Progress 08/15/08 to 08/14/11
Outputs
OUTPUTS: Over the course of the entire project, the most significant advancements were made in (1) the characterization of the effects of the stunter1 mutation on male and female gametophyte development and post-fertilization seed development and (2) the fine mapping of the maternal effect lethal1 allele and identification of a second allele. Objective 1: Fine structure mapping of maternal effect lethal1 (mel1) and characterization of the expression patterns of the affected gene(s). The mapping interval for the mel1 mutation was narrowed to include 3 annotated candidate genes and 5 putative transcripts. The 3 annotated genes were sequenced from a mel1 heterozygous individual. A Ds transposable element near the mapping interval was mobilized to generate a large population of transposition events. Individuals were screened for mel1-like seed phenotypes to identify a second allele of mel1 to aid in mapping efforts. For the six other maternal effect mutants, mapping populations have been generated for future mapping studies. Additionally, efforts have been made in the field to begin generating and identifying second alleles of baseless1 (bsl1) and stunter1 (stt1). For heirless1 (hrl1), defective kernel x1 (dkx1), and no legacy1 (nol1), Mu or Ac/Ds flanking sequences were isolated and submitted for next generation SOLiD sequencing to identify flanking sequences in the mapping interval that co-segregate with the defective kernel phenotypes. Objective 2: Characterization of the genetic and phenotypic effects of the mutations on the gametophytes and their defective progeny. For the maternal effect mutants where the chromosome locations of the mutations are known (mel1, stt1, bsl1, and san scion1 (ssc1)) we have tested whether the mutations are dominant, if dosage of the gene affects the phenotype, and have linked visible seed markers (e.g. waxy1 and a2) to the mutations to follow the mutations more easily and aid in pollen analysis. Tissue has been collected for each maternal effect mutant to analyze embryo sac morphology by confocal laser scanning microscopy (CLSM). The seed and gametophyte (embryo sac and pollen) phenotypes associated with the stt1 mutant have been well characterized. Fertilization rates of stt1 embryo sacs were determined by CLSM. Trisomic stt1 stocks were generated to determine whether stt1 is a dominant or recessive maternal effect mutant. Translocation stocks for the chromosome arm on which stt1 is located were used to generate hypoploid (stt1 over a deletion) stt1 embryos and endosperm, and novel phenotypes were assessed by CLSM. Objective 3: Determination if the genes affected by the mutations are themselves imprinted or if the mutations affect imprinting of other genes and examination of the expression patterns of common embryo, endosperm, and embryo sac specific genes for deviations in spatial and temporal gene expression. Each of the maternal effect mutants were tested for global imprinting effects using the imprinted R1-r:standard (R1-r:st) allele of the r1 gene. Additionally one candidate gene in the mel1 mapping interval has been tested to determine if it is imprinted during seed development. PARTICIPANTS: Individuals: Dr. Allison Phillips (self) as the principle investigator of the project conducted the majority of the research to date. Dr. Matthew Evans served as a mentor for the project. Training Opportunities: Three undergraduates participated in the project by conduction independent research during the summer months. Anisha Patel helped with the stt1 mapping efforts, Tiffany Shih helped with the analysis of imprinting of the mel1 candidate gene, and Sejal Parekh helped with confocal analysis of stt1 embryo sacs. All three were under the guidance of Drs Phillips and Evans. Professional Development Opportunities: Through grant funding, Dr. Phillips was able to attend the annual Maize Meeting for both years of the project allowing for lots of networking and professional development opportunities. TARGET AUDIENCES: Research is conducted in the interest of the scientific community in general and developmental plant biology and maize researches in particular. