Co-segregation of the gynomonoecious sex form1 gene (gsf1) of Tripsacum dactyloides (Poaceae) with molecular markers.

The only monogenic trait in Tripsacum to date was first identified in the prolific sex form variant Tripsacum dactyloides (L.) L. forma prolificum Dayton et Dewald. The expression of this trait is controlled by the presence of a single-gene, recessive pistillate mutation hereby designated the gynomonoecious sex form1 gene (gsf1), after the registered plant germplasm accession GSF-I (PI483447) from which it was first identified. This trait confers a high degree of feminization to the primarily male floral structure of the Tripsacum rachis. Two molecular markers were found to co-segregate with the gsf1 gene in a diploid (2n = 36) F2 population of Tripsacum dactyloides, where the female parent (GSF-I) had been previously determined to be homozygous recessive for the gene. Phenotypic scoring data were compared with restriction fragment length polymorphism data and linkage relationships were determined. The gsf1 gene is located ~7 cM from tda48, a Tripsacum-derived molecular marker, and ~9 cM from npi286, a maize-derived molecular marker. The marker npi286 also maps within ~5 cM of the tassel seed2 locus (ts2) of maize, which confers a similar change in the inflorescence of the maize tassel.     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Cytological and molecular evaluation of the reproductive behavior of Tripsacum andersonii and a female fertile derivative (Poaceae)

Research was conducted to characterize the reproductive behavior of the highly sterile Tripsacum andersonii Gray and its viable progeny through breeding, cytological, and molecular studies. Four progeny were obtained from open-pollinated seeds of clones (M-34445, M-34450 and M-34455) of T. andersonii maintained at the USDA-ARS National Germplasm Repository, Miami, Florida. One of the progeny had 64 chromosomes, which is typical of T. andersonii, and probably resulted from apomictic reproduction. Karyotypes of the other three progeny indicated a tetraploid Tripsacum genomic constitution (2n = 4x = 72) plus a haploid set of Zea (1n = 1x = 10) chromosomes. Two of these progeny were completely sterile, whereas one (95-51) produced ～5% seed set when crossed with diploid (2n = 36) T. dactyloides (L.)L. The partially fertile 95-51 produced four progeny, one with 2n = 72 (elimination of 10 Zea chromosomes), two with 2n = 82 (apomictic reproduction) and one with 2n = 100 (sexual polyploidization). Polymerase Chain Reaction - Random Amplified Polymerase DNA analysis verified that T. andersonii accessions from seven countries were genetically uniform, and that its progeny were derived through apomixis and sexual polyploidization. This analysis also confirmed that chromosome elimination, apomixis, and sexual polyploidization reproductive behaviors occur in the T. andersonii derivative 95-51.     

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.)

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 μm) was applied to 27 microtillers for 3–5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually. Received: 12 February 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998     

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac, parthenogenetic embryo development from the egg cell, and endosperm formation either autonomously, or due to fertilization of polar nuclei by the sperm (under pseudogamous form of apomixis). In the latter case, an obstacle to the normal endosperm development is disturbance of maternal (m) -to-paternal (p) genomic ratio 2m: 1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms. Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m: 2p that provides formation of kernels with plump endosperm. Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions. These kernels had hybrid endosperm and diploid maternaltype embryo or hybrid embryo with different ploidy level (2n, 3n, 4n). The frequencies of plump kernels on the ear ranged from 0.2-0.3% to 5.7-6.2% counting from the number of ovaries. Maternal-type plants were found in two maize lines, their frequency varying from 10.7 to 37.5% of the progeny plants. In CMS-lines of sorghum pollinated with tetraploid sorghum accessions, the frequency of plump kernels ranged from 0.6 to 14.0% counting from the number of ovaries; the frequency of maternal-type plants varied from 33.0 up to 96.1%. The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins. These data testify to variable modes of seed formation under diploid × tetraploid crosses in maize and sorghum both by amphi- and by apomixis. Therefore, usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.     

Inheritance and mapping of 2n-egg production in diploid alfalfa.

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.     

Aberrant microspore development in hybrids of maize x Tripsacum dactyloides.

