Cosegregation Groups Research Articles

Construction of AFLP-based cosegregation groups of tetraploid Plicatula species and identification of markers linked to apomixis

Abstract Most species of Plicatula are important native forages. This work aimed to build framework cosegregation groups of the apomictic tetraploid race of Paspalum guenoarum cv. Rojas and localize the locus controlling apomixis in the species. An interspecific population derived from crossing a completely sexual tetraploid plant of P. plicatulum and an apomictic tetraploid individual of P. guenoarum cv. Rojas was used. Both, disomic and tetrasomic inheritance were detected in both parental genotypes. In P. guenoarum, ten cosegregation groups were built, including 50 markers expanding for 583 cM. The estimated genome coverage was 63.95%. The apomixis locus was located in the linkage group M8, together with seven other loci (four paternal and three biparental markers). The group extended for 59 cM. The four paternal markers showed strong linkage to apomixis, and two of them mapped at 4 and 7 cM at both sides of the locus. Five female linkage groups were constructed with markers segregating from P. plicatulum. One of them (F3) being homologous to the male group carrying apomixis. The linkage groups presented here constitute the first genetic frame for species of Plicatula group. Moreover, molecular markers linked to apomixis in P. guenoarum can assist fundamental research and breeding programs.

Identification of QTLs for sex expression in dioecious and monoecious hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.) is a diploid species including both dioecious and monoecious cultivars with hetero- and homomorphic sex chromosomes, respectively. It displays a high plasticity of sex expression, i.e., the ratio of female and male flowers. In this study, we investigated the role of sex chromosomes in the genetic determinism of sex expression in dioecious and monoecious hemp. The experimental materials were three F1 segregating populations, two dioecious (C1 and C2: ‘Carmagnola’ ♀ × ‘Carmagnola’ ♂), and one monoecious (UF: ‘Uso 31’ × ‘Fedora 17’). A ‘sex’ phenotypic marker was mapped in C1 and C2. In total, 23, 42, and 26 AFLP markers (71 markers in total) were mapped to three, nine, and three co-segregation groups putatively located on sex chromosomes in C1, C2, and UF, respectively. Recombination rates with sex ranged from 0 to 0.5. Five sex-linked markers were detected in UF, revealing homologies between the X chromosomes of monoecious hemp and the X and Y chromosomes of dioecious hemp. Five QTLs associated with quantitative variations in sex expression were identified in each map. Four markers associated with variations in sex expression in UF segregated with sex or accounted for a putative QTL in C1 or C2. Two QTLs and three of these markers were mapped in UF in a region homologous to the sex-locus region of the dioecious maps. Given these results, conducting further research on the genetic determinism of sex expression in hemp using a quantitative approach appears relevant.

A novel linkage map of sugarcane with evidence for clustering of retrotransposon-based markers.

BackgroundThe development of sugarcane as a sustainable crop has unlimited applications. The crop is one of the most economically viable for renewable energy production, and CO2 balance. Linkage maps are valuable tools for understanding genetic and genomic organization, particularly in sugarcane due to its complex polyploid genome of multispecific origins. The overall objective of our study was to construct a novel sugarcane linkage map, compiling AFLP and EST-SSR markers, and to generate data on the distribution of markers anchored to sequences of scIvana_1, a complete sugarcane transposable element, and member of the Copia superfamily.ResultsThe mapping population parents (‘IAC66-6’ and ‘TUC71-7’) contributed equally to polymorphisms, independent of marker type, and generated markers that were distributed into nearly the same number of co-segregation groups (or CGs). Bi-parentally inherited alleles provided the integration of 19 CGs. The marker number per CG ranged from two to 39. The total map length was 4,843.19 cM, with a marker density of 8.87 cM. Markers were assembled into 92 CGs that ranged in length from 1.14 to 404.72 cM, with an estimated average length of 52.64 cM. The greatest distance between two adjacent markers was 48.25 cM. The scIvana_1-based markers (56) were positioned on 21 CGs, but were not regularly distributed. Interestingly, the distance between adjacent scIvana_1-based markers was less than 5 cM, and was observed on five CGs, suggesting a clustered organization.ConclusionsResults indicated the use of a NBS-profiling technique was efficient to develop retrotransposon-based markers in sugarcane. The simultaneous maximum-likelihood estimates of linkage and linkage phase based strategies confirmed the suitability of its approach to estimate linkage, and construct the linkage map. Interestingly, using our genetic data it was possible to calculate the number of retrotransposon scIvana_1 (~60) copies in the sugarcane genome, confirming previously reported molecular results. In addition, this research possibly will have indirect implications in crop economics e.g., productivity enhancement via QTL studies, as the mapping population parents differ in response to an important fungal disease.

