Mapping QTLs in breeding for drought tolerance in maize (Zea mays L.)

Summary Grain yield in the maize (Zea mays L) plant is sensitive to drought in the period three weeks either side of flowering. Maize is well-adapted to the use of restriction fragment length polymorphisms (RFLPs) to identify a tight linkage between gene(s) controlling the quantitative trait and a molecular marker. We have determined the chromosomal locations of quantitative trait loci (QTLs) affecting grain yield under drought, anthesis-silking interval, and number of ears per plant. The F₃ families derived from the cross SD34(tolerant) × SD35 (intolerant) were evaluated for these traits in a two replicated experiment. RFLP analysis of the maize genome included non-radioactive DNA-DNA hybridization detection using chemiluminescence. To identify QTLs underlying tolerance to drought, the mean phenotypic performances of F₃ families were compared based on genotypic classification at each of 70 RFLP marker loci. The genetic linkage map assembled from these markers was in good agreement with previously published maps. The phenotypic correlations between yield and other traits were highly significant. In the combined analyses, genomic regions significantly affecting tolerance to drought were found on chromosomes 1,3,5,6, and 8. For yield, a total of 50% of the phenotypic variance could be explained by five putative QTLs. Different types of gene action were found for the putative QTLs for the three traits.     

The European Prunus mapping project Progress in the almond linkage map

Summary Six European research groups are collaborating to develop genetic markers and linkage maps for use inPrunus breeding programmes. A basic map with 200 RFLPs and 50 more markers including isozymes and RAPDs will be constructed using two highly segregating populations: an interspecific peach × almond F₂ and a cherry F₂. Then, the parents of eleven almond, cherry, peach or plum breeding progenies segregating for target characters will be screened for polymorphisms at the marker loci, and a set of reduced maps, one per progeny, will be constructed with markers spaced 20–30 cM and covering the whole genome. Cosegregation analysis of markers and characters of interest will allow us to find linkages between markers and major genes or quantitative trait loci responsible for the expression of these traits. A map with 72 markers, 7 isozymes and 65 RFLPs, has been developed at the IRTA-Cabrils laboratory using an intraspecific almond progeny, ‘Ferragnes’ × ‘Mono’. Probes for the analysis of RFLPs were obtained from almond genomic and cDNA libraries. The level of polymorphism for RFLPs and the distribution of markers in the chromosomes of almond are discussed.     

Genetic mapping of quantitative trait loci in rye (Secale cereale L.)

Using the marker information of 275 F₂ plants quantitative traits determining morphological and yield characters were studied analyzing F₃progenies grown in four different experiments at three sites. The map constructed contains 113 markers including the major dwarfing gene Ddw1 with an average distance of about 10 cM between adjacent markers. Of the 21 QTLs detected ten were found to map on chromosome 5RL in the region of Ddw1. Beside the expected effects on plant height and peduncle length that are most probably due to the presence of the major dwarfing gene, additional effects on yield characters and flowering time were discovered in that region which may be caused by pleiotropic effects of Ddw1. An additional supposed gene cluster consisting of four QTLs controlling flowering time and yield components was discovered in the centromere region of chromosome 2R. Further loci are distributed on chromosomes 1R (1), 4R (1) 6R (3) and 7R (1). The map positions of the quantitative trait loci detected in rye are discussed in relation to major genes or QTLs determining agronomically important traits in other cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Construction of a molecular functional map of rapeseed (Brassica napus L.) using differentially expressed genes between hybrid and its parents

Brassica napus L. is an important oilseed and fodder crop with significant heterosis for seed yield and other agronomic traits, but very little is known about the molecular basis of heterosis. As an initial step towards understanding the molecular events associated with this phenomenon, a molecular functional map of rapeseed was constructed using differentially expressed genes in hybrid identified by microarrays. Single-strand conformational polymorphism (SSCP) analysis was applied for genetic mapping in an F ₂ population of 184 individuals resulting from crossing ‘`SI-1300 × Eagle’'. A total of 162 markers including 154 loci corresponding to 98 differentially expressed genes assigned to 17 functional categories and 8 SSR markers were grouped into 21 linkage groups (LGs), covering a total map distance of 2267.3 cM. Subsequently, this map was aligned with Arabidopsis thaliana in silico. Comparative mapping shows that genes localized on each Arabidopsis chromosome have orthologs dispreading in different B. napus LGs. Similarly, a majority of LGs were made of homologous genes from different Arabidopsis chromosomes. In addition, a total of 25 syntenic regions were identified in B. napus, in most of which the gene order was not consistent between the two species, and each of the conserved regions in the A. thaliana genome was homologous to 1--5 distinct regions in the B. napus genome. These results indicate that it is not easy to exploit A. thaliana information for B. napus based on synteny.     

