Ga1 Locus Research Articles

Pectin methylesterase activities in reproductive tissues of maize plants with different haplotypes of the Ga1 and Ga2 cross incompatibility systems

Key messageTotal PME activity in reproductive tissues was related to haplotypes at maize cross incompatibility loci, suggesting that these loci function by controlling PME activity.In maize, the pollination outcome depends on the haplotypes of the interacting male gametophyte (germinated pollen) and female sporophyte (silk) at several cross-incompatibility loci. Functional alleles (-S haplotypes) of the cross-incompatibility loci Ga1 and Ga2, both encode two pectin methylesterases (PMEs), one that is expressed in silk and the other in pollen. We examined total PME activity in reproductive tissues containing functional and null haplotypes at the Ga1 or Ga2 loci. In pollinated silks, there was a correlation between total PME activity and the -S haplotype pollen in both Ga1 and Ga2 systems. We did not detect a significant relationship between PME activity and pollination outcome of either system. We re-examined previously reported active site amino acid substitutions in PMEs encoded by cross incompatibility loci. We observed that different active site substitutions are present in the pollen and silk PMEs of cross incompatibility loci and these differences are conserved across Ga1, Ga2 and Tcb-1. This work establishes a relationship between total PME activity and the haplotypes of the Ga1 locus in pollinated silks.

The Ga1 locus of the genus Zea is associated with novel genome structures derived from multiple, independent non-homologous recombination events.

The Ga1 locus controls cross incompatibility between field corn and popcorn. The Ga1-S haplotype contains two types of pectin methylesterase (PME) genes, ZmPme3 and several copies of ZmGa1P, that are expressed in silk and pollen, respectively. The ga1 haplotype contains non-functional tandem repeat sequences related to ZmPme3 and ZmGa1P. This haplotype can cross pollinate freely and is widely present in field corn. The primary objective of this study is to characterize the repeat sequences from a diverse collection of maize and teosinte lines and use this information to understand the evolution of the Ga1 locus. First, we characterized the complexity of the Ga1 genome region in high quality maize genome assemblies which led to their categorization into five groups based on the number and type of PME-like sequences found at this region. Second, we studied duplication events that led to the ga1 and Ga1-S repeats using maximum-likelihood phylogenetic reconstruction. Divergence estimates of the ga1 haplotype suggest that the duplication events occurred more than 600 kya whereas those in Ga1-S occurred at three time points i.e., > 600 kya, ∼ 260 kya and ∼ 100 kya. These estimates suggest that the ga1 and Ga1-S tandem duplication events occurred independently. Finally, analysis of ZmPme3 and ZmGa1P homologs in Zea and Tripsacum genomes suggests that ga1 and Ga1-S repeats originated from an ancestral pair of PME genes that duplicated and diverged through two evolutionary branches prior to the domestication of maize.

A single silk- and multiple pollen-expressed PMEs at the Ga1 locus modulate maize unilateral cross-incompatibility.

The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross-incompatibility (UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene (PME) (ZmGa1F) and eight pollen-expressed PMEs (ZmGa1P and ZmGa1PL1-7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

A pollen expressed PME gene at Tcb1 locus confers maize unilateral cross-incompatibility.

A pollen expressed PME gene at Tcb1 locus confers maize unilateral cross-incompatibility.

Three types of genes underlying the Gametophyte factor1 locus cause unilateral cross incompatibility in maize

Unilateral cross incompatibility (UCI) occurs between popcorn and dent corn, and represents a critical step towards speciation. It has been reported that ZmGa1P, encoding a pectin methylesterase (PME), is a male determinant of the Ga1 locus. However, the female determinant and the genetic relationship between male and female determinants at this locus are unclear. Here, we report three different types, a total of seven linked genes underlying the Ga1 locus, which control UCI phenotype by independently affecting pollen tube growth in both antagonistic and synergistic manners. These include five pollen-expressed PME genes (ZmGa1Ps-m), a silk-expressed PME gene (ZmPME3), and another silk-expressed gene (ZmPRP3), encoding a pathogenesis-related (PR) proteins. ZmGa1Ps-m confer pollen compatibility. Presence of ZmPME3 causes silk to reject incompatible pollen. ZmPRP3 promotes incompatibility pollen tube growth and thereby breaks the blocking effect of ZmPME3. In addition, evolutionary genomics analyses suggest that the divergence of the Ga1 locus existed before maize domestication and continued during breeding improvement. The knowledge gained here deepen our understanding of the complex regulation of cross incompatibility.