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The mel1 maternal effect mutation in maize results in defective miniature seeds with abnormal or absent embryos and a reduced endosperm. The mutation does not cause gross morphological defects in the embryo sacs, but does affect pollen tube growth. Fine structure mapping has allowed us to narrow the mapping interval to a 100 Kb region on the short arm of chromosome 2 with three candidate genes. Of these candidate genes, one is of particular interest based on previous mutational analyses and expression studies in Arabidopsis and maize. There is transcript evidence that this gene is expressed in embryo sacs, pollen, and early endosperm in maize kernels. We have sequenced the complete coding region of this gene from a mel1 heterozygous individual and have not identified a lesion. To aid in mapping efforts we mobilized a Ds transposable element located near the mel1 mapping interval to generate a population of transposition events. We have identified 10 individuals, based on mel1-like seed phenotypes, that are candidates for a second allele of mel1; however, sequenced flanking regions for each did not map to the region of mel1. stt1 is a recessive, maize maternal effect mutant that displays miniature kernels. Kernels that inherit stt1 through the female gametophyte exhibit a reduced but normal endosperm with a smaller but otherwise normal embryo. The mutant displays incomplete and variable penetrance of the kernel phenotype and reduced transmission of the mutation through the male and female parents. The stt1 mutation causes significant changes in the sizes of both the male and female gametophytes. stt1 pollen grains are smaller than wild type, have reduced germination efficiency, and reduced pollen tube growth. The reduction in size of stt1 embryo sacs is largely due to a decrease in size of the central cell. The antipodal cells of mutant embryo sacs are also abnormal, being larger, more vacuolated, and fewer in number than wild type. After fertilization, the embryo and endosperm of stt1 seeds develop more slowly and are smaller than wild type. Analysis of seeds carrying a mutant maternal allele of stt1 over a deletion of the paternal allele demonstrates that both alleles are active zygotically in the endosperm and embryo and that early embryo development in maize is independent of endosperm development. The results suggest that stt1 is a novel maternal effect mutant and that the morphology of mutant embryo sacs influences endosperm development, leading to the production of miniature kernels. The nol1 maternal effect mutation in maize results in defective seeds with abnormal or absent embryos and a reduced endosperm. The nol1 mutation causes changes in the placement of the polar nuclei within the central cell of the embryo sacs, where the nuclei are either off center of the central cytoplasmic strand or adjacent to either wall of the embryo sac. This misplacement of the polar nuclei is phenotypically similar to the embryo sac defects seen in the bsl1 maternal effect mutant, which also conditions defective seeds. For both mutants, the morphological embryo sac defects likely are the direct cause for the seed defects.
Publications
- Phillips, A.R. and M.M. Evans. 2011. Analysis of stunter1, a maize mutant with reduced gametophyte size and maternal effects on seed development. Genetics 187(4):1085-1097.
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Progress 08/15/09 to 08/14/10
Outputs
OUTPUTS: Objective 1: Fine structure mapping of maternal effect lethal1 (mel1) and characterization of the expression patterns of the affected gene(s). The mapping interval for the mel1 mutation was narrowed to include 3 annotated candidate genes and 5 putative transcripts. The 3 annotated genes were sequenced from a mel1 heterozygous individual. A Ds transposable element near the mapping interval was mobilized to generate a large population of transposition events. Individuals were screened for mel1-like seed phenotypes to identify a second allele of mel1 to aid in mapping efforts. For the six other maternal effect mutants, mapping populations have been generated for future mapping studies. Additionally, efforts have been made in the field to begin generating and identifying second alleles of baseless1 (bsl1) and stunter1 (stt1). For heirless1 (hrl1), defective kernel x1 (dkx1), and no legacy1 (nol1), Mu or Ac/Ds flanking sequences were isolated and submitted for next generation SOLiD sequencing to identify flanking sequences in the mapping interval that co-segregate with the defective kernel phenotypes. Objective 2: Characterization of the genetic and phenotypic effects of the mutations on the gametophytes and their defective progeny. For the maternal effect mutants where the chromosome locations of the mutations are known (mel1, stt1, bsl1, and san scion1 (ssc1)) we have tested whether the mutations are dominant, if dosage of the gene affects the phenotype, and have linked visible seed markers (e.g. waxy1 and a2) to the mutations to follow the mutations more easily and aid in pollen analysis. Tissue has been collected for each maternal effect mutant to analyze embryo sac morphology by confocal laser scanning microscopy (CLSM). The seed and gametophyte (embryo sac and pollen) phenotypes associated with the stt1 mutant have been well characterized. Fertilization rates of stt1 embryo sacs were determined by CLSM. Trisomic stt1 stocks were generated to determine whether stt1 is a dominant or recessive maternal effect mutant. Translocation stocks for the chromosome arm on