Molecular mapping of powdery mildew resistance genes in wheat: A review

Powdery mildew, caused by Blumeria graminis f. sp. tritici (syn. Erysiphe graminis f. sp. tritici), is one of the most important diseases of common wheat (Triticum aestivum L.) worldwide. Molecular mapping and cloning of genes for resistance to powdery mildew in hexaploid wheat will facilitate the study of molecular mechanisms underlying resistance to powdery mildew diseases and help understand the structure and function of powdery mildew resistance genes, and permit marker-assisted selection in breeding programs. So far, 48 genes/alleles for resistance to powdery mildew at 32 loci have been identified and located on 16 different chromosomes, of which 21 resistance genes/alleles have been tagged by restriction fragment length polymorphisms (RFLPs), random-amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), sequence characterized amplified regions (SCARs), sequence-tagged sites (STS) or simple sequence repeats (SSRs). Several quantitative trait loci (QTLs) for adult plant resistance (APR) to powdery mildew have been associated with molecular markers. The detailed information on chromosomal location and molecular mapping of these genes has been reviewed. Isolation of powdery mildew resistance genes and development of valid molecular markers for pyramiding resistance genes in breeding programs is also discussed.     

Meta-QTL analysis and identification of candidate genes related to root traits in maize

Maize root system architecture determines key functions of uptake of water and nutrients in plants. A large number of quantitative trait loci (QTLs) of root-related traits have been found in different populations of maize. Identification of consistent QTLs across diverse genetic backgrounds could be instrumental on marker-assisted selection of traits and identification of candidate functional genes. In this study, 20 published papers were investigated regarding on reported results of QTLs related to root traits of maize, and in total 428 individual QTLs for 23 root-related traits were used for meta-analysis, resulting in 53 Meta-QTLs (MQTLs) retrieved over ten maize chromosomes. Among these MQTLs regions, in total 45 maize homologs were considered as candidate genes affecting maize root traits by comparing with 7 genes from rice and 36 genes from Arabidopsis. Three maize genes (GRMZM5G813206, GRMZM2G167220 and GRMZM2G467069) identified from MQTL_8-5 could play important roles on lateral root and crown root development of maize. Two of the MQTLs, i.e. MQTL_7-2 and MQTL_9-1, involved in nitrogen (N) and phosphorus (P) stress responses and both of them with small physical distance (less than 3 Mb), could be used for abiotic stress improvement of maize root traits. These MQTLs and candidate genes will be helpful for future gene cloning and marker-assisted selection in maize.     

Association of isozyme markers with quantitative trait loci in random single seed descent derived lines of lentil (Lens culinaris Medik.)

Summary Polymorphism at isozyme loci was used to locate factors responsible for variation in quantitative traits of lentil. Eight sets of random single seed descent (RSSD) derived lines were developed by advancing individual F₃ plants of interspecific (L. culinaris Medik. × L. orientalis Boiss.) hybrids to the F₆. The RSSD lines in each of the eight sets differed for alleles at 2–8 isozyme loci. In each set, association of isozyme loci with variation in seven quantitative traits (days to flower, days to mature, plant height, biomass, seed yield, harvest index, seed weight) was determined for each pairwise combination of a quantitative trait with a marker locus. Loci affecting variation in all seven quantitative traits were detected by their association with 14 isozyme markers (Aat-c, Aat-m, Aat-p, Adh-1, Fk, Gal-1, Gal-2, Lap-1, Lap-2, Pgd-p, Pgi, Pgm-c, Pgm-p, Skdh). The known position of 10 the 14 isozyme loci on the lentil genetic map was used to mark the genomic regions for possible location of associated quantitative trait loci (QTL). Detected QTL were found to be located in six of the seven linkage groups on lentil genetic map. Regions of the genome represented by linkage groups, 1, 5 and 7 appeared to affect a greater number of traits than other genomic regions represented by linkage groups 2, 3 and 4. Results indicated that the mean expression of quantitative traits at segregating marker locus classes can be used to locate the genetic factors in lentil which influence the behavior of economically important traits.     