A genetic linkage map of tetraploid orchardgrass (Dactylis glomerataL.) and quantitative trait loci for heading date

Orchardgrass (Dactylis glomerata L.), or cocksfoot, is indigenous to Eurasia and northern Africa, but has been naturalized on nearly every continent and is one of the top perennial forage grasses grown worldwide. To improve the understanding of genetic architecture of orchardgrass and provide a template for heading date candidate gene search in this species, the goals of the present study were to construct a tetraploid orchardgrass genetic linkage map and identify quantitative trait loci associated with heading date. A combination of SSR markers derived from an orchardgrass EST library and AFLP markers were used to genotype an F1 population of 284 individuals derived from a very late heading Dactylis glomerata subsp. himalayensis parent and an early to mid-heading Dactylis glomerata subsp. aschersoniana parent. Two parental maps were constructed with 28 cosegregation groups and seven consensus linkage groups each, and homologous linkage groups were tied together by 38 bridging markers. Linkage group lengths varied from 98 to 187 cM, with an average distance between markers of 5.5 cM. All but two mapped SSR markers had homologies to physically mapped rice (Oryza sativa L.) genes, and six of the seven orchardgrass linkage groups were assigned based on this putative synteny with rice. Quantitative trait loci were detected for heading date on linkage groups 2, 5, and 6 in both parental maps, explaining between 12% and 24% of the variation.

Genetic analysis of the sugarcane (Saccharum spp.) cultivar ‘LCP 85-384’. I. Linkage mapping using AFLP, SSR, and TRAP markers

Sugarcane hybrids are complex aneu-polyploids (2n = 100-130) derived from inter-specific hybridization between ancestral polyploid species, namely S. officinarum L. and S. spontaneum L. Efforts to understand the sugarcane genome have recently been enhanced through the use of new molecular marker technologies. A framework genetic linkage map of Louisiana's popular cultivar LCP 85-384 was constructed using the selfed progeny and based on polymorphism derived from 64 AFLP, 19 SSR and 12 TRAP primer pairs. Of 1,111 polymorphic markers detected, 773 simplex (segregated in 3:1 ratio) and 182 duplex (segregate in 77:4 ratio) markers were used to construct the map using a LOD value of ≥ 4.0 and recombination threshold of 0.44. The genetic distances between pairs of markers linked in the coupling phase was computed using the Kosambi mapping function. Of the 955 markers, 718 simplex and 66 duplex markers were assigned to 108 co-segregation groups (CGs) with a cumulative map length of 5,617 cM and a density of 7.16 cM per marker. Fifty-five simplex and 116 duplex markers remained unlinked. With an estimated genome size of 12,313 cM for LCP 85-384, the map covered approximately 45.6% of the genome. Forty-four of the 108 CGs were assigned into 9 homo(eo)logous groups (HGs) based on information from locus-specific SSR and duplex markers, and repulsion phase linkages detected between CGs. Meiotic behavior of chromosomes in cytogenetic studies and repulsion phase linkage analysis between CGs in this study inferred the existence of strong preferential chromosome pairing behavior in LCP 85-384. This framework map marks an important beginning for future mapping of QTLs associated with important agronomic traits in the Louisiana sugarcane breeding programs.

Analysis of genome-wide linkage disequilibrium in the highly polyploid sugarcane

Linkage disequilibrium (LD) in crops, established by domestication and early breeding, can be a valuable basis for mapping the genome. We undertook an assessment of LD in sugarcane (Saccharum spp), characterized by one of the most complex crop genomes, with its high ploidy level (>or=8) and chromosome number (>100) as well as its interspecific origin. Using AFLP markers, we surveyed 1,537 polymorphisms among 72 modern sugarcane cultivars. We exploited information from available genetic maps to determine a relevant statistical threshold that discriminates marker associations due to linkage from other associations. LD is very common among closely linked markers and steadily decreases within a 0-30 cM window. Many instances of linked markers cannot be recognized due to the confounding effect of polyploidy. However, LD within a sample of cultivars appears as efficient as linkage analysis within a controlled progeny in terms of assigning markers to cosegregation groups. Saturating the genome coverage remains a challenge, but applying LD-based mapping within breeding programs will considerably speed up the localization of genes controlling important traits by making use of phenotypic information produced in the course of selection.

