Single-dose Restriction Fragments Research Articles

SSR-enriched genetic linkage maps of bermudagrass (Cynodon dactylon×transvaalensis), and their comparison with allied plant genomes.

We report SSR-enriched genetic maps of bermudagrass that: (1) reveal partial residual polysomic inheritance in the tetraploid species, and (2) provide insights into the evolution of chloridoid genomes. This study describes genetic linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), that integrate heterologous microsatellite markers from sugarcane into frameworks built with single-dose restriction fragments (SDRFs). A maximum likelihood approach was used to construct two separate parental maps from a population of 110 F1 progeny of a cross between the two parents. The T89 map is based on 291 loci on 34 cosegregating groups (CGs), with an average marker spacing of 12.5cM. The T574 map is based on 125 loci on 14 CGs, with an average marker spacing of 10.7cM. Six T89 and one T574 CG(s) deviated from disomic inheritance. Furthermore, marker segregation data and linkage phase analysis revealed partial residual polysomic inheritance in T89, suggesting that common bermudagrass is undergoing diploidization following whole genome duplication (WGD). Twenty-six T89 CGs were coalesced into 9 homo(eo)logous linkage groups (LGs), while 12 T574 CGs were assembled into 9 LGs, both putatively representing the basic chromosome complement (x = 9) of the species. Eight T89 and two T574 CGs remain unassigned. The marker composition of bermudagrass ancestral chromosomes was inferred by aligning T89 and T574 homologs, and used in comparisons to sorghum and rice genome sequences based on 108 and 91 significant blast hits, respectively. Two nested chromosome fusions (NCFs) shared by two other chloridoids (i.e., zoysiagrass and finger millet) and at least three independent translocation events were evident during chromosome number reduction from 14 in the polyploid common ancestor of Poaceae to 9 in Cynodon.

Using a linkage mapping approach to identify QTL for day‐neutrality in the octoploid strawberry

AbstractA linkage mapping approach was used to identify quantitative trait loci (QTL) associated with day‐neutrality in the commercial strawberry, Fragaria × ananassa (Duch ex Rozier). Amplified Fragment Length Polymorphism (AFLP) markers were used to build a genetic map with a population of 127 lines developed by crossing the day‐neutral (DN) ‘Tribute’ with the short‐day (SD) ‘Honeoye’. The population was genotyped with AFLP markers and 429 single dose restriction fragments (SDRF) were placed on a consensus map of 1541 cM with 43 linkage groups. Individuals from the mapping population were observed for their flowering habit throughout the growing season in Michigan (MI), Minnesota (MN), Maryland (MD), Oregon (OR) and California (CA). Eight QTL were found that were either location specific or shared among locations. None of these QTL explained >36% of the phenotypic variation, indicating that the inheritance of day‐neutrality is likely a polygenic trait.

A framework linkage map of bermudagrass (Cynodon dactylon × transvaalensis) based on single-dose restriction fragments

This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps.

Analysing diversity in sugarcane resistance gene analogues

As resistance genes have been shown to contain conserved motifs and cluster in many plant genomes, the identification of resistance gene analogues can be used as a strategy for both the discovery of DNA markers linked to disease resistance loci and the map-based cloning of disease resistance genes. Sugarcane suffers from many important diseases and an analysis of resistance gene analogues offers a means to identify DNA markers linked to resistance loci. However, sugarcane has the most complex genome of any crop plant and initially it is important to understand the extent of resistance gene analogue diversity in the sugarcane genome before genetic analysis. We review herein how more than 100 expressed sequence tags with homology to different resistance genes have been identified in sugarcane with many mapped as single-dose restriction fragment length polymorphism markers. Importantly, some of these resistance gene analogues have been shown to be linked to disease resistance genes or disease quantitative trait loci. In an attempt to more efficiently analyse additional resistance gene analogues in sugarcane, we report on experiments aimed at investigating the molecular diversity of several resistance gene analogue families using a modified form of a technique termed Ecotilling. Using Ecotilling, we were able to rapidly detect single nucleotide polymorphisms in fragments amplified by PCR from four different resistance gene analogue families, SoRP1D, SoPTO, SoXa21 and SoHs1pro-1. An analysis of a diverse set of sugarcane varieties, including modern sugarcane cultivars and several S. officinarum and S. spontaneum clones, indicated that all amplicons, apart from SoHs1pro-1, contained significant polymorphism within the gene region studied. However, a comparison among these sugarcane clones, including between the parents of two sugarcane mapping populations, indicated that most polymorphisms were multi-dose, not single-dose, preventing their genetic map location or association with disease susceptibility or resistance from being determined.