Molecular markers in Brassica oilseed breeding: current status and future possibilities

As PCR techniques have developed over the last 15 years, a wealth of new DNA marker technologies have arisen which have enabled the generation of high‐density molecular maps for all the major Brassica crop species. Molecular markers have also been heavily used in analyses of genetic diversity in Brassica crops. The majority of the work utilizing molecular markers in Brassica oilseed breeding has to date been based on genetic mapping using various DNA marker systems in segregating populations generated for specific investigations of particular traits of interest. For numerous qualitative traits, traditional mapping approaches have led to the development of marker‐assisted selection strategies in oilseed Brassica breeding, and in some cases to map‐based cloning of the responsible genes. For quantitative traits, however, it has become apparent that traditional mapping of quantitative trait loci (QTL) is often not sufficient to develop effective markers for trait introgression or for identification of the genes responsible. In this case, allele‐trait association studies in non‐structured genetic populations represent an interesting new approach, provided the degree of gametic phase disequilibrium between the QTL and the marker loci is sufficient. Because Brassica species represent the closest crop plant relatives to the model plant Arabidopsis thaliana, significant progress will be achieved in the coming years through integration of candidate gene approaches in crop brassicas, using the detailed information now available for the Arabidopsis genome. Integration of information from the model plant with the increasing supply of data from physical mapping and sequencing of the diploid Brassica genomes will undoubtedly give great insight into the genetics underlying both simple and complex traits in oilseed rape. This review describes the current use of available genetic marker technologies in oilseed rape breeding and provides an outlook for use of new technologies, including single‐nucleotide polymorphism markers, candidate gene approaches and allele‐trait association studies.     

A SNP genetic linkage map based on the ‘Hamilton’ by ‘Spencer’ recombinant inbred line population identified QTL for seed isoflavone contents in soybean

Soybean is one of the most important crops worldwide for its protein and oil as well as the health beneficial phytoestrogens or isoflavone. This study reports a relatively dense single nucleotide polymorphism (SNP)‐based genetic map based on ‘Hamilton’ by ‘Spencer’ recombinant inbred line population and quantitative trait loci (QTL) for seed isoflavone contents. The genetic map is composed of 1502 SNP markers and covers about 1423.72 cM of the soybean genome. Two QTL for seed isoflavone contents have been identified in this population. One major QTL that controlled both daidzein (qDZ1) and total isoflavone contents (qTI1) was found on LG C2 (Chr 6). And a second QTL for glycitein content (qGT1) was identified on the LG G (Chr 18). These two QTL in addition to others identified in soybean could be used in soybean breeding to optimize isoflavone content. This newly assembled soybean linkage map is a useful tool to identify and map QTL for important agronomic traits and enhance the identification of the genes involved in these traits.     

QTL mapping of improving forage maize starch degradability in European elite maize germplasm

Improving maize starch content is of great importance for both forage and grain yield. In this study, 13 starch degradability traits were analysed including percentage of the seedling area, floury endosperm, hard endosperm of total grain area, percentage of the floury endosperm surface and vitreousness ratio surface hard: floury endosperm surface, etc. We mapped quantitative trait loci (QTL) in a biparental population of 309 doubled haploid lines based on field phenotyping at two locations. A genetic linkage map was constructed using 168 SSR (simple sequence repeat) markers, which covered 1508 cM of the maize genome, with an average distance of 9.0 cM. Close phenotypic and genotypic correlations were found for all traits, and were all statistically significant (p = 0.01) at two locations. Major QTL for more than two traits were detected, especially in two regions in bins 4.05–4.06 and 7.04–7.05, associated with 13 and 9 traits, respectively. This study contributes to marker‐assisted breeding and also to fine mapping candidate genes associated with maize starch degradability.     

Integration of AFLP markers into an RFLP-based map of durum wheat

Amplified fragment length polymorphism (AFLP) is a powerful technique which can readily be applied to a wide range of species for mapping purposes. AFLPs were added to a linkage map of durum wheat constructed using restriction fragment length polymorphisms (RFLPs). The mapping population included 65 recombinant inbred lines derived from a cross between the durum wheat cultivar ‘Messapia’ and accession ‘MG4343’ of the wild Triticum turgidum ssp. dicoccoides (Körn.). Genomic DNA was digested with MseI (4‐cutter) and Sse83871 (8‐cutter). Using a silver‐staining protocol, 14 primer combinations revealed 421 clearly scorable amplicons including 100 polymorphisms. The presence of nine pairs of bands linked in repulsion phase with each pair generated by one primer combination suggested the presence of codominant alleles; sequence analysis of four band pairs confirmed their codominant nature. The integration of 80 AFLP loci extended the map in several telomeric regions, reduced the size of four large gaps present in the previous map, and eliminated one gap. The new map obtained after the inclusion of the 80 AFLP loci and eight additional RFLP loci spans 2063cM which represent a 52.6% increment compared with the previous map. Compared with the distribution of RFLPs, no significant clustering of AFLP markers was observed.     