which stt1 is located were used to generate hypoploid (stt1 over a deletion) stt1 embryos and endosperm, and novel phenotypes were assessed by CLSM. Objective 3: Determination if the genes affected by the mutations are themselves imprinted or if the mutations affect imprinting of other genes and examination of the expression patterns of common embryo, endosperm, and embryo sac specific genes for deviations in spatial and temporal gene expression. Each of the maternal effect mutants were tested for global imprinting effects using the imprinted R1-r:standard (R1-r:st) allele of the r1 gene. Additionally one candidate gene in the mel1 mapping interval has been tested to determine if it is imprinted during seed development. PARTICIPANTS: Individuals: Dr. Allison Phillips (self) as the principle investigator of the project conducted the majority of the research to date. Dr. Matthew Evans served as a mentor for the project. Training Opportunities: Three undergraduates participated in the project by conduction independent research during the summer months. Anisha Patel helped with the stt1 mapping efforts, Tiffany Shih helped with the analysis of imprinting of the mel1 candidate gene, and Sejal Parekh helped with confocal analysis of stt1 embryo sacs. All three were under the guidance of Drs Phillips and Evans. TARGET AUDIENCES: Research is conducted in the interest of the scientific community in general and developmental plant biology and maize researches in particular. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The maternal effect lethal1 (mel1) maternal effect mutation in maize results in defective miniature seeds with abnormal or absent embryos and a reduced endosperm. The mutation does not cause any gross morphological defects in the embryo sacs, but does affect pollen tube growth. Fine structure mapping has allowed us to narrow the mapping interval to a 100 Kb region on the short arm of chromosome 2 with three candidate genes. Of these candidate genes, one is of particular interest based on previous mutational analyses and expression studies in Arabidopsis and maize. There is transcript evidence that this gene is expressed in embryo sacs, pollen, and early endosperm in maize kernels. We have sequenced the complete coding region of this gene from a mel1 heterozygous individual and have not identified a lesion. To aid in mapping efforts we identified a Ds transposable element located near the mel1 mapping interval and mobilized the element to generate a population of transposition events. We have identified approximately 10 individuals, based on mel1-like seed phenotypes, that are candidates for a second allele of mel1. stunter1 (stt1) is a recessive, maize maternal effect mutant that displays miniature kernels. Kernels that inherit stt1 through the female gametophyte exhibit a reduced but normal endosperm with a smaller but otherwise normal embryo. The mutant displays incomplete and variable penetrance of the kernel phenotype and reduced transmission of the mutation through the male and female parents. The stt1 mutation causes significant changes in the sizes of both the male and female gametophytes. stt1 pollen grains are smaller than wild type, have reduced germination efficiency, and reduced pollen tube growth. The reduction in size of stt1 embryo sacs is largely due to a decrease in size of the central cell. The antipodal cells of mutant embryo sacs are also abnormal, being larger, more vacuolated, and fewer in number than wild type. After fertilization, the embryo and endosperm of stt1 seeds develop more slowly and are smaller than wild type. Analysis of seeds carrying a mutant maternal allele of stt1 over a deletion of the paternal allele demonstrates that both alleles are active zygotically in the endosperm and embryo and that early embryo development in maize is independent of endosperm development. The results suggest that stt1 is a novel maternal effect mutant and that the morphology of mutant embryo sacs influences endosperm development, leading to the production of miniature kernels. The no legacy1 (nol1) maternal effect mutation in maize results in defective seeds with abnormal or absent embryos and a reduced endosperm. The nol1 mutation causes changes in the placement of the polar nuclei within the central cell of the embryo sacs, where the nuclei are either off center of the central cytoplasmic strand or adjacent to either wall of the embryo sac. This misplacement of the polar nuclei is phenotypically similar to the embryo sac defects seen in the baseless1 (bsl1) maternal effect mutant, which also conditions defective seeds. For both mutants, the morphological embryo sac defects likely are the direct cause for the seed defects.
Publications
- Phillips, A.R. and M.S.S. Evans. (2010) The maize stunter1 maternal effect mutation affects male and female gametophytes. Submitted to Development.
