AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.)

Rye (Secale cereale L.) is considered to be the most aluminum (Al)-tolerant species among the Triticeae. It has been suggested that aluminum tolerance in rye is controlled by three major genes (Alt genes) located on rye chromosome arms 3RL, 4RL, and 6RS, respectively. Screening of an F₆ rye recombinant inbred line (RIL) population derived from the cross between an Al-tolerant rye (M39A-1–6) and an Al-sensitive rye (M77A-1) showed that a single gene controls aluminum tolerance in the population analyzed. In order to identify molecular markers tightly linked to the gene, we used a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis techniques to evaluate the F₆ rye RIL population. We analyzed approximately 22,500 selectively amplified DNA fragments using 204 primer combinations and identified three AFLP markers tightly linked to the Alt gene. Two of these markers flanked the Alt locus at distance of 0.4 and 0.7 cM. Chromosomal localization using cloned AFLP and a restriction fragment length polymorphism (RFLP) marker indicated that the gene was on the long arm of rye chromosome 4R. The RFLP marker (BCD1230) co-segregated with the Alt gene. Since the gene is on chromosome 4R, the gene was designated as Alt3. These markers are being used as a starting point in the construction of a high resolution map of the Alt3 region in rye. Received: 29 March 2000 / Accepted: 9 July 2001     

Molecular markers linked to the aluminium tolerance gene Alt1 in rye (Secale cereale L.)

 Rye has one of the most efficient group of genes for aluminium (Al) tolerance among cultivated species of Triticeae. This tolerance is controlled by at least two independent and dominant loci (Alt1 and Alt3) located on chromosomes 6RS and 4R. We used two pooled DNA samples, one of Al-tolerant individuals and another of Al-sensitive plants from one F₂ that segregated for the Alt1 locus. We also used two pooled DNA samples, one with genotypes 11 and another with genotypes 22 for the Lap1 locus (leucin aminopeptidase) from another F₂ progeny that segregated for this locus, located on the 6RS chromosome arm. We identified several RAPD markers associated with the pooled Al-tolerant plants and also with one of the bulks for the Lap1 locus. The RAPD fragments linked to Alt1 and Lap1 genes were transformed into SCAR markers to confirm their chromosomal location and linkage data. Two SCARs (ScR01 ₆₀₀ and ScB15 ₇₉₀₀ ) were closely linked to the Alt1 locus, ScR01 ₆₀₀ located 2.1 cM from Alt1 and ScB15 ₇₉₀ located 5.5 cM from Alt1, on the 6RS chromosome arm. These SCAR markers can aid in the transfer of Al tolerance genes into Al-sensitive germplasms. Received: 9 December 1997 / Accepted: 12 May 1998     

From the rye Alt3 and Alt4 aluminum tolerance loci to orthologous genes in other cereals

The major limit to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which limit growth by inhibiting root elongation. Aluminum tolerance in rye is controlled by (at least) four independent loci (Alt1, Alt2, Alt3 and Alt4) located on chromosome arms 6RS, 3RS, 4RL and 7RS, respectively. In this work, we analyzed several F₂ populations in which two different Alt loci were segregating. We constructed a map of chromosome 7R, which contains the Alt4 locus and microsatellite and PCR-markers (B1, B4, B11, B26 and BCD1230). These markers were mapped to the S arm of 7R using wheat-rye addition lines. Our results show that all these markers are linked to the Alt4 locus already known to be on 7RS. In addition, the OPS14 ₇₀₅ RAPD marker was linked to the Alt3 locus using bulked segregant analysis. This RAPD marker was transformed into a SCAR (ScOPS14 ₇₀₅ ) and was localized to arm 4RL using wheat-rye addition lines. Finally, this SCAR was linked to the Alt3 locus at a genetic distance of 23.4 cM. In light of the current findings, and taking into account the synteny relationships in cereals, we propose candidate Alt3 and Alt4 orthologues in other cereals.     

Development of codominant markers for identifying species hybrids

Herein we describe a simple method fordeveloping species-diagnostic markers thatwould permit the rapid identification of hybridindividuals. Our method relies on amplifiedlength polymorphism (AFLP) and single strandconformation polymorphism (SSCP) technologies,both of which can be performed in any molecularbiology facility using standard laboratoryequipment. We demonstrate the utility of theAFLP-SSCP method by developing threetaxon-specific markers that will be suitablefor monitoring introgression in endangeredKlamath basin suckers.     