Breeding drought tolerant cowpea: constraints,accomplishments, and future prospects

This review presents an overview of accomplishments on different aspects of cowpea breeding for drought tolerance. Furthermore it provides options to enhance the genetic potential of the crop by minimizing yield loss due to drought stress. Recent efforts have focused on the genetic dissection of drought tolerance through identification of markers defining quantitative trait loci (QTL) with effects on specific traits related to drought tolerance. Others have studied the relationship of the drought response and yield components, morphological traits and physiological parameters. To our knowledge, QTLs with effects on drought tolerance have not yet been identified in cowpea. The main reason is that very few researchers are working on drought tolerance in cowpea. Some other reasons might be related to the complex nature of the drought stress response, and partly to the difficulties associated with reliable and reproducible measurements of a single trait linked to specific molecular markers to be used for marker assisted breeding. Despite the fact that extensive research has been conducted on the screening aspects for drought tolerance in cowpea only very few—like the ‘wooden box’ technique—have been successfully used to select parental genotypes exhibiting different mechanisms of drought tolerance. Field and pot testing of these genotypes demonstrated a close correspondence between drought tolerance at seedling and reproductive stages. Some researchers selected a variety of candidate genes and used differential screening methods to identify cDNAs from genes that may underlie different drought tolerance pathways in cowpea. Reverse genetic analysis still needs to be done to confirm the functions of these genes in cowpea. Understanding the genetics of drought tolerance and identification of DNA markers linked to QTLs, with a clear path towards localizing chromosomal regions or candidate genes involved in drought tolerance will help cowpea breeders to develop improved varieties that combine drought tolerance with other desired traits using marker assisted selection.

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Molecular markers linked to rhizomania resistance in sugar beet, Beta vulgaris, from two different sources map to the same linkage group

Rhizomania, one of the most important diseases of sugar beet, is caused by beet necrotic yellow vein virus, a Furovirus vectored by the fungus Polymyxa betae Keskin. Reduction of the production losses caused by this disease can only be achieved by using tolerant cultivars. The objective of this study was the identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to a rhizomania resistance gene. The RAPD markers were identified using bulked segregant analysis in a segregating population of 62 individuals derived by intercrossing plants of the resistant commercial hybrid GOLF, and the resistance locus was positioned in a molecular marker linkage map made with a different population of 50 GOLF plants. The resistance locus, Rr1, was mapped to linkage group III of our map of Beta vulgaris L. ssp. vulgaris, which consisted of 76 RAPDs, 20 restriction fragment length polymorphisms (RFLPs), three sequence characterized amplified regions (SCARs) and one sequence tagged site (STS). In total, 101 molecular markers were mapped over 14 linkage groups which spanned 688.4 cM with an average interval length of 8.0 cM. In the combined map, Rr1 proved to be flanked by the RAPD loci RA411₁₈₀₀ and AS7₁₁₀₀ at 9.5 and 18.5cM, respectively. Moreover, in our I₂ population, we found that a set of markers shown by Barzen et al. (1997) to be linked to the ‘Holly’ type resistance gene was also linked to the ‘GOLF’-type resistance gene. These results appeared to indicate that the rhizomania resistance gene present in the GOLF hybrid could be the same gene underlying resistance in ‘Holly’-based resistant genotypes. Two other explanations could be applied: first, that two different alleles at the same locus could have been selected; second, that two different genes at two different but clustered loci underwent the selection process.     

