前景选择和背景选择,前景选择是对供体基因的选择,选择携带有供体基因个体参加配种,从而使该基因在回交过程中不会丢失,并在横交过程中能尽快固定,背景选择是对受体遗传背景的选择,选择那些含有受体基因组比例较高的个体参加配种,从而加快恢复受体遗传背景的速度。本研究通过计算机模拟对不同的前景选择方法和不同的背景选择方法进行了比较。前景选择方法包括对受体基因的直接选择（假设该基冈可以直接测定）、利用单个连锁标记的间接选择和利用两侧标记的间接选择3种,背景选择方法包括随机选择、基因组相似性选择、指数选择和标记辅助BLUP（MBLUP）选择4种。研究结果表明,对于前景选择来说,对供体基因的直接选择能保证该基因在回交的各个世代中保持一个稳定的频率（0．25）并在横交阶段迅速固定（2个世代）,用两侧标记的间接选择也能得到类似的结果,但如果仅利用单个连锁标记进行选择,则会导致供体基因的频率在回交阶段中有所下降,并在横交阶段不能被固定。对于背景选择来说,如果最终的目的是要完全恢复受体的遗传背景,基因组相似性选择或标记指数选择是最好的选择方法,它们可使受体的遗传背景在回交3个世代后就恢复到98％以上,而随机选择或MBLUP选择需要至少5个世代的回交才能达到这个水平。但如果最终的目的只是要恢复受体的某些优良性状,则MBLUP选择是值得推荐的方法,它可使影响这些性状的受体基因频率在回交3个世代后就达到99％以上,而且还能在整个基因导入过程中给这些性状带来最大的遗传进展。虽然用标记指数选择也有相似的结果,但与之相比,MBLUP的成本要低得多,更具有实际可行性。     

Comparison of Different Foreground and Background Selection Methods in Marker-Assisted Introgression

Three different methods for foreground selection and four different methods for background selection were compared in terms of the efficiency of marker-assisted introgression of a QTL allele from a donor line into a recipient line and also in terms of the recovery of the recipient genetic background. The results showed that for the introgression of a donor QTL allele, a direct selection on the QTL itself (when the QTL genotype can be directly identified) would ensure that the allele is successfully introgressed and rapidly fixed. However, when a direct selection on the QTL is not feasible, an indirect selection using two closely linked flanking markers can be used, which also shows similar results. For the recovery of the recipient genetic background, if the goal is to recover the whole genetic background of the recipient, genomic similarity selection or marker index selection would be the best choice: Only three generations of backcrosses were required to recover over 98% of the recipient genome. Whereas if the goal is to recover certain background traits of the recipient, MBLUP selection would give the best results, which achieved not only over 99% recovery of the recipient QTL alleles for the background traits after three generations of backcrosses, but also showed the best genetic improvement of these traits.     

Selection strategies for the development of rye introgression libraries

Computer simulations can be employed to find optimal procedures for developing introgression libraries in rye with marker-assisted backcrossing. Our objectives were to investigate the effects of the employed (1) breeding scheme, (2) selection strategy, and (3) population sizes on the donor genome coverage of the library, the number of introgression lines carrying additional donor chromosome segments outside the target regions, and the number of required marker data points. With respect to these target criteria, a BC₃S₂ breeding scheme and increasing population sizes from early to advanced generations were superior to a BC₂S₃ breeding scheme and constant population sizes. The smallest number of donor segments outside the target regions was reached with a three-stage selection strategy, which consists on selection for the target segment, selection for recombination at flanking markers and selection for recurrent parent alleles across the entire genome. Omitting the selection for flanking markers in generation BC₁ reduced considerably the number of required marker data points. A pre-selection of chromosomes consisting completely of donor genome in BC₁ was advantageous, if the effort in the breeding nursery should kept minimum. Adopting the described designs can help rye breeders to successfully develop introgression libraries.     

The construction of a substitution library of recombinant backcross lines in Brassica oleracea for the precision mapping of quantitative trait loci.