Identification of Male‐Specific AFLP Markers in Dioecious Texas Bluegrass

Dioecy is a breeding system that promotes cross‐pollination in plants. The transfer of this trait into economically important self‐pollinated cereal crops would revolutionize the production of hybrids in these species and provide a means for increasing yields because of heterosis. Texas bluegrass (Poa arachnifera Torr.) (2n = 8x = 56) is a polymorphic dioecious species that provides an opportunity to genetically map the dioecy locus. In this study, amplified fragment length polymorphism (AFLP)‐based linkage maps were constructed for both the maternal and paternal plants used to develop a mapping population of Texas bluegrass. The maternal map contained 126 single dose restriction fragments (SDRFs), 31 linkage groups, 1744 cM, and an average marker spacing of 13.8 cM. The paternal map contained 210 SDRFs, 46 linkage groups, 2699 cM, and an average marker spacing of 12.9 cM. Approximately 76 to 81% of the Texas bluegrass genome was covered. Two AFLP markers (txbg7 and txbg154) mapped equidistantly (9.5 cM) on opposite sides of the dioecy locus (PDio1) on the paternal map. These markers provide a preliminary tool for studying sex determination and a framework for further characterization of the genomic region conferring dioecy in Texas bluegrass.

QTL Associated with Day-neutrality in Strawberry

While it is of great significance for strawberry breeders to know the genetics of day-neutrality (DN), evidence for inheritance of the trait is still contradictory. A linkage mapping approach is being used to determine how many QTL regulate DN and the proportion of the variability explained by each. A preliminary genetic linkage map was constructed for 125 individuals of the day neutra× short day (SD) cross `Tribute' × `Honeoye' using single dose restriction fragments (SDRFs) of amplified fragment length polymorphic (AFLP) markers. Over 500 SDRFs from 55 AFLP primer combinations were used to build the map using the software tool Join Map 3.0 at a LOD score of 3.0. Single marker analysis using WinQTL cartographer software previously determined 27 SDRF markers to co-segregate with DN for 57 individuals of the mapping population phenotyped in the field for the years 2002 and 2003, indicating putative QTL for DN. These markers were included in the linkage analysis and seven of them mapped to five different linkage groups that may indicate the quantitative nature of the trait. For determining QTL and percentage of phenotype governed by each QTL, however, accurate phenotypic evaluation is critical. Therefore, controlled environment (growth chamber) studies were used to obtain flowering response data under SD and long day (LD) conditions with two day/night temperatures. This study was conducted for the entire mapping population (over 400 individuals) so that QTL detected can be confirmed by fine mapping the QTL regions. We will also test how robust the QTL detected are, by analyzing the same segregating population at six different field locations in the United States (California, Maryland, Michigan, Minnesota, New York, and Oregon) for their flowering response under SD and LD conditions.

Investigation of genomic organization in switchgrass (Panicum virgatum L.) using DNA markers

We report an early investigation into genomic organization and chromosomal transmission in switchgrass based on restriction fragment length polymorphism (RFLP) markers. The segregation of 224 single dose restriction fragments (SDRF) in 85 full-sib progeny of a cross between the genotypes Alamo (AP13) and Summer (VS16) was used to determine linkage associations in each parent. In the seed parent AP13, 11 cosegregation groups were identified by 45 SDRF markers with a cumulative recombination length of 412.4 cM. In the pollen parent VS16, 57 SDRF markers were assigned to 16 cosegregation groups covering a length of 466.5 cM. SDRF markers identified by the same probes and mapping to different cosegregation groups were used to combine the two maps and identify homology groups. Eight homology groups were identified among the nine haploid linkage groups expected in switchgrass. The high incidence of repulsion phase associations indicates that preferential pairing between homologous chromosomes is predominant in switchgrass. Based on marker distribution in the paternal map (VS16), we estimated the recombinational length of switchgrass genome to be 4,617 cM. In order to link 95% of the genome to a marker at a 15-cM distance, a minimum of 459 markers will be required. Using information from the ratio of repulsion to coupling linkages, we infer that switchgrass is an autotetraploid with a high degree of preferential pairing. The information presented in this study establishes a foundation for extending genetic mapping in this crop and constitutes a framework for basic and applied genetic studies.

A Model Selection-Based Interval-Mapping Method for Autopolyploids

While extensive progress has been made in quantitative trait locus (QTL) mapping for diploid species, similar progress in QTL mapping for polyploids has been limited due to the complex genetic architecture of polyploids. To date, QTL mapping in polyploids has focused mainly on tetraploids with dominant and/or codominant markers. Here, we extend this view to include any even ploidy level under a dominant marker system. Our approach first selects the most likely chromosomal marker configurations using a Bayesian selection criterion and then fits an interval-mapping model to each candidate. Profiles of the likelihood-ratio test statistic and the maximum-likelihood estimates (MLEs) of parameters including QTL effects are obtained via the EM algorithm. Putative QTL are then detected using a resampling-based significance threshold, and the corresponding parental configuration is identified to be the underlying parental configuration from which the data are observed. Although presented via pseudo-doubled backcross experiments, this approach can be readily extended to other breeding systems. Our method is applied to single-dose restriction fragment autotetraploid alfalfa data, and the performance is investigated through simulation studies.