Generation of PCR-based markers for the detection of rye chromatin in a wheat background

Oligonucleotide primers were developed to detect the presence of four rye sequences using a PCR assay. These assays give a rye-specific signal from wheat DNA template which contains various rye chromosomes or chromosome segments. The sequences identified were associated with the nucleolar organiser region, the 5S-Rrna-R1 locus, the telomere, and a widely dispersed, rye-specific repetitive element Ris-1. The primers amplified from the well-established loci Nor-R1 and 5S-Rrna-R1 on rye chromosome arm 1RS, and also located a 5s-Rrna locus on chromosome 3R. The telomere-associated sequence was present on every rye chromosome, and was also present, at a low copy number, in both wheat and barley. These assays will be particularly useful for introgression programmes aimed at reducing the rye content of the 1BL.1RS wheat-rye translocation. When multiplexed, the primers will enable a rapid, simultaneous assay for a number of distinct rye loci, which can be derived from a small portion of mature endosperm tissue.     

EST derived PCR-based markers for functional gene homologues in cotton.

We investigated the utility of the Gossypium arboreum EST sequences in the GenBank database for developing PCR-based markers targeting known-function genes in cultivated tetraploid cottons, G. hirsutum and G. barbadense. Four hundred sixty-five randomly selected ESTs from this library were subjected to BLASTn search against all GenBank databases, of which putative function was assigned to 93 ESTs based on high nucleotide homology to previously studied genes. PCR primers were synthesized for 89 of the known-function ESTs. A total of 57 primer pairs amplified G. arboreum genomic DNA, but only 39 amplified in G. hirsutum and G. barbadense, suggesting that sequence divergence may be a factor causing non-amplification for some sites. DNA sequence analysis showed that most primer pairs were targeting the expected homologous loci. While the amplified products that were of larger size than the corresponding EST sequences contain introns, the primer pairs with a smaller amplicon than predicted from the flanking EST sequences did not amplify the expected orthologous gene sequences. Among the 39 primer pairs that amplified tetraploid cotton DNA, 3 detected amplicon size polymorphisms and 10 detected polymorphisms after digestion with one of six restriction enzymes. Ten of the polymorphic loci were subsequently mapped to an anchor RFLP map. Digestion of PCR-amplified sequences offers one means by which cotton genes can be mapped to their chromosomal locations more quickly and economically than by RFLP analysis.     

Development of conserved ortholog set markers linked to the restorer gene Rfp1 in rye

Restoration of male fertility is a prerequisite for hybrid rye breeding and currently the most straightforward approach to minimize ergot infection in hybrid rye varieties. Molecular markers are important tools for the efficient introgression and management of restorer genes like Rfp1 originating from unadapted genetic resources. Furthermore, closely linked markers flanking Rfp1 are indispensible for identifying and selecting individuals with haplotypes showing recombination between Rfp1 and other gene(s) that reside in close proximity and have a negative influence on yield. We identified orthologous gene sets in rice, Brachypodium, and Sorghum and used these gene models as templates to establish conserved ortholog set (COS) markers for the restorer gene Rfp1 on the long arm of rye chromosome 4R. The novel co-dominant markers delimit Rfp1 within a 0.7-cM interval and allow prediction of Rfp1 genotypes with a precision not feasible before. The COS markers enabled an alignment of the improved genetic map of rye chromosome 4R with wheat and barley maps and allowed identification of regions orthologous to Rfp1 in wheat and barley on the short arms of chromosomes 6D and 6H, respectively. Results obtained in this study revealed that micro-collinearity around the Rfp1 locus in rye is affected by rearrangements relative to other grass genomes. The impact of the novel COS markers for practical hybrid rye breeding is discussed.     