Cytogenetic investigations of meiosis in hybrids between maize and Tripsacum have been well documented; however, the inherent problem of male and female sterility has not been addressed either on a genetic or cytogenetic level. The purpose of this cytological study was to identify some of the probable causes of male sterility in maize x Tripsacum dactyloides hybrids. Disturbances in pollen development of maize x T. dactyloides hybrids, derived from both diploid (2n) and tetraploid (4n) Tripsacum sources, were commonly observed. Anomalies in the development of the microspore apparently occurred because of a failure of the chromosomes to congregate at the metaphase plate, development of a tripolar spindle, and failure of cytokinesis at the first and second meiotic divisions. Phenotypic features of abnormal microspore development were the maturation of large pollen grains, "Siamese" pollen grains, the occurrence of variable invaginations, and a nuclear budding-type behavior. These abnormalities were not observed in the 56-chromosome amphidiploid or the 38-chromosome backcross generations.     

Fitness differences among diploids, tetraploids, and their triploid progeny in Chamerion angustifolium: mechanisms of inviability and implications for polyploid evolution

Abstract. Theoretical models indicate that the evolution of tetraploids in diploid populations will depend on both the relative fitness of the tetraploid and that of the diploid-tetraploid hybrids. Hybrids are believed to have lower fitness due to imbalances in either the ploidy (endosperm imbalance) or the ratio of maternal to paternal genomes in their endosperm (genomic imprinting). In this study we created diploids, tetraploids, and hybrid triploids of Chamerion angustifolium from crosses between field-collected diploid and tetraploid plants and evaluated them at six life stages in a greenhouse comparison. Diploid offspring (from 2 x × 2 x crosses) had significantly higher seed production and lower biomass than tetraploid offspring (from 4 x × 4 x crosses). Relative to the diploid, the cumulative fitness of tetraploids was 0.67. In general, triploids (from 2 x × 4 x , 4 x × 2 x crosses) had significantly lower seed production, lower pollen viability, and higher biomass than diploid individuals. Triploid offspring derived from diploid maternal parents had lower germination rates, but higher pollen production than those with tetraploid mothers. Relative to diploids, the cumulative fitness of 2 x × 4 x triploids and 4 x × 2 x triploids was 0.12 and 0.06, respectively, providing some support for effect of differing maternal:paternal ratios and endosperm development as a mechanism of hybrid inviability. Collectively, the data show that tetraploids exhibit an inherent fitness disadvantage, although the partial viability and fertility of triploids may help to reduce the barrier to tetraploid establishment in sympatric populations.     

Detection of the apomictic mode of reproduction in maize-Tripsacum hybrids using maize RFLP markers

Polyploid plants in the genus Tripsacum, a wild relative of maize, reproduce through gametophytic apomixis of the diplosporous type, an asexual mode of reproduction through seed. Moving gene(s) responsible for the apomictic trait into crop plants would open new areas in plant breeding and agriculture. Efforts to transfer apomixis from Tripsacum into maize at CIMMYT resulted in numerou intergeneric F₁ hybrids obtained from various Tripsacum species. A bulk-segregant analysis was carried out to identify molecular markers linked to diplospory in T. dactyloides. This was possible because of numerous genome similarities among related species in the Andropogoneae. On the basis of maize RFLP probes, three restriction fragments co-segregating with diplospory were identified in one maize-Tripsacum dactyloides F₁ population that segregated 1∶1 for the mode of reproduction. The markers were also found to be linked in the maize RFLP map, on the distal end of the long arm of chromosome 6. These results support a simple inheritance of diplospory in Tripsacum. Manipulation of the mode of reproduction in maize-Tripsacum backcross generations, and implications for the transfer of apomixis into maize, are discussed.     

