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Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize.

Kelliher T, Starr D, Wang W, McCuiston J, Zhong H, Nuccio ML, Martin B - Front Plant Sci (2016)

Bottom Line: CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility.CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids.In contrast, many of the complemented CENH3 (-∕-) lines produced haploids at low frequencies.

View Article: PubMed Central - PubMed

Affiliation: Biology Research, Syngenta Crop Protection, Research Triangle Park Durham, NC, USA.

ABSTRACT
Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3 (-∕-) and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3 (-∕-) lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

No MeSH data available.


Related in: MedlinePlus

AcGREEN-tailswap-CENH3 localizes to centromeres. These images are representative of those found in all events checked from all four constructs. (A) CENH3 localization has a punctate pattern in the scutellar nuclei of a transgene-hemizygous individual from event *32A. Arrow indicates a nucleus containing several CENH3+ foci corresponding to centromeres. (B) Close up of (A). Arrow indicates one CENH3+ centromere. (C) CENH3 localizes to nuclei in the epidermal and endothecial cells of the pre-meiotic (~1.0 mm long) anther from a transgene-hemizygous individual from event *32A. Arrow indicates one epidermal cell nucleus with several discrete CENH3+ centromeres. (D) CENH3 was not observed in a transgene- individual from event *32A. Arrow indicates one scutellar nucleus with no CENH3+ centromeres visible. PI, propidium iodide; ep, epidermal cells; en, endothecial cells.
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Figure 2: AcGREEN-tailswap-CENH3 localizes to centromeres. These images are representative of those found in all events checked from all four constructs. (A) CENH3 localization has a punctate pattern in the scutellar nuclei of a transgene-hemizygous individual from event *32A. Arrow indicates a nucleus containing several CENH3+ foci corresponding to centromeres. (B) Close up of (A). Arrow indicates one CENH3+ centromere. (C) CENH3 localizes to nuclei in the epidermal and endothecial cells of the pre-meiotic (~1.0 mm long) anther from a transgene-hemizygous individual from event *32A. Arrow indicates one epidermal cell nucleus with several discrete CENH3+ centromeres. (D) CENH3 was not observed in a transgene- individual from event *32A. Arrow indicates one scutellar nucleus with no CENH3+ centromeres visible. PI, propidium iodide; ep, epidermal cells; en, endothecial cells.

Mentions: To establish that the fusion proteins localized to centromeres, one transgene-negative and two transgene-positive T1 individuals were imaged from at least two events from each of the four constructs. In every transgene-positive sample checked, multiple cells in the scutellum (Figures 2A,B), and meiotic staged anther (Figure 2C) had several intra-nuclear AcGREEN+ spots, consistent with a centromeric localization. This pattern was not found in any of the negative control samples checked (Figure 2D), but it was found in transgene-hemizygous and -homozygous individuals, regardless of the presence or absence of the native CENH3 wild-type allele. Only ~40% of nuclei in a given tissue sample had this pattern, while the remaining nuclei did not have any fluorescent signal in the AcGREEN channel. This is consistent with the idea that CENH3 loading on centromeres is cell-cycle dependent. In most nuclei that had the signal, between 8 and 10 dots were clearly distinguished, but some nuclei had up to 20 dots, most likely due to endo-reduplication or because those cells were in the G2 phase of the cell cycle.


Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize.

Kelliher T, Starr D, Wang W, McCuiston J, Zhong H, Nuccio ML, Martin B - Front Plant Sci (2016)

AcGREEN-tailswap-CENH3 localizes to centromeres. These images are representative of those found in all events checked from all four constructs. (A) CENH3 localization has a punctate pattern in the scutellar nuclei of a transgene-hemizygous individual from event *32A. Arrow indicates a nucleus containing several CENH3+ foci corresponding to centromeres. (B) Close up of (A). Arrow indicates one CENH3+ centromere. (C) CENH3 localizes to nuclei in the epidermal and endothecial cells of the pre-meiotic (~1.0 mm long) anther from a transgene-hemizygous individual from event *32A. Arrow indicates one epidermal cell nucleus with several discrete CENH3+ centromeres. (D) CENH3 was not observed in a transgene- individual from event *32A. Arrow indicates one scutellar nucleus with no CENH3+ centromeres visible. PI, propidium iodide; ep, epidermal cells; en, endothecial cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814585&req=5

Figure 2: AcGREEN-tailswap-CENH3 localizes to centromeres. These images are representative of those found in all events checked from all four constructs. (A) CENH3 localization has a punctate pattern in the scutellar nuclei of a transgene-hemizygous individual from event *32A. Arrow indicates a nucleus containing several CENH3+ foci corresponding to centromeres. (B) Close up of (A). Arrow indicates one CENH3+ centromere. (C) CENH3 localizes to nuclei in the epidermal and endothecial cells of the pre-meiotic (~1.0 mm long) anther from a transgene-hemizygous individual from event *32A. Arrow indicates one epidermal cell nucleus with several discrete CENH3+ centromeres. (D) CENH3 was not observed in a transgene- individual from event *32A. Arrow indicates one scutellar nucleus with no CENH3+ centromeres visible. PI, propidium iodide; ep, epidermal cells; en, endothecial cells.
Mentions: To establish that the fusion proteins localized to centromeres, one transgene-negative and two transgene-positive T1 individuals were imaged from at least two events from each of the four constructs. In every transgene-positive sample checked, multiple cells in the scutellum (Figures 2A,B), and meiotic staged anther (Figure 2C) had several intra-nuclear AcGREEN+ spots, consistent with a centromeric localization. This pattern was not found in any of the negative control samples checked (Figure 2D), but it was found in transgene-hemizygous and -homozygous individuals, regardless of the presence or absence of the native CENH3 wild-type allele. Only ~40% of nuclei in a given tissue sample had this pattern, while the remaining nuclei did not have any fluorescent signal in the AcGREEN channel. This is consistent with the idea that CENH3 loading on centromeres is cell-cycle dependent. In most nuclei that had the signal, between 8 and 10 dots were clearly distinguished, but some nuclei had up to 20 dots, most likely due to endo-reduplication or because those cells were in the G2 phase of the cell cycle.

Bottom Line: CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility.CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids.In contrast, many of the complemented CENH3 (-∕-) lines produced haploids at low frequencies.

View Article: PubMed Central - PubMed

Affiliation: Biology Research, Syngenta Crop Protection, Research Triangle Park Durham, NC, USA.

ABSTRACT
Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3 (-∕-) and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3 (-∕-) lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

No MeSH data available.


Related in: MedlinePlus