Microsatellite markers flanking a stem solidness gene on chromosome 3BL in durum wheat

Sawfly (Cephus cinctus Norton) is a major insect pest of wheat (Triticum spp.). The development of durum wheat (Triticum turgidum L. var durum) with stem solidness for resistance to sawfly is a strategy to minimize loss from this insect. This study was undertaken to identify a DNA marker linked to stem solidness and sawfly cutting in durum wheat for use in marker-assisted selection. A set of 151 doubled haploid lines developed from the cross of Kyle*2/Biodur sel. (solid stemmed) and Kofa (hollow stemmed) were evaluated for stem solidness and sawfly cutting. Microsatelite primers that generated polymorphisms between the parental genotypes were tested on the whole population, and primers that followed a 1:1 ratio of parental bands were used in linkage analysis with least squares mean stem solidness scores. Three microsatellite markers, Xgwm247, Xgwm181 and Xgwm114 located on chromosome 3BL, were shown to be associated with the stem solidness locus and with sawfly cutting. The Xgwm114 marker was located on one side of the stem solidness locus with Xgwm247 and Xgwm181 on the opposing side. Recombinant inbred line populations G9580B-FE1C/AC Navigator and Golden Ball/DT379//STD65 segregating for the stem solidness trait confirmed the association between the markers and the stem solidness gene. The Golden Ball/DT379//STD65 population was also tested with the Xwmc632 microsatellite marker, which showed a polymorphism associated with stem solidness. The results also indicated the stem solidness trait was controlled by a single locus in both doubled haploid and recombinant inbred line populations. The markers should be useful in breeding programs for the identification and selection of stem solidness.     

Dominant vs. codominant genetic markers in the estimation of male mating success

Although a major component of fitness, male reproductive success is generally extremely difficult to estimate. As a result, genetic methods and maximum likelihood models have been developed to estimate male parentage, but all are limited in practice by the degree of genetic variation observable. Scoring individuals phenotypically at a large number of random loci exhibiting dominance (e.g. RAPD markers) may provide a means of detecting sufficient genetic variation. Dominance, however, represents a loss of information and therefore greater variation in the estimate of paternity. A mixture model describing mating in a population is presented to quantify the trade-off between marker types when estimates of male fertility are sought. A sample size 1.5-2.0 times greater is required for dominant markers under some conditions to obtain the same confidence in fertility estimates as for codominant markers, although with large sample sizes the fertility estimates are similar for either marker type. Since the number of dominant DN A markers is not limited in the same manner as is the number of codominant protein markers, one's confidence in the estimates can be increased above that possible from proteins by surveying additional loci. However, for a fixed sample size a trade-off exists between the number of progeny assayed per female and the number of loci surveyed. In many cases more progeny per female provide better estimates of fertility than more loci.     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999     

Development of PCR-based markers for thermosensitive genetic male sterility gene tms3(t) in rice (Oryza sativa L.)

Development of simple and reliable PCR-based markers is an important component of marker-aided selection (MAS) activities for agronomically important genes in rice breeding. In order to develop PCR-based markers for a rice thermosensitive genetic male sterility gene tms3(t), located on chromosome 6, the nucleotide sequences of four linked RAPD markers OPF18(2600), OPAC3(640), OPB19(750) and OPM7(550) were used to design and synthesize several pairs of specific primers for PCR amplification of the genomic DNA of both the parents IR32364TGMS (sterile) and IR68 (fertile), involved in mapping this gene. For the RAPD marker OPF 18(2600), two pairs of specific primer pair combination from different positions of the sequence resulted in generation of two codominant STS (Sequence Tagged Sites) markers. In case of markers OPAC3(640), OPB19(750) and OPAA7(550) the first two could generate dominant polymorphism, while the last one could not be successful in PCR amplification. Both the codominant STSs with primer combinations F18F/F18RM and F18FM/F18RM were found to be tightly linked to the tms3(t) gene with a genetic distance of 2.7 cM. The sizes of the different alleles in case of F18F/F18RM, F18FM/F18RM combinations were 2300 bp, 1050 bp, and 1900 bp, 1000 bp respectively. The efficiency of marker-assisted selection for this trait was estimated as 84.6%. Polymorphism survey of 12 elite rice lines, indicated that these PCR-based markers for tms3(t) can now be used in selecting TGMS plants at seeding stage in the segregating populations in environment independent of controlled temperature regime.     