Tripsacum-maize interaction: a novel cytogenetic system

The genera Zea and Tripsacum cross readily when they are not isolated by gametophytic barriers, and it has been postulated that intergeneric introgression played a role in the evolution of maize. The basic x = 9 Tripsacum and x = 10 Zea genomes have little cytological affinity for each other in hybrids that combine 10 Zea with 18 Tripsacum chromosomes. However, one to four Tripsacum chromosomes sometimes associate with Zea chromosomes in hybrids between Z. mays (2n = 20) and T. dactyloides (2n = 72). These hybrids with 10 Zea and 36 Tripsacum chromosomes frequently produce functional female gametes with 36 Tripsacum chromosomes only. When they are pollinated with maize, their offspring again have 36 Tripsacum and 10 maize chromosomes, but the Tripsacum genome is contaminated with maize genetic material. In these individuals, intergenome pairing is the rule, and when they are pollinated with maize, their offspring have 36 Tripsacum and 10, 12, 14, 16, 18, or 20 Zea chromosomes. Plants with 36 Tripsacum and 20 Zea chromosomes behave cytologically as alloploids, although the Tripsacum genome is contimated with maize, and one basic maize genome is contaminated with with Tripsacum genetic material. When they are pollinated with maize, offspring with 18 Tripsacum and 20 Zea chromosome are obtained. Further successive backcrosses with maize selectively eliminate Tripsacum chromosomes, and eventually plants with 2n = 20 Zea chromosomes are recovered. Many of these maize plants are highly "tripsacoid." Strong gametophytic selection for essentially pure Zea gametes, however, eliminates all obvious traces of Tripsacum morphology within a relatively few generations.     

Ploidy barrier to endosperm development in maize

Maize kernels inheriting the indeterminate gametophyte mutant (ig) on the female side had endosperms that ranged in ploidy level from diploid (2x) to nonaploid (9x). In crosses with diploid males, only kernels of the triploid endosperm class developed normally. Kernels of the tetraploid endosperm class were half-sized but with well-developed embryos that regularly germinated. Kernels of endosperm composition other than triploid or tetraploid were abortive.-Endosperm ploidy level resulting from mating ig/ig x tetraploid Ig similarly was variable. Most endosperms started to degenerate soon after pollination and remained in an arrested state. Hexaploid endosperm was exceptional; it developed normally during the sequence of stages studied and accounted for plump kernels on mature ears. Since such kernels have diploid maternal tissues (pericarp) but triploid embryos, the present finding favors the view that endosperm failure or success in such circumstances is governed by conditions within the endosperm itself.-Whereas tetraploid endosperm consisting of three maternal genomes and one paternal genome is slightly reduced in size but supports viable seed development, that endosperm having two maternal and two paternal chromosome sets was highly defective and conditioned abortion. Thus, development of maize endosperm evidently is affected by the parental source of its sets of chromosomes.     

Numerical analysis of isozyme variation in Glycine tomentella

Isozyme variation among 114 accessions of the Glycine tomentella Hayata was analysed by single linkage cluster analysis and the unweighted pair group centroid method (UPGMC). The diploid accessions fell into six distinct, well defined groups, which conformed with differences in chromosome number (2n − 38 or 2n − 40) or in geographic origin. The majority of the tetraploid accessions belonged to a large, geographically widespread group, predominantly aneuploid (2n − 78) group. The remaining four tetraploid groups were distinct on the basis of morphology or geographic distribution. The validity of tetraploid isozyme groupings for reflecting subspecific differentiation was supported by the published reports of hybrid fertility. All of the nineteen crosses between isozyme groups have yielded sterile hybrids, whereas five crosses within groups have yielded fertile hybrids. The relationship between diploids and tetraploids was examined either as the similarity between individual accessions, or that between isozyme groups. These analyses indicated that each tetraploid group is closely related to only one or two of the diploid groups or subgroups.     

Genome-wide analysis of maize cytoplasmic male sterility-S based on QTL mapping

Three types of sterile cytoplasm in cytoplasmic-male-sterility (CMS) maize, T, C and S, can be identified according to their fertility-restoration and mitochondrial DNA RFLP patterns. CMS-S, which is the least stable among the three types of CMS, is controlled by sterile cytoplasm interactions with certain nuclear-encoded factors. We constructed a high-resolution map of loci associated with male-restoration of CMS-S in BC1 populations of maize. The map covers 1730.29 cM, including 32 RFLP, 51 SSR 62 RAPD and 21 AFLP markers. Genome-wide QTL analysis detected 6 QTLs with significant effects on male fertility as assessed by their starch-filled pollen ratios. Four QTLs out of six were located between the SSR markers MSbnlg1633-Mrasg20, MSbnlg1662-Msume1126, MSume1230-MSumc1525, and RAPD marker MraopQ07-2-MraopK06-2 on chromosome 2. Two other minor loci were mapped between MraopK16-1-Mraopi4-1, on chromosome 9, and between Msuncbnlg1139-MraopR10-2, on chromosome 6. The Rf3 nuclear restoring gene was precisely located on the chromosome 2, 2.29 cM to the left of umc1525 and 8.9 cM to the right of umc1230. The results provide important markers for marker-assisted selection of stable CMS-S maize.     