Functional integrated genetic linkage map based on EST-markers for a sugarcane (Saccharum spp.) commercial cross

The growing availability of ESTs provides a potentially valuable source of new DNA markers. The authors examined the SUCEST database and developed EST-derived markers. Thus to enhance the resolution of an existing linkage map and to identify putative functional polymorphic gene loci in a sugarcane commercial cross, 149 EST-SSRs and 10 EST-RFLPs were screened in the SP80-180 × SP80-4966 mapping population. With the markers already analyzed in the previous map, 2303 polymorphic markers were generated, of which 1669 (72.5%) were single-dose (SD) markers. Out of these 1669 SD markers, 664 (40%) were scattered onto 192 co-segregation groups (CGs) with a total estimated length of 6.261,1 cM. Using both genomic and EST-derived SSR and RFLP markers, 120 out of the 192 CGs were formed into fourteen putative homology groups (HGs). The EST-derived markers were subjected to BLASTX search in the SUCEST database, of which putative function was assigned to 113 EST-SSRs and six EST-RFLPs based on high nucleotide homology to previously studied genes. The integration of EST-derived markers improved the map, making it possible to consider additional fine mapping of the genome, and providing the means for developing ‘perfect markers’ associated with key QTL. To summarize, this paper deals with the construction of a genetic linkage map of sugarcane that is populated by functionally associated markers.

A genetic map of tetraploid Paspalum notatum Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F1 family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.

Genetic mapping in sugarcane, a high polyploid, using bi-parental progeny: identification of a gene controlling stalk colour and a new rust resistance gene

Modern sugarcane cultivars (Saccharum spp) are highly polyploïd and aneuploid interspecific hybrids (2n = 100-130). Two genetic maps were constructed using a population of 198 progeny from a cross between R570, a modern cultivar, and MQ76-53, an old Australian clone derived from a cross between Trojan (a modern cultivar) and SES528 (a wild Saccharum spontaneum clone). A total of 1,666 polymorphic markers were produced using 37 AFLP primer combinations, 46 SSRs and 9 RFLP probes. Linkage analysis led to the construction of 86 cosegregation groups for R570 and 105 cosegregation groups for MQ76-53 encompassing 424 and 536 single dose markers, respectively. The cumulative length of the R570 map was 3,144 cM, while that of the MQ76-53 map was 4,329 cM. Here, we integrated mapping information obtained on R570 in this study with that derived from a previous map based on a selfed R570 population. Two new genes controlling Mendelian traits were localized on the MQ76-53 map: a gene controlling the red stalk colour was linked at 6.5 cM to an AFLP marker and a new brown rust resistance gene was linked at 23 cM to an AFLP marker. Besides another previously identified brown rust resistance gene (Bru1), these two genes are the only other major genes to be identified in sugarcane so far.

Development of an integrated genetic map of a sugarcane (Saccharum spp.) commercial cross, based on a maximum-likelihood approach for estimation of linkage and linkage phases

Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars ('SP80-180' x 'SP80-4966') using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane.

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.

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.

Linkage mapping in tetraploid willows: segregation of molecular markers and estimation of linkage phases support an allotetraploid structure for Salix alba x Salix fragilis interspecific hybrids.

Salix alba-Salix fragilis complex includes closely related dioecious polyploid species, which are obligate outcrossers. Natural populations of these willows and their hybrids are represented by a mixture of highly heterozygous genotypes sharing a common gene pool. Since nothing is known about their genomic constitution, tetraploidy (2n=4x=76) in willow species makes basic and applied genetic studies difficult. We have used a two-way pseudotestcross strategy and single-dose markers (SDMs) to construct the first linkage maps for both pistillate and staminate willows. A total of 242 amplified fragment length polymorphisms (AFLPs) and 50 selective amplifications of microsatellite polymorphic loci (SAMPL) markers, which showed 1:1 segregation in the F(1) mapping populations, were used in linkage analysis. In S. alba, 73 maternal and 48 paternal SDMs were mapped to 19 and 16 linkage groups covering 708 and 339 cM, respectively. In S. fragilis, 13 maternal and 33 paternal SDMs were mapped in six and 14 linkage groups covering 98 and 321 cM, respectively. For most cosegregation groups, a comparable number of markers linked in coupling and repulsion was identified. This finding suggests that most of chromosomes pair preferentially as occurs in allotetraploid species exhibiting disomic inheritance. The detection of 10 pairs of marker alleles from single parents showing codominant inheritance strengthens this hypothesis. The fact that, of the 1122 marker loci identified in the two male and female parents, the vast majority (77.5%) were polymorphic and as few as 22.5% were shared between parental species highlight that S. alba and S. fragilis genotypes are differentiated. The highly difference between S. alba- and S. fragilis-specific markers found in both parental combinations (on average, 65.3 vs 34.7%, respectively) supports the (phylogenetic) hypothesis that S. fragilis is derived from S. alba-like progenitors.