Linked markers flanking the gene for multiple endocrine neoplasia type 2A

The inherited cancer syndrome multiple endocrine neoplasia type 2A (MEN2A) has recently been mapped to chromosome 10. We have typed 29 families with this disorder with DNA markers from the pericentromeric region of chromosome 10. Two markers, RBP3 and MCK2, were tightly linked to the MEN2A gene at recombination fractions of less than 3%. Multipoint analysis of the linkage data suggests that the gene is located within a 3-cM interval defined by the markers RBP3/MCK2 on one side and TB14.34 on the other. No evidence for locus heterogeneity was detected in any of the 27 families from 14 countries who were informative for the markers tested. The data confirm and refine the original assignment and provide the basis for presymptomatic screening for this disorder.     

Inference of individual ploidy level using codominant markers

A significant portion of plant species are polyploids, with ploidy levels sometimes varying among individuals and/or populations. Current techniques to determine the individual ploidy, e.g., flow cytometry, chromosome counting or genotyping‐by‐sequencing, are often cumbersome. Based on the genotypic probabilities for polysomic inheritance under double‐reduction, we developed a model to estimate allele frequency and infer the ploidy status of individuals from the allelic phenotypes of codominant genetic markers. The allele frequencies are estimated by an expectation‐maximization algorithm in the presence of null alleles, false alleles, negative amplifications and self‐fertilization, and the posterior probabilities are used to assign individuals into different levels of ploidy. The accuracy of this method under different conditions is evaluated. Our methods are freely available in a new software package, ploidyinfer , for use by other researchers which can be downloaded from http://github.com/huangkang1987/ploidyinfer .     

Candidate gene identification of an aluminum-activated organic acid transporter gene at the Alt4 locus for aluminum tolerance in rye (Secale cereale L.)

Among cereal crops, rye is one of the most tolerant species to aluminum. A candidate gene approach was used to determine the likely molecular identity of an Al tolerance locus (Alt4). Using PCR primers designed from a wheat aluminum tolerance gene encoding an aluminum-activated malate transporter (TaALMT1), a rye gene (ScALMT1) was amplified, cloned and sequenced. Subsequently, the ScALMT1 gene of rye was found to be located on 7RS by PCR amplification using the wheat–rye addition lines. SNP polymorphisms for this gene were detected among the parents of three F₂ populations that segregate for the Alt4 locus. A map of the rye chromosome 7R, including the Alt4 locus ScALMT1 and several molecular markers, was constructed showing a complete co-segregation between Alt4 and ScALMT1. Furthermore, expression experiments were carried out to clarify the function of this candidate gene. Briefly, the ScALMT1 gene was found to be primarily expressed in the root apex and upregulated when aluminum was present in the medium. Five-fold differences in the expression were found between the Al tolerant and the Al non-tolerant genotypes. Additionally, much higher expression was detected in the rye genotypes than the moderately tolerant “Chinese Spring” wheat cultivar. These results suggest that the Alt4 locus encodes an aluminum-activated organic acid transporter gene that could be utilized to increase Al tolerance in Al sensitive plant species. Finally, TaALMT1 homologous sequences were identified in different grasses and in the dicotyledonous plant Phaseolus vulgaris. Our data support the hypothesis of the existence of a common mechanism of Al tolerance encoded by a gene located in the homoeologous group four of cereals. G. Fontecha and J. Silva-Navas contributed equally to this work.     

Characterization of AFLP markers in damselflies: prevalence of codominant markers and implications for population genetic applications.

Amplified fragment length polymorphism (AFLP) analysis is becoming increasingly popular as a method for generating molecular markers for population genetic applications. For practical considerations, it is generally assumed in population studies that AFLPs segregate as dominant markers, i.e., that present and absent are the only possible states of a given locus. We tested the assumption of dominance in natural populations of the damselfly Nehalennia irene (Hagen) (Odonata: Coenagrionidae). Electro-blotted AFLP products from 21 samples were probed with individual markers. Eleven markers were analyzed, of which two were monomorphic and nine were polymorphic. Only two of the polymorphic markers behaved in a strictly dominant manner. The remaining seven polymorphic markers displayed various degrees of codominance, with 2-10 visible alleles in the sample. Of the three markers displaying the highest degree of variability, two contained microsatellite repeat tracts. Our results suggest that the assumption of dominance is unfounded. As a result, AFLP analysis may be unsuitable for estimating several important population genetic parameters, including genetic diversity.