Revisiting pollen-pistil interaction and cross incompatibility in maize

The review addressed aspects of plant fertilisation and the phenomenon of genetic cross-incompatibility in maize controlled by the Gametophyte1 locus. This phenomenon determines the failure to accomplish successful fertilisation and a full seed set when pollen grains carrying the ga1 allele pollinate female inflorescences carrying the Ga1-strong (Ga-1s) allele in the homozygous state (Ga1-s/Ga1-s). We divided the review work into several topics — first, the introduction of sexual plant reproduction. Second, pollen-pistil interactions in plants. Third, reproductive barriers during plant reproduction. Third, Incompatibility in plants. Fourth, fine mapping of the Ga1 locus in maize. Fifth, recent researches on Ga1-related cross-incompatibility in maize.

A PECTIN METHYLESTERASE gene at the maize Ga1 locus confers male function in unilateral cross-incompatibility

Unilateral cross-incompatibility (UCI) is a unidirectional inter/intra-population reproductive barrier when both parents are self-compatible. Maize Gametophyte factor1 (Ga1) is an intraspecific UCI system and has been utilized in breeding. However, the mechanism underlying maize UCI specificity has remained mysterious for decades. Here, we report the cloning of ZmGa1P, a pollen-expressed PECTIN METHYLESTERASE (PME) gene at the Ga1 locus that can confer the male function in the maize UCI system. Homozygous transgenic plants expressing ZmGa1P in a ga1 background can fertilize Ga1-S plants and can be fertilized by pollen of ga1 plants. ZmGa1P protein is predominantly localized to the apex of growing pollen tubes and may interact with another pollen-specific PME protein, ZmPME10-1, to maintain the state of pectin methylesterification required for pollen tube growth in Ga1-S silks. Our study discloses a PME-mediated UCI mechanism and provides a tool to manipulate hybrid breeding.

A Pectin Methylesterase ZmPme3 Is Expressed in Gametophyte factor1-s (Ga1-s) Silks and Maps to that Locus in Maize (Zea mays L.).

The ga1 locus of maize confers unilateral cross incompatibility, preventing cross pollination between females carrying the incompatible Ga1-s allele and males not carrying a corresponding compatible allele. To characterize this system at the molecular level, we carried out a transcript profiling experiment in which silks from near isogenic lines carrying the Ga1-s and ga1 alleles were compared. While several differentially expressed genes were identified, only one mapped to the known location of ga1. This gene is a pectin methylesterase (PME), which we designated as ZmPme3, and is present and expressed only in Ga1-s genotypes. While a functional ZmPME3 is not present in the ga1 genotypes examined, a pectin methylesterase gene cluster is found in ga1 genotypes. The gene cluster in W22 contains 58 tandem full-length or partial PME pseudo genes. These data combined with a wealth of previously published data on the involvement of PMEs in pollen tube growth suggest a role for cell wall modification enzymes in the pollen exclusion component of Ga1-s gametophytic incompatibility. Consistent with this role, a third allele which lacks the female function of Ga1-s, Ga1-m, has a mutationally inactivated version of ZmPme3.

Identification of resistance to the Ga1-m gametophyte factor in maize

Due to maize’s wind-driven pollination, non-target pollen contamination is problematic for producers and breeders. Maize gametophyte factors, specifically gametophyte factor 1 (ga1), have long been used to produce selectively pollinating phenotypes. The use of these factors is a cornerstone of commercial popcorn production, and they are used for a large range of other purposes, including preventing contamination by genetically modified pollen in organic production. However this system is at great risk from another allele at the ga1 locus, Ga1-m, which overcomes the selectively pollinating phenotypes. To further complicate this problem, the risk posed by this allele has been under-assessed. Here we reinterpret the key study on Ga1-s and report genetic resistance to the Ga1-m allele in maize lines that carry dominant gametophyte factors. We identified genetic resistance to the allele segregating in lines derived from four landraces, showed the resistance is heritable, and that it acts in full-strength and attenuated versions. Additionally, we have suggested the validity of evolutionary-based inquiry into our plant genetic resources, and provided some validation of this effort. Our results provide the first report of effective genetic resistance to pollination by the Ga1-m allele, providing an option to continue the use of genetic barriers to non-target pollination. A source of resistance to the Ga1-m allele allows research to be conducted about the allele itself, allowing for research into the possible existence of multiple versions of the allele and their distributions. We anticipate our research will be a starting point for identification of additional sources of resistance to the Ga1-m allele, specifically in popcorn production, where it is most immediately needed to prevent pollen contamination, as well as the eventual localization and mapping of the resistance alleles. We also believe the suggestion of evolutionary-based inquiry into plant genetic resources will provide a highly effective method for identification of specific traits, but will need more extensive validation.