Mapping QTL for Day-neutrality in Strawberry

While it is important for strawberry breeders to know the genetics of day-neutrality, evidence for inheritance of the trait is still contradictory. It is not known how many genes govern the trait, to what extent each gene affects phenotype and how the environment influences gene expression. Several recent studies point toward a polygenic threshold model and a rejection of the single gene model. A linkage mapping approach is being used to determine if day neutrality can be mapped to several different quantitative trait loci (QTL) that may represent different genes. To confirm that a linkage mapping approach is the method of choice for QTL detection, a small population of the cross `Honeoye' x `Tribute' consisting of 57 progeny segregating for the trait was genotyped with single dose restriction fragment (SDRF) markers and a preliminary genetic map was created using Join Map 3.0. Results separated the molecular markers into at least 24 linkage groups and several putative QTL for day neutrality were identified indicating that the technique will be successful. However, due to the complexity of the octoploid genome of strawberry, over 200 progeny need to be genotyped to build a complete map that includes the 56 linkage groups of the genome. Furthermore, for determining QTL, an accurate phenotypic evaluation is critical. Individuals of the population above were phenotyped under field conditions (East Lansing, Mich.) in 2002 and 2003, and are now being analyzed under controlled temperature and photoperiod conditions for confirmation of the QTL detected for the trait. A larger population of the same cross with over 200 progeny has also been generated and will be mapped using molecular markers after determining their phenotype under the same environmental conditions.

Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (Fragaria x ananassa) using AFLP mapping.

A two-way pseudo-testcross strategy, combined with Single Dose Restriction Fragment (SDRF) marker analysis, was used for genetic mapping in the octoploid cultivated strawberry Fragaria x ananassa (2n = 8 x = 56). Based on a 113 full-sib progeny from a cross between the variety Capitola and the clone CF1116, we generated two parental maps using Amplified Fragment Length Polymorphism (AFLP) markers. Ninety two percent of the markers (727 out of 789) showed ratios corresponding to simplex markers (the majority being SDRF markers), and 8% (62 out of 789) fitted a multiplex ratio. Linkage maps were first established using SDRF markers in coupling phase. The female map comprised 235 markers distributed among 43 co-segregation groups, giving a map size of 1,604 cM. On the male map, 280 markers were assigned to 43 co-segregation groups, yielding a map size of 1,496 cM. Once the co-segregation groups were established, their association was tested using repulsion-phase markers. In total, taking into account associations representing the same linkage groups, 30 linkage groups were detected on the female side and 28 on the male side. On the female map, 68.3% of the pairwise marker linkages were in coupling versus 31.7% in repulsion phase, and the corresponding figures on the male map were 72.2% and 27.8%, respectively. In addition, both groups linked only in the coupling phase and groups linked in the repulsion phase were characterized. The observations suggest that the meiotic behavior of the F. x ananassa genome is neither fully disomic nor fully polysomic, but rather mixed. The genome may not be as completely diploidized as previously assumed.

Mapping Genetic Factors Associated with Winter Hardiness, Fall Growth, and Freezing Injury in Autotetraploid Alfalfa

Winter hardiness is a complex trait and one of the most important adaptations for alfalfa (Medicago sativa L.) grown in northern climates. In the absence of winter hardiness data, alfalfa breeders predict the potential of genotypes with component traits related to winter hardiness. This research was undertaken to identify and compare some of the genomic regions that control winter injury (WI) and two component traits, fall growth (FG) and freezing injury (FI). Two plants, B17 and P13, representing the extremes for each trait were crossed, and a F1 plant was backcrossed to each parent to create two populations of 101 individuals each. Each population was scored for 82 single dose restriction fragment loci, and 17 or 19 two‐allele loci and evaluated for FG, FI, and WI in 2 yr of replicated field trials. Trait measures over the 2 yr were significantly correlated (r = 0.71, r = 0.42, and r = 0.76 for FG, FI, and WI, respectively). Significant correlations also existed between WI and FG (r = 0.50 and 0.56) and FI (r = 0.34 and 0.58) for each year. One to six single dose restriction fragment were significant factors in multiple regression models that explained 6.3 to 52.2% of the phenotypic variation for each trait in each year and the average of 2 yr. More of the phenotypic variation was explained in the backcross to B17 (the winter hardy, fall dormant parent) than in the backcross to P13 (the winter sensitive, non‐fall dormant parent) and for FG than for FI and WI. Partial dominance was detected for P13 alleles at most loci associated with FG and for B17 at loci associated with FI. Additive gene action predominated for loci associated with WI. Severe winter kill and the association of FG with plant vigor may have masked identification of quantitative trait loci (QTL) in the P13 backcross. In the B17 backcross, genomic regions that contain QTL affecting FG, FI, and WI were identified on linkage groups 5 and 8, but QTL affecting only FG and FI were identified on linkage groups 1 and 3. These data indicate that there is a genetic basis for the use of predictor traits in the absence of winter hardiness data. However, they also suggest that genetic components of fall dormancy and winter hardiness can be manipulated independently, and they reveal regions that may be useful for marker‐assisted selection with this material.

