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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

CENH3-based haploid induction in maize. (A) Photograph of the ear crossed by pollen from individual * A004A:3, which exhibited a 3.6% haploid induction rate (3 haploids found out of 84 embryos). Such elevated levels of induction were always found coincident with low levels of embryo abortion (the white kernels in the photo) scattered around the ear. (B) Progeny from this ear were germinated on media, sampled, and identified by PCR as either haploid or diploid. Based on the PCR calls, a few putative haploid and diploid plants were then transplanted to soil and grown until the diploid plants were shedding pollen. Putative haploids (such as plant * A004A:3-104, right) were male and female sterile. They were also shorter and had thinner leaves than the putative diploids (such as plant * A004A:3-103, left). (C,D) Adult leaf samples were tested to confirm ploidy status. The diploid 2N peak was set at 200 units (X-axis) by adjusting the gain while running cells extracted from a known diploid plant through the flow cube. (C) The sample from the putative diploid plant *A004A:3-103 had a 2N peak at 200 units and a smaller 4N peak at 400 units. (D) The putative haploid plant *A004A:3-104 had a 2N peak at 100 units and a smaller 4N peak at 200 units. The image in (D) is representative of the histograms found for all putative haploids.
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Figure 4: CENH3-based haploid induction in maize. (A) Photograph of the ear crossed by pollen from individual * A004A:3, which exhibited a 3.6% haploid induction rate (3 haploids found out of 84 embryos). Such elevated levels of induction were always found coincident with low levels of embryo abortion (the white kernels in the photo) scattered around the ear. (B) Progeny from this ear were germinated on media, sampled, and identified by PCR as either haploid or diploid. Based on the PCR calls, a few putative haploid and diploid plants were then transplanted to soil and grown until the diploid plants were shedding pollen. Putative haploids (such as plant * A004A:3-104, right) were male and female sterile. They were also shorter and had thinner leaves than the putative diploids (such as plant * A004A:3-103, left). (C,D) Adult leaf samples were tested to confirm ploidy status. The diploid 2N peak was set at 200 units (X-axis) by adjusting the gain while running cells extracted from a known diploid plant through the flow cube. (C) The sample from the putative diploid plant *A004A:3-103 had a 2N peak at 200 units and a smaller 4N peak at 400 units. (D) The putative haploid plant *A004A:3-104 had a 2N peak at 100 units and a smaller 4N peak at 200 units. The image in (D) is representative of the histograms found for all putative haploids.

Mentions: Slightly elevated induction rates were found when the AcGREEN-tailswap-CENH3 line was homozygous on either side of the induction cross (3/2330 as a female and 3/2342 as a male) (Table 4). Similarly, when this transgene was hemizygous and used as the female in the induction cross, a total of 8 out of 3991 embryos were identified as haploids, for an HIR of 0.20%, which is about three-fold above the background. When the AcGREEN-tailswap-CENH3 transgene was used as the male in the induction cross, 28 confirmed haploids were recovered out of 3252 embryos, for an average HIR of 0.86% (Table 4). This represents a statistically significant 13.2-fold increase over the control. The highest HIR on an ear was 3.6%, which was found four times in this set. One of the ears, a cross by pollen from individual *A004A:3, was photographed and showed ~11% embryo abortion (43/390 kernels) (Figure 4A). Some progeny from this ear were germinated and an example of a haploid and diploid were photographed at maturity (Figure 4B). Their ploidy status was confirmed via Flow Cytometry (Figures 4C,D).


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)

CENH3-based haploid induction in maize. (A) Photograph of the ear crossed by pollen from individual * A004A:3, which exhibited a 3.6% haploid induction rate (3 haploids found out of 84 embryos). Such elevated levels of induction were always found coincident with low levels of embryo abortion (the white kernels in the photo) scattered around the ear. (B) Progeny from this ear were germinated on media, sampled, and identified by PCR as either haploid or diploid. Based on the PCR calls, a few putative haploid and diploid plants were then transplanted to soil and grown until the diploid plants were shedding pollen. Putative haploids (such as plant * A004A:3-104, right) were male and female sterile. They were also shorter and had thinner leaves than the putative diploids (such as plant * A004A:3-103, left). (C,D) Adult leaf samples were tested to confirm ploidy status. The diploid 2N peak was set at 200 units (X-axis) by adjusting the gain while running cells extracted from a known diploid plant through the flow cube. (C) The sample from the putative diploid plant *A004A:3-103 had a 2N peak at 200 units and a smaller 4N peak at 400 units. (D) The putative haploid plant *A004A:3-104 had a 2N peak at 100 units and a smaller 4N peak at 200 units. The image in (D) is representative of the histograms found for all putative haploids.
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Related In: Results  -  Collection

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

Figure 4: CENH3-based haploid induction in maize. (A) Photograph of the ear crossed by pollen from individual * A004A:3, which exhibited a 3.6% haploid induction rate (3 haploids found out of 84 embryos). Such elevated levels of induction were always found coincident with low levels of embryo abortion (the white kernels in the photo) scattered around the ear. (B) Progeny from this ear were germinated on media, sampled, and identified by PCR as either haploid or diploid. Based on the PCR calls, a few putative haploid and diploid plants were then transplanted to soil and grown until the diploid plants were shedding pollen. Putative haploids (such as plant * A004A:3-104, right) were male and female sterile. They were also shorter and had thinner leaves than the putative diploids (such as plant * A004A:3-103, left). (C,D) Adult leaf samples were tested to confirm ploidy status. The diploid 2N peak was set at 200 units (X-axis) by adjusting the gain while running cells extracted from a known diploid plant through the flow cube. (C) The sample from the putative diploid plant *A004A:3-103 had a 2N peak at 200 units and a smaller 4N peak at 400 units. (D) The putative haploid plant *A004A:3-104 had a 2N peak at 100 units and a smaller 4N peak at 200 units. The image in (D) is representative of the histograms found for all putative haploids.
Mentions: Slightly elevated induction rates were found when the AcGREEN-tailswap-CENH3 line was homozygous on either side of the induction cross (3/2330 as a female and 3/2342 as a male) (Table 4). Similarly, when this transgene was hemizygous and used as the female in the induction cross, a total of 8 out of 3991 embryos were identified as haploids, for an HIR of 0.20%, which is about three-fold above the background. When the AcGREEN-tailswap-CENH3 transgene was used as the male in the induction cross, 28 confirmed haploids were recovered out of 3252 embryos, for an average HIR of 0.86% (Table 4). This represents a statistically significant 13.2-fold increase over the control. The highest HIR on an ear was 3.6%, which was found four times in this set. One of the ears, a cross by pollen from individual *A004A:3, was photographed and showed ~11% embryo abortion (43/390 kernels) (Figure 4A). Some progeny from this ear were germinated and an example of a haploid and diploid were photographed at maturity (Figure 4B). Their ploidy status was confirmed via Flow Cytometry (Figures 4C,D).

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