Single-Pollen Genotyping

Isagi, Yuji; Suyama, Yoshihisa

doi:10.1007/978-4-431-53901-8

Cited by 8 publications

(3 citation statements)

References 149 publications

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“…Twenty pollen grains were isolated per genotype. After collection, individual pollen grains were transferred to a DNA-free PCR tube (0.2 ml capacity) containing 2 μl of extraction buffer (Isagi and Suyama, 2011): 0.01% sodium dodecyl sulfate, SDS, 0.1 g/l proteinase K, 0.01 M Tris-HCl pH 7.8 and 0.01 M EDTA. Following this step, the presence of a single pollen grain in the drop of buffer was checked under the stereo microscope.…”

Section: Methodsmentioning

confidence: 99%

Polyploidy in Fruit Tree Crops of the Genus Annona (Annonaceae)

et al. 2019

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Genome duplication or polyploidy is one of the main factors of speciation in plants. It is especially frequent in hybrids and very valuable in many crops. The genus Annona belongs to the Annonaceae, a family that includes several fruit tree crops, such as cherimoya (Annona cherimola), sugar apple (Annona squamosa), their hybrid atemoya (A. cherimola × A. squamosa) or pawpaw (Asimina triloba). In this work, genome content was evaluated in several Annona species, A. triloba and atemoya. Surprisingly, while the hybrid atemoya has been reported as diploid, flow cytometry analysis of a progeny obtained from an interspecific cross between A. cherimola and A. squamosa showed an unusual ploidy variability that was also confirmed karyotype analysis. While the progeny from intraspecific crosses of A. cherimola showed polyploid genotypes that ranged from 2.5 to 33%, the hybrid atemoyas from the interspecific cross showed 35% of triploids from a total of 186 genotypes analyzed. With the aim of understanding the possible implications of the production of non-reduced gametes, pollen performance, pollen size and frequency distribution of pollen grains was quantified in the progeny of this cross and the parents. A large polymorphism in pollen grain size was found within the interspecific progeny with higher production of unreduced pollen in triploids (38%) than in diploids (29%). Moreover, using PCR amplification of selected microsatellite loci, while 13.7% of the pollen grains from the diploids showed two alleles, 41.28% of the grains from the triploids amplified two alleles and 5.63% showed up to three alleles. This suggests that the larger pollen grains could correspond to diploid and, in a lower frequency, to triploid pollen. Pollen performance was also affected with lower pollen germination in the hybrid triploids than in both diploid parents. The results confirm a higher percentage of polyploids in the interspecific cross, affecting pollen grain size and pollen performance. The occurrence of unreduced gametes in A. cherimola, A. squamosa and their interspecific progeny that may result in abnormalities of ploidy such as the triploids and tetraploids observed in this study, opens an interesting opportunity to study polyploidy in Annonaceae.

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Section: Methodsmentioning

confidence: 99%

Polyploidy in Fruit Tree Crops of the Genus Annona (Annonaceae)

et al. 2019

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“…Angiosperm pollen contains both vegetative and generative sperm nuclei, which can be structurally and morphologically different (Van Tuyl et al, 1989; Bino et al, 1990; Dewitte et al, 2009). The genotyping of individualized pollen grain nuclei opens up new opportunities in different areas of research such as the ecology of pollination, genetic, and genomic studies (Isagi and Suyama, 2010). In addition, genotyping of individual pollen grains can be useful for the determination of the haplotypes of the male parent and meiotic recombination patterns (Mase et al, 2014; Dreissig et al, 2017) and also allows performing studies on the genetic structures of pollen grain populations as compared with those originated at the plant level, without interferences due to a potential cross-incompatibility (Gu et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification

et al. 2019

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Flow cytometry is widely used to determine genome size and ploidy level in plants. This technique, when coupled with fluorescence-activated cell sorting (FACS), whole genome amplification and genotyping (WGA), opens up new opportunities for genetic studies of individualized nuclei. This strategy was used to analyze the genetic composition of single pollen nuclei of different citrus species. The flow cytometry and microscope observations allowed us to differentiate the populations of pollen nuclei present in the diploid and euploid genotypes analyzed, showing that citrus has binuclear pollen. We have identified in the “CSO” tangor an additional nuclei population composed by the vegetative plus generative nuclei. Genotyping of this nuclei population revealed that vegetative and generative nuclei show the same genetic configuration. In addition, we have demonstrated the presence of unreduced gametes in the diploid genotype “Mexican lime.” Genomic amplification is a robust method for haploid nuclei genotyping with several molecular markers, whereas in diploid nuclei using heterozygous markers showed a bias towards one of the two alleles, limiting the use of this tool in this type of nuclei. We further discuss the importance and applications of single pollen genotyping in citrus genetic studies.

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“…Total DNA extraction from a small sample of Metacalanus sp. was performed using the whole body according to the method described by Suyama (2011). DNA was quantified using a NanoDrop 2000 instrument (Thermo-Fisher, Waltham, MA, USA) and then adjusted to 1 ng μL -1 with sterilized water for PCR amplification.…”

Section: Dna Extraction Pcr and Sequencingmentioning

confidence: 99%

A new species of Pilarella (Copepoda, Calanoida, Arietellidae) from the hyperbenthic layer of Japan, with a molecular phylogenetic analysis of some representative genera of the Arietellidae

Komeda¹,

Adachi²,

Ohtsuka³

2021

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A new species of the continental shelf hyperbenthic genus Pilarella is described, the first from the Indo-Pacific. This is the second species of Pilarella known, and the first description of a male in the genus. The new species is easily distinguished from other species of Pilarella (P. longicornis) based on: (1) short caudal rami, approximately 1.5 times longer than wide; (2) 2 setae on the mandibular endopod; (3) 6 setae on the maxillular coxal epipodite; and (4) in the female, a short left antennule reaching the posterior border of the genital double-somite. The new diagnosis of Pilarella differs from Metacalanus in the separation of ancestral segments IX–XII and XIV–XV of the antennule, and the presence of 5–6 setae on the maxillular praecoxal arthrite. Pilarella is also separated from Metacalanalis based on the absence of a seta on the third ancestral segment of the antennary exopod, the symmetry of legs 1–3, the presence of a medial basal seta on the female leg 5, and 2 lateral exopodal spines on the female leg 5. A molecular phylogenetic analysis of some representative genera of the family Arietellidae, including the present new species, recovers two arietellid clades (Metacalanus- and Arietellus-clades) as in previous studies. Dichotomous keys for the genera of Arietellidae and the species of Pilarella are included.

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Single-Pollen Genotyping

Cited by 8 publications

References 149 publications

Polyploidy in Fruit Tree Crops of the Genus Annona (Annonaceae)

Polyploidy in Fruit Tree Crops of the Genus Annona (Annonaceae)

Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification

A new species of Pilarella (Copepoda, Calanoida, Arietellidae) from the hyperbenthic layer of Japan, with a molecular phylogenetic analysis of some representative genera of the Arietellidae

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