Comparative fine mapping of fruit quality QTLs on chromosome 4 introgressions derived from two wild tomato species

Despite their unsuitability for agricultural production, the wild relatives of crop species represent a largely untapped resource of novel QTLs potentially useful for crop plant improvement. In this regard, previous introgression studies, involving several different wild tomato species, have shown that the long arm of chromosome 4 contains QTLs for many horticulturally important traits including soluble solids content, fruit shape, lycopene content and biochemical composition. However, these earlier studies were unable to determine how many genes control these traits and whether genes affecting the same character from different wild species are allelic or not. In an effort to shed light on these issues,we have constructed a series of lines containing small, overlapping introgressions for portions of the long arm of chromosome 4 from L. peruvianum and L. hirsutum and tested these lines in replicated field trials. The results provide evidence for multiple, non-allelic loci controlling soluble solids and fruit weight. They also show that the loci controlling some traits (e.g. fruit shape, fruit weight, epidermal reticulation) co-localize to the same portions of chromosome 4, a result that maybe attributed to pleiotropy and/or gene dense areas with lower than average recombination. The implications of these finding for molecular breeding and utilization of exotic germplasm are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mitochondrial DNA variation in foxtail millet, Setaria italica (L.) P. Beauv.

Mitochondrial DNA (mtDNA) wascharacterized by RFLPs in 94 strains offoxtail millet, Setaria italica (L.)P. Beauv. Three RFLP patterns wereobserved by using rice atp6 as aprobe, and were designated as types I-III. Difference between types I and II seems tobe attributed to recombination between twoatp6 genes. In East and SoutheastAsia and Afghanistan, both of types I andII were found, while type I was predominantin India, Central Asia and Europe. InChina, type III was also found. Chinesestrains showed higher gene diversity thanthose from other regions. This resultcoincided the previous studies on isozymesand nuclear RFLPs.     

QTL analysis of variation for vigour in rose

The improvement of energy efficiency in the greenhouse production of cut rose and pot rose can be achieved through the use of rose cultivars having vigorous growth. A better understanding of the inheritance of vigour and its related traits will assist the breeding activities. Quantitative trait locus (QTL) analyses were performed with the help of an integrated linkage map of a diploid rose population originating from a cross between Rosa multiflora-derived genotypes. The underlying datasets for ten vigour-related traits were collected in an evaluation study of this population in two greenhouse experiments with suboptimal temperatures for growth. We identified ten chromosomal regions, scattered over the seven linkage groups, containing QTLs for these traits. Considering each trait separately, we detected a total of 42 QTLs. Among these QTLs, 24 were found in both of the experiments, eight and ten were specific to either of the two experiments. The number of QTLs for individual traits varied from three to five with a respective contribution to the phenotypic variation from 12 to 35%. QTLs for highly correlated traits frequently co-localized, indicating a common genetic basis. Clustering of QTLs for different traits was noted in some chromosome regions, for instance, one on chromosome 2 included major QTLs for eight of ten traits under study, suggesting co-localization of several separate genes or/and the occurrence of various genes having pleiotropic effects. The discovery of markers associated to QTL regions is in roses the first step towards marker-assisted selection for vigour improvement enabling the transfer of useful QTL-alleles of R. multiflora to pot and cut roses.     

Comparative genetics and disease resistance in wheat

The hexaploid wheat genome is too complex for direct map-basedcloning and model genomes have to be used to isolate genes from wheat.Comparative genomic analysis at the genetic map level has shown extensiveconservation of the gene order between the different grass genomes inmany chromosomal regions. However, little is known about the geneorganization in grass genomes at the microlevel. We have investigated themicrocollinearity at Lrk gene loci in the genomes of four grass species:wheat, barley, maize and rice. The Lrk genes, which encodereceptor-like kinases, were found to be consistently associated with anothertype of receptor-like kinase (Tak) on chromosome groups 1 and 3 inTriticeae and on chromosomes homoeologous to Triticeae group 3 in theother grass genomes. On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form acluster of genes. Comparison of the gene composition at orthologous Lrk loci in wheat, barley and rice revealed a maximal gene density of onegene per 5 kb. We conclude that small and large grass genomes containregions which are highly enriched in genes. Microrearrangements betweendifferent grass genomes have been found and therefore, the choice of agood model genome is critical. We have recently started to work on theT. monococcum model genome and confirmed its usefulness foranalysis of the Lr10 leaf rust disease resistance locus in wheat.     

A genetic linkage map in southern‐by‐spring oat identifies multiple quantitative trait loci for adaptation and rust resistance

We developed 178 recombinant inbred lines from a southern‐by‐spring oat population designated as “TxH.” These lines were genotyped to generate a high‐quality linkage map that resolved 6,902 markers into 21 linkage groups that matched closely with the latest hexaploid oat consensus map. Three major quantitative trait loci (QTLs) affecting heading date were found in locations that are consistent with known QTLs and candidate genes, and two other QTLs affecting heading date were found in novel locations. Five QTLs affecting plant height were found. Both sets of QTLs are responsible for transgressive segregation observed for these two traits. Four QTLs affecting resistance to crown rust, caused by the pathogen Puccinia coronata f. sp. avenae, were identified. Two of these QTLs are consistent with known clusters of rust resistance genes, while two may represent new locations of novel rust resistance genes. A complete set of SNP sequences suitable for generating markers for molecular selection is provided.     