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

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

Genetic linkage map in sour cherry using RFLP markers

Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS) and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS×EB. The RS linkage map consists of 126 single-dose restriction fragment (SDRF, Wu et al. 1992) markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95 SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifty three markers mapped in both parents were used as bridges between both maps and 13 sets of homologous linkage groups were identified. Homoeologous relationships among the sour cherry linkage groups could not be determined because only 15 probes identified duplicate loci. Fifty nine of the markers on the linkage maps were detected with probes used in other Prunus genetic linkage maps. Four of the sour cherry linkage groups may be homologous with four of the eight genetic linkage groups identified in peach and almond. Twenty one fragments expected to segregate in a 1 : 1 ratio segregated in a 2 : 1 ratio. Three of these fragments were used in the final map construction because they all mapped to the same linkage group. Six fragments exhibited segregation consistent with the expectations of intergenomic pairing and/or recombination.

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4.

RFLP linkage map of asparagus

A population resulting from a double pseudotestcross of two outbred-derived asparagus (Asparagus officinalis L.) clones was evaluated by RFLP (restriction fragment length polymorphism) analysis to produce individual maps of the male and female parents. An asparagus PstI genomic library was created and used as the source of probes. Scoring of bands was done by examining SDRFs (single dose restriction fragments) that are present in one parent and absent in the other and segregate 1:1 in the progeny. The data were analyzed as a backcross population; inversion or recoding allowed for the detection of repulsion phase linkage. The male parent map consisted of 33 loci in 10 groups, while the female parent map had 48 loci arranged in 14 groups. Segregation distortion was minimal (5%), and 17% of the markers were found to be unlinked. Loci of the configuration a/b x a/b and a/c x b/c were used to bridge seven homologous linkage groups between the two parents. The sex locus was not found to be associated with any linkage group. Key words : RFLP, bridge loci, repulsion phase linkage, double pseudotestcross.

The detection and estimation of linkage in polyploids using single-dose restriction fragments

Restriction fragment length polymorphism (RFLP) linkage maps have been constructed in several major diploid crops. However, construction of RFLP maps directly in polyploids has lagged behind for several reasons: (1) there are a large number of possible genotypes for each DNA probe expected in a segregating population, and these genotypes cannot always be identified readily by their banding phenotypes; and (2) the genome constitutions (allopolyploidy versus autopolyploidy) in many high polyploids are not clearly understood. We present here an analysis of these problems and propose a general method for mapping polyploids based on segregation of single-dose restriction fragments (SDRFS). SDRFs segregate 1:1 (presence: absence) in gametes of heterozygous plants. Hypothetical allopolyploid and autopolyploid species with four ploidy levels of 2n = 4x, 6x, 8x, and 10x, are used to illustrate the procedures for identifying SDRFs, detecting linkages among SDRFs, and distinguishing allopolyploid versus autopolyploids from polyploids of unknown genome constitution. Family size required, probability of linkage, and attributes of different mapping populations are discussed. We estimate that a population size of 75 is required to identify SDRFs with 98% level of confidence for the four ploidy levels. This population size is also adequate for detecting and estimating linkages in the coupling phase for both allopolyploids and autopolyploids, but linkages in the repulsion phase can be estimated only in allopolyploids. For autopolyploids, it is impractical to estimate meaningful linkages in repulsion because very large family sizes (>750) are required. For high-level polyploids of unknown genome constitution, the ratio between the number of detected repulsion versus coupling linkages may provide a crude measurement of preferential chromosome pairing, which can be used to distinguish allopolyploidy from autopolyploidy. To create a mapping population, one parent (P1) should have high heterozygosity to ensure a high frequency of SDRFs, and the second parent (P2) should have a low level of heterozygosity to increase the probability of detecting polymorphic fragments. This condition could be satisfied by choosing outcrossed hybrids as one parental type and inbreds, haploids, or doubled haploids as the other parental type.