The currently available methods for locating quantitative trait loci (QTLs) and measuring their effects in segregating populations lack precision unless individual QTLs have very high heritabilities. The use of recombinant backcross lines containing short regions of donor chromosome introgressed into a constant recipient background permits QTLs to be located with greater precision. The present paper describes the use of molecular markers to introgress defined short regions of chromosome from a donor doubled haploid calabrese line of Brassica oleracea (var. italica) into a recipient short generation variety (Brassica oleracea var. alboglabra). We demonstrate that in just two or three generations of backcrossing, combined with selection for mapped molecular markers, the generation of a library of recombinant backcross lines is feasible. The possible use and refinement of these lines are discussed. Key words : backcrossing, Brassica oleracea, introgression, molecular markers, near-isogenic lines, QTL mapping, recombinant backcross lines, substitution lines.     

Precise mapping Fhb5, a major QTL conditioning resistance to Fusarium infection in bread wheat (Triticum aestivum L.)

Qfhi.nau-5A is a major quantitative trait locus (QTL) against Fusarium graminearum infection in the resistant wheat germplasm Wangshuibai. Genetic analysis using BC(3)F(2) and BC(4)F(2) populations, derived from selfing two near-isogenic lines (NIL) heterozygous at Qfhi.nau-5A that were developed, respectively, with Mianyang 99-323 and PH691 as the recurrent parent, showed that Qfhi.nau-5A inherited like a single dominant gene. This QTL was thus designated as Fhb5. To fine map it, these two backcross populations and a recombinant inbred line (RIL) population derived from Nanda2419?×?Wangshuibai were screened for recombinants occurring between its two flanking markers Xbarc56 and Xbarc100. Nineteen NIL recombinants were identified from the two backcross populations and nine from the RIL population. In the RIL recombinant selection process, selection against Fhb4 present in the RIL population was incorporated. Genotyping these recombinant lines with ten markers mapping to the Xbarc56-Xbarc100 interval revealed four types of Mianyang 99-323-derived NIL recombinants, three types of PH691-derived NIL recombinants, and four types of RIL recombinants. In different field trials, the percentage of infected spikes of these lines displayed a distinct two-peak distribution. The more resistant class had over 55% less infection than the susceptible class. Common to these resistant genotypes, the 0.3-cM interval flanked by Xgwm304 and Xgwm415 or one of these two loci was derived from Wangshuibai, while none of the susceptible recombinants had Wangshuibai chromatin in this interval. This interval harboring Fhb5 was mapped to the pericentromeric C-5AS3-0.75 bin through deletion bin mapping. The precise localization of Fhb5 will facilitate its utilization in marker-assisted wheat breeding programs.     

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.     

Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata.

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.     

Molecular mapping of the novel powdery mildew resistance gene <Emphasis Type="Italic">Pm36</Emphasis> introgressed from <Emphasis Type="Italic">Triticum turgidum</Emphasis> var. <Emphasis Type="Italic">dicoccoides</Emphasis> in durum wheat

Powdery mildew, caused by Blumeria graminis f.sp. tritici, is one of the most important wheat diseases in many regions of the world. Triticum turgidum var. dicoccoides (2n=4x=AABB), the progenitor of cultivated wheats, shows particular promises as a donor of useful genetic variation for several traits, including disease resistances. The wild emmer accession MG29896, resistant to powdery mildew, was backcrossed to the susceptible durum wheat cultivar Latino, and a set of backcross inbred lines (BC(5)F(5)) was produced. Genetic analysis of F(3) populations from two resistant introgression lines (5BIL-29 x Latino and 5BIL-42 x Latino) indicated that the powdery mildew resistance is controlled by a single dominant gene. Molecular markers and the bulked segregant analysis were used to characterize and map the powdery mildew resistance. Five AFLP markers (XP43M32((250)), XP46M31((410)), XP41M37((100)), XP41M39((250)), XP39M32((120))), three genomic SSR markers (Xcfd07, Xwmc75, Xgwm408) and one EST-derived SSR marker (BJ261635) were found to be linked to the resistance gene in 5BIL-29 and only the BJ261635 marker in 5BIL-42. By means of Chinese Spring nullisomic-tetrasomic, ditelosomic and deletion lines, the polymorphic markers and the resistance gene were assigned to chromosome bin 5BL6-0.29-0.76. These results indicated that the two lines had the same resistance gene and that the introgressed dicoccoides chromosome segment was longer (35.5 cM) in 5BIL-29 than that introgressed in 5BIL-42 (less than 1.5 cM). As no powdery mildew resistance gene has been reported on chromosome arm 5BL, the novel resistance gene derived from var. dicoccoides was designated Pm36. The 244 bp allele of BJ261635 in 5BIL-42 can be used for marker-assisted selection during the wheat resistance breeding process for facilitating gene pyramiding.     