Construction of a genetic linkage map and QTL analysis of fiber-related traits in upland cotton (Gossypium hirsutum L.)

A genetic linkage map with 70 loci (55 SSR, 12 AFLP and 3 morphological loci) was constructed using 117 F₂ plants obtained from a cross between two upland cotton cultivars Yumian 1 and T586, which have relatively high levels of DNA marker polymorphism and differ remarkably in fiber-related traits. The linkage map comprised of 20 linkage groups, covering 525 cM with an average distance of 7.5 cM between two markers, or approximately 11.8% of the recombination length of the cotton genome. The present genetic linkage map was used to identify and map the quantitative trait loci (QTLs) affecting lint percentage and fiber quality traits in 117 F_2:3 family lines. Sixteen QTLs for lint percentage and fiber quality traits were identified in six linkage groups by multiple interval mapping: four QTLs for lint percentage, two QTLs for fiber 2.5% span length, three QTLs for fiber length uniformity, three QTLs for fiber strength, two QTLs for fiber elongation and two QTLs for micronaire reading. The QTL controlling fiber-related traits were mainly additive, and meanwhile including dominant and overdominant. Several QTLs affecting different fiber-related traits were detected within the same chromosome region, suggesting that genes controlling fiber traits may be linked or the result of pleiotropy.     

Occurrence, inheritance and use of reproductive mutants in alfalfa improvement

The widespread occurrence of 2ngametes (i.e. gametes with the somatic chromosome number) in the Medicago sativa-coerulea-falcata complex supports the concept that gene flow from diploid to tetraploid species occurs continuously in nature and plays a key role in alfalfa evolution. Breeders realized early that gene transfer between ploidy levels via 2n pollen and 2n eggs would have had potential use in cultivated alfalfa improvement. Cytological investigations provided insights into the types of meiotic abnormalities responsible for the production of 2n gametes. Alterations were defined as genetically equivalent to first (FDR) or second division restitution(SDR) mechanisms. For breeding purposes,data have proven that 2n gametes of the FDR type are more advantageous than those obtained by SDR for transferring parental heterozygosity and retaining epistatic interactions. The use of diploid meiotic mutants that produce 2ngametes is now recognized as one of the most effective methods available for exploiting heterosis and introgressing wild germplasm traits into cultivated tetraploid alfalfa via unilateral (USP) and bilateral sexual polyploidization (BSP) schemes. Both2n egg and 2n pollen producers could be used for direct gene transfer from wild diploid relatives into cultivated alfalfa by means of 2x-4x and4x-2x crosses. Although data have shown that forage yield improvement can be achieved when plants are sexually tetraploidized, problems related to reduced plant fertility and seed production remain largely unexplained. Apomixis has the potential of cloning plants through seed and thus provides a unique opportunity for developing superior tetraploid cultivars with permanently fixed heterosis and epistatic effects. A main goal in alfalfa breeding could be the introduction of functional apomixis (i.e. Apomeiosis and parthenogenesis) in cultivated alfalfa stocks. In the future, the efficiency of alfalfa breeding programs based on the use of reproductive mutants could be improved by direct selection at the genotype level using RFLPs and PCR-based markers. Suitable DNA markers and detailed linkage maps of alfalfa mutants should help to discover apomictic mutants and address basic genetic issues such as the extent of genomicre combination in polyploid hybrids and the effect of sexual polyploidization on heterosis. Molecular markers have recently been used in alfalfa for studying the inheritance of 2n gamete formation and identifying polymorphisms associated to genes involved in meiotic abnormalities. Molecular tagging of 2n egg and 2n pollen formation not only should explain the genetic control and regulation of these traits, but may also be an essential step towards marker-assisted selection of 2n gamete producers and implementation of USP and BSP breeding schemes. Future perspectives include strategies for the map-based cloning of genomic DNA markers,and screening of EST mini-libraries related to flowers at different developmental stages from meiotic mutants and wild-type scan lead to the identification of mRNAs and thus of candidate genes that control 2n gamete formation in alfalfa. This revised version was published online in July 2006 with corrections to the Cover Date.