Genetic dissection of a modern sugarcane cultivar (Saccharum spp.). I. Genome mapping with AFLP markers

Sugarcane cultivars are polyploid, aneuploid clones derived from interspecific hybridization between Saccharum officinarum and S. spontaneum. Their genome has recently started to be unravelled as a result of the development of molecular markers. We constructed an AFLP genetic map based on a selfing population of a specific cultivar, R570.Using 37 AFLP primer pairs, we detected 1,185 polymorphic markers of which 939 were simplex (segregated 3:1); these were used to construct the map. Of those 939, 887 were distributed on 120 cosegregation groups (CGs) based on linkages in coupling, while 52 remained unlinked. The cumulative length of all the groups was 5,849 cM, which is probably around one-third of the total genome length. Comparison with reference S. officinarum clones enabled us to assign 11 and 79 CGs to S. spontaneum and S. officinarum,respectively, whereas 11 CGs were probably derived from recombination between chromosomes of the two ancestral species. The patchy size of the groups, which ranges from 1 to 232 cM, illustrates the difficulty to access large portions of chromosomes, particularly those inherited from S. officinarum. Repulsion phase linkages suggested a high preferential pairing for 13 CG pairs. Out of the 120 CGs, 34 could be assigned to one of the 10 homo(eo)logy groups already defined in a previous RFLP map owing to the use of a small common marker set. The genome coverage was significantly increased in the map reported here. Implications for quantitative trait loci (QTL) research and marker-assisted breeding perspectives are discussed.

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.

RFLP mapping in cultivated sugarcane (Saccharum spp.): genome organization in a highly polyploid and aneuploid interspecific hybrid.

Sugarcane cultivars are polyploid, aneuploid, interspecific hybrids between the domesticated species Saccharum officinarum and the wild relative S. spontaneum. Cultivar chromosome numbers range from 100 to 130 with approximately 10% contributed by S. spontaneum. We have undertaken a mapping study on the progeny of a selfed cultivar, R570, to analyze this complex genome structure. A set of 128 restriction fragment length polymorphism probes and one isozyme was used. Four hundred and eight markers were placed onto 96 cosegregation groups, based on linkages in coupling only. These groups could tentatively be assembled into 10 basic linkage groups on the basis of common probes. Origin of markers was investigated for 61 probes and the isozyme, leading to the identification of 80 S. officinarum and 66 S. spontaneum derived markers, respectively. Their distribution in cosegregation groups showed better map coverage for the S. spontaneum than for the S. officinarum genome fraction and occasional recombination between the two genomes. The study of repulsions between markers suggested the prevalence of random pairing between chromosomes, typical of autopolyploids. However, cases of preferential pairing between S. spontaneum chromosomes were also detected. A tentative Saccharum map was constructed by pooling linkage information for each linkage group.

A molecular approach to unraveling the genetics of sugarcane, a complex polyploid of the Andropogoneae tribe

Modern sugarcane varieties are complex aneuploids and typically have chromosome numbers in the 100-125 range with about 5-10% of them contributed by wild relatives, mainly Saccharum spontaneum, and the rest by S. officinarum. This particular genomic constitution was found favorable for mapping the S. spontaneum genome, using maize as a diploid reference for comparison. We conducted an analysis of 32 individuals derived from the selfing of variety SP 701006 using four isozymes and 53 maize probes which covered the whole maize genome. A total of 348 segregating bands were generated. Highly significant cosegregations enabled us to place 94 markers into 25 cosegregation groups. Eighteen of these groups involved S. spontaneum specific markers and might therefore mark S. spontaneum chromosomes in segregation. On the basis of probes in common, the 25 cosegregation groups could be assembled into eight tentative linkage groups, of which seven describe S. spontaneum chromosomes. A large degree of synteny between sugarcane and maize could be inferred, with a much lower rate of recombination in sugarcane.