Genotype Selection to Rapidly Breed Congenic Strains

Congenic strains can now be constructed guided by the transmission of DNA markers. This allows not only selection for transmission of a desired, donor-derived differential region but also selection against the transmission of unwanted donor origin genomic material. The additional selection capacity should allow congenic strains to be produced in fewer generations than is possible with random backcrosses. Here, we consider modifications of a standard backcross breeding scheme to produce congenic mice by the inclusion of genotype-based selective breeding strategies. Simulation is used to evaluate the consequences of each strategy on the number of chromosomes that contain unwanted, donor-derived genetic material and the average length of this unwanted donor DNA for each backcross generation. Our prototypic strategy was to choose a single mouse to sire each generation using criteria designed to select against the transmission of chromosomes, other than the one containing the replacement genomic region, that contain any donor origin sequence at all. This chromosome elimination strategy resulted in an average of 16.4 chromosomes free of donor DNA in mice of the third backcross (N(3)) generation. A strategy based solely on positive selection for the replacement region required six backcross generations to achieve the same results.     

Mapping of a locus for adult plant resistance to downy mildew in broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> convar. <Emphasis Type="Italic">italica</Emphasis>)

The identification of the gene Pp523, conferring downy mildew resistance to adult plants of broccoli (Brassica oleracea convar. italica), led to the construction of a genetic map that included this resistance locus, 301 amplified fragment length polymorphisms, 55 random amplified polymorphic DNAs, 46 inter-simple sequence repeats, three simple sequence repeats, four other PCR markers and a flower colour locus, all gathered into nine major linkage groups. Nineteen additional molecular markers were clustered into one group of four markers, one group of three markers and six pairs of markers. The map spans over 731.9 cM, corresponding to 89.5% of the 818 cM estimated to be the total genome length. A significant number of the mapped markers, 19.3%, showed distorted segregation. The average distance between mapped adjacent markers is 1.64 cM, which places this map among the densest published to date for this species. Using bulked segregant analysis, we identified a group of molecular markers flanking and closely linked in coupling to the resistance gene and included these in the map. Two markers linked in coupling, OPK17_980 and AT.CTA_133/134, are located at 3.1 cM and 3.6 cM, respectively, at each side from the resistance gene. These markers can be used for marker-assisted selection in breeding programs aiming at the introgression of this gene in susceptible B. oleracea genotypes. The fine mapping of the genomic region surrounding the Pp523 resistance gene is currently being carried out, a basic condition for its isolation via positional cloning.     

Application of a simplified marker-assisted backcross technique for hybrid breeding in rice

Hybrid rice has contributed greatly to the self-sufficiency of the food supply in China. However, bacterial blight is a major disease that limits hybrid rice production in China. The study was conducted to develop an efficient breeding technique to improve the bacterial blight resistance in hybrid rice. A marker-assisted backcross breeding technique was adopted to improve HN189, an elite restorer line containing the Pi1 gene. This breeding technique was simplified to foreground selection with only one generation of backcrossing and background selection based on phenotypic selection. A novel bacterial blight resistance gene, Xa23, was introgressed into HN189. Two improved restorer lines, HBH145 (with one generation of backcrossing) and HBH146 (with two generations of backcrossing), were obtained that had a significant bacterial blight resistance advantage over HN189. The corresponding hybrid combination Tianyou H145 (Tianfeng A / HBH145) was certified one year earlier than Qianyou H146 (Qianjiang 1A / HBH146). The use of the marker-assisted backcross breeding technique with one generation of backcrossing and without background selection in rice breeding programs shortened the breeding period of the rice.