Transmission genetics of chromatin from a synthetic amphidiploid to cultivated peanut (Arachis hypogaea L.). broadening the gene pool of a monophyletic polyploid species

Polyploidy creates severe genetic bottlenecks, contributing to the genetic vulnerability of leading crops. Cultivated peanut is thought to be of monophyletic origin, harboring relatively little genetic diversity. To introduce variability from diploid wild species into tetraploid cultivated Arachis hypogaea, a synthetic amphidiploid [[A. batizocoi K9484 x (A. cardenasii GKP10017 x A. diogoi GKP10602)](4x)] was used as donor parent to generate a backcross population of 78 progeny. Three hundred seventy RFLP loci were mapped onto 23 linkage groups, spanning 2210 cM. Chromatin derived from the two A-genome diploid ancestors (A. cardenasii and A. diogoi) comprised mosaic chromosomes, reflecting crossing over in the diploid A-genome interspecific F(1) hybrid. Recombination between chromosomes in the tetraploid progeny was similar to chromosome pairing reported for A. hypogaea, with recombination generally between chromosomes of the same subgenomic affinity. Segregation distortion was observed for 25% of the markers, distributed over 20 linkage groups. Unexpectedly, 68% of the markers deviating from expected segregation showed an excess of the synthetic parent allele. Genetic consequences, relationship to species origins, and significance for comparative genetics are discussed.     

The role of tetraploids in the sexual-asexual cycle in dandelions (Taraxacum)

Apomictic plants often produce pollen that can function in crosses with related sexuals. Moreover, facultative apomicts can produce some sexual offspring. In dandelions, Taraxacum, a sexual-asexual cycle between diploid sexuals and triploid apomicts, has been described, based on experimental crosses and population genetic studies. Little is known about the actual hybridization processes in nature. We therefore studied the sexual-asexual cycle in a mixed dandelion population in the Netherlands. In this population, the frequencies of sexual diploids and triploids were 0.31 and 0.68, respectively. In addition, less than 1% tetraploids were detected. Diploids were strict sexuals, triploids were obligate apomicts, but tetraploids were most often only partly apomictic, lacking certain elements of apomixis. Tetraploid seed fertility in the field was significantly lower than that of apomictic triploids. Field-pollinated sexual diploids produced on average less than 2% polyploid offspring, implying that the effect of hybridization in the 2x-3x cycle in Taraxacum will be low. Until now, 2x-3x crosses were assumed to be the main pathway of new formation of triploid apomicts in the sexual-asexual cycle in Taraxacum. However, tetraploid pollen donors produced 28 times more triploid offspring in experimental crosses with diploid sexuals than triploid pollen donors. Rare tetraploids may therefore act as an important bridge in the formation of new triploid apomicts.     

High resolution genetic mapping by genome sequencing reveals genome duplication and tetraploid genetic structure of the diploid Miscanthus sinensis

We have created a high-resolution linkage map of Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.     

Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999     

Glutamate Oxaloacetic Transaminase and Malate Dehydrogenase Isozymes of Zea mays L. × Tripsacum dactyloides L. Hybrids and Parents

Electrophoretic patterns of glutamate oxaloacetic transaminase (Got) and malate dehydrogenase (Mdh) of Zea mays L. × Tripsacum dactyloides L. hybrids and their parents have been compared. The results suggested that Got and Mdh isozymes may be used as markers for genic regions on 5 S and 6 L maize chromosomes and for linkage groups D and L on T. dactyloides chromosomes, syntenic to genic regions on 5 S and 6 L maize chromosomes. The latter have a regulatory effect on fertility and on the apomictic mode of reproduction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extraordinary tertiary constrictions of Tripsacum dactyloides chromosomes: implications for karyotype evolution of polyploids driven by segmental chromosome losses

Tripsacum dactyloides (2n = 2x = 36) is an ancient tetraploid species. Here we report that T. dactyloides chromosomes contain an extraordinary tertiary constriction, which causes a radical and distant separation of a terminal segment from the chromosome. The relationships between extraordinary tertiary constriction and segmental chromosome loss as well as karyotype evolution of polyploid species are discussed.