Frequency of Polyploids of Solanum tuberosum Dihaploids in 2X × 2X Crosses

When breeding diploid potatoes, tetraploid progeny can result from the union of 2n eggs and 2n pollen in 2x-2x crosses. Thirty-three crosses were made to examine tetraploid progeny frequency in 2x-2x crosses. All crosses were between S. tuberosum dihaploids and diploid self-compatible donors, M6 and DRH S6-10-4P17. Using chloroplast counting for ploidy determination, the frequency of tetraploid progeny was as high as 45% in one of the 33 crosses. Based upon single nucleotide polymorphism (SNP) genotyping, the tetraploid progeny were attributed to bilateral sexual polyploidization (BSP), which is caused by the union of 2n egg and 2n pollen. Dihaploids were identified that produce lower frequencies of 2n eggs. The results of this study suggest that S. tuberosum dihaploids with a high frequency of 2n eggs should be avoided in 2x - 2x crosses for diploid breeding programs.

Assessment of the genetic composition of triploid hybrid Populus using SSR markers with low recombination frequencies

The induction of nonreduced gametes, whether from first-division restitution (FDR), second-division restitution (SDR), or both, is an important approach for polyploidization. However, an accurate method for determining the genetic constitution of polyploid hybrids is not available. In this study, based on both flow cytometric analysis and somatic chromosome counting, 164 triploid hybrids of the female parent Populus pseudo-simonii × P. nigra ‘Zheyin3#’ (2n = 2x = 38, abbreviated ZY3) and male parent P. × beijingensis (2n = 2x = 38, abbreviated BJY) were produced by high-temperature treatment during ZY3 megasporogenesis. Using six simple sequence repeat (SSR) markers with low recombination frequencies to reduce the impact of recombination, we analyzed the allelic configurations of the obtained triploid hybrids. Although the allelic configurations were not always consistent at all six loci, by combining the allelic configurations at the six loci, we inferred that 40 triploids originated from FDR 2n eggs of ZY3, whereas the others originated from SDR 2n eggs. In conclusion, our study provides a novel and effective tool for analyzing 2n gametes and performing early selection to improve triploid poplar breeding programs.

Induction of Diploid Eggs With Colchicine During Embryo Sac Development in Populus

Diploid (2n) eggs were induced by treating developing embryo sacs of Populus with colchicine solution, in order to produce triploid plants. The optimal pollinated time of female catkins was confirmed as timing point for each treatment. When female catkins of P. pseudo-simonii x P. nigra ‘Zheyin3#’ had become 5.62 ± 0.13 cm long 84 h after they emerged from their bract scales and all stigmas were exposed, pistils all over the entire catkin had optimal stigma receptivity. Observation of paraffin sections showed that embryo sac development of ‘Zheyin3#’, which initiated 12 h before pollination and finished 132 h after pollination, was a successive and asynchronous process. Generative cell division of pollen of the male parent P. x beijingensis took place 3-16 h after pollination. Catkins of 18-96 h after pollination of ‘Zheyin3#’ were treated with colchicine solution. In the progeny, twenty three triploids were detected by chromosome counting and the highest rate of triploids was 66.7% in one treatment. The rate of triploid yield was positively correlated with the frequency of four-nucleate embryo sacs (r = 0.6721, p = 0.0981) and was not significantly correlated with the percentages of uni-, twoand eight-nucleate embryo sac (r = -0.1667, p = 0.7210, r = -0.3069, p = 0.5031 and r = 0.0189, p = 0.9679, respectively), suggesting that the third mitotic division of embryo sac may be the effective stage to induce 2n eggs. Through this approach, completely homozygous 2n eggs can be produced. Its significance for plant breeding is discussed.

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.Key words: Medicago spp., meiotic mutants, molecular markers.