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Objective 1: Fine structure mapping of maternal effect lethal1 (mel1) and characterization of the expression patterns of the affected gene(s). In order to narrow the mapping interval for the mel1 mutation, a larger mapping population was generated, new insertion/deletion markers were tested, new dCAPs markers were designed from the genomic sequences, and these new markers were tested on the mapping population. In the narrowed region, candidate genes were identified based on synteny with rice and the new maize genome annotations. In addition, efforts have been made to identify homozygous mel1 plants and/or kernels for sequencing of candidate genes in order to identify the associated lesion. For the six other maternal effect mutants, mapping populations have been generated for future mapping studies. Objective 2: Characterization of the genetic and phenotypic effects of the mutations on the gametophytes and their defective progeny. Field experiments have been conducted to test the phenotypic expression of each mutant in various inbred backgrounds. Male and female transmission rates and penetrance rates of the seed phenotypes have been determined for large populations of each mutant. For those maternal effect mutants where the chromosome location of the mutations are known (mel1, stunter1 (stt1), baseless1 (bsl1), and san scion1) we have tested whether the mutations are dominant, if dosage of the gene affects the phenotype, and have linked visible seed markers (e.g. waxy1 and a2) to the mutations to follow the mutations more easily. Additionally, efforts have been made in the field to begin generating and identifying second alleles of bsl1, mel1, and stt1. The seed and gametophyte phenotypes associated with the stt1 mutant have been characterized. Confocal laser scanning microscopy was utilized to examine embryo sac morphology throughout development and gross embryo and endosperm morphology at several time points post fertilization. The morphology of the endosperm of developing seeds was analyzed in more detail using endosperm stains and B-glucuronidase (GUS) fusion endosperm markers to differentiate three of the four regions of the mature endosperm (aleurone, basal endosperm transfer layer, and the central starchy endosperm). The waxy1 locus was placed in repulsion to stt1 using the appropriate waxy-marked reciprocal translocation stock and stt1 pollen was then examined by pollen staining and germination assays. Similar analyses have been initiated for the other maternal effect mutants. Objective 3: Determination if the genes affected by the mutations are themselves imprinted or if the mutations affect imprinting of other genes and examination of the expression patterns of common embryo, endosperm, and embryo sac specific genes for deviations in spatial and temporal gene expression. Thus far we have tested stt1 and heirless1 for global imprinting effects using the imprinted R1-r:standard (R1-r:st) allele of the r1 gene and have begun generating the stocks to test the remaining mutants. Additionally one candidate gene in the mel1 mapping interval has been tested to determine if it is imprinted during seed development. PARTICIPANTS: Individuals: Dr. Allison Phillips (self) as the principle investigator of the project conducted the majority of the research to date. Dr. Matthew Evans served as a mentor for the project. Training Opportunities: Two undergraduates participated in the project by conducting independent research during the summer months. Anisha Patel helped with the stunter1 mapping efforts and Tiffany Shih helped with the analysis of imprinting of the mel1 candidate gene. Both were under the guidance of Drs Phillips and Evans. TARGET AUDIENCES: Target audience: Research is conducted in the interest of the scientific community in general and developmental plant biology and maize researchers in particular. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The maternal effect lethal1 (mel1) maternal effect mutation in maize results in defective miniature seeds with abnormal or absent embryos and a reduced endosperm. The mutation does not cause any gross morphological defects in the embryo sacs, but does affect pollen tube growth. Fine structure mapping has allowed us to narrow the mapping interval to a 120 Kb region on the short arm of chromosome 2 with approximately seven candidate genes. Of these candidate genes, one is of particular interest based on previous mutational analyses and expression studies in Arabidopsis and maize. There is EST evidence that this gene is expressed in embryo sacs and early endosperm in maize kernels. Additionally, a previous study has shown that when one of the four Arabidopsis orthologs is disrupted, embryo and endosperm development arrests at the 1-cell zygotic stage when the mutation is inherited from the maternal parent. Recently we determined that this candidate gene is imprinted during early endosperm development in maize. We are working to identify whether or not there is a lesion in this gene in mel1 by sequencing fragments of this gene. To facilitate this we are trying to isolate homozygous mel1 tissue or identify polymorphisms between the wild-type alleles in two different inbreds to distinguish the mutated allele from the wild-type allele in a heterozygote. stunter1 (stt1) is a maternal effect mutant that displays defective maize kernels. The small, stunted kernels exhibit a reduced but normal endosperm with a relatively normal embryo. In an attempt to identify the genetic lesion and better characterize the stt1 mutant, we have used map-based cloning and conducted phenotypic analyses of the male and female gametophytes and the developing seed. The lesion is located in a 1.5 cM region on the long arm of Chromosome 2. The mutant displays incomplete and variable penetrance of the kernel phenotype and reduced transmission of the mutation through the male and female parents. In addition, the stt1 mutation causes significant changes in the sizes of both the male and female gametophytes. The stt1 pollen grains are smaller than wild type, and although the pollen nuclei appear normal, the stt1 pollen grains do not germinate as well as wild-type grains. The reduction in size of stt1 embryo sacs is largely due to differences in the central cell, which is approximately half the size in the mutant embryo sacs compared to wild type. Additionally, the antipodal cells of mutant embryo sacs appear larger, less cytoplasmically dense, and fewer in number than wild type. After fertilization, the embryo and endosperm of stt1 seeds develop more slowly than in wild-type seeds. The results strongly suggest that stt1 is a maternal effect mutant and that the morphology of the mutant embryo sacs prior to fertilization influences endosperm development, ultimately leading to the production of miniature kernels from mutant embryo sacs.
Publications
- No publications reported this period
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