Genotype × environment interactions in populations possessing Ga1-s and ga1 alleles for cross incompatibility in maize

Use of cross incompatibility in corn (Zea mays L.) by the Ga1-s allele may reduce cross-fertilization in specialty and conventional organic corn with pollen from genetically-modified (GM) corn. For effective use, information about environment, and genotype × environment effects on cross-fertilization by ga1 as well as heritability of cross incompatibility in maize is necessary. Our objective was to obtain this information. Four population pairs differing in their genotype at ga1 were evaluated for cross incompatibility with ga1 pollen in different environments. Populations were derived by crossing the recurrent parents B116, PHG35, ARZM16035:S19, and (CHZM05015:Mo17)Mo17 to Ga1-s donor parent Mo508W/Mo506W. Two replicates of each treatment were grown in the center of 952 m2 fields planted with purple corn as an adventitious source of ga1/ga1 pollen. Open pollination was allowed and the amount of cross-fertilization estimated by averaging the percentage of purple seeds. Environment and genotype × environment effects were not significant. Contrasts to evaluate differences in cross-fertilization between Ga1-s and ga1 populations revealed that mean percentages of cross-fertilization in Ga1-s populations of B116, ARZM16035:S19, and (CHZM05015:Mo17)Mo17 were significantly lower than in ga1 populations. The estimated broad-sense heritability on an entry-mean basis for cross incompatibility was 0.81. Results suggest differences in genotype at ga1 played a major role in cross-fertilization of populations differing in their genotype at the ga1 locus. Incompatibility may be selected effectively over different environments and the Ga1-s system may be of value to reduce cross-fertilization with GM corn pollen.

A new method for the identification and isolation of genes essential for Arabidopsis thaliana seed germination.

Seed viability, dormancy and germination efficiency are very important aspects of the life cycle of plants and their potential to survive and spread in the environment. To characterize the genes controlling these processes, we have devised a technique for the selection of mutants impaired in seed germination. Selection for such a trait is complicated by physiological factors that interact with these processes and affect seed germination efficiency. The distinction between low seed germination potential due to physiological factors that interfere with seed maturation or germination and germination deficiency due to genetic factors was based on screening for tagged mutations. Arabidopsis thaliana T-DNA primary transformants obtained by an in planta transformation technique are all heterozygotes. We screened for lack of germination of 1/4 of the seeds in the progeny of independent transformants, and simultaneously for the abnormal segregation (2:1 instead of 3:1) of a kanamycin resistance marker carried by the T-DNA inserted into the genome of these primary transformants in the plants that germinate. This yielded several mutants affected in the germination processes. One of the mutants, designated ABC33, was further characterized. Once the viable embryos from non-germinating seeds were removed from their testa, they grew and displayed a dwarf phenotype which could be complemented by providing gibberellic acid. A genetic and molecular analysis, based on the characterization of the flanking genomic sequences of the T-DNA insert, showed that ABC33 is a new loss-of-function allele at the GA1 locus.

Transmission and yield effects of a gibberellin mutant allele in potato

A selected clone ofSolanum tuberosum Group Andigena PI 347773 (clone Adg 11.1) and the cultivar Superior had previously been shown to be heterozygous at a gibberellin related dwarfing locus, ga1. Experiments were conducted to test for gametophytic selection for alleles at the ga1 locus in reciprocal crosses and for the effect of genotype on tuber yield. The genotypes of individuals from superior-Adg 11.1 reciprocal F1 families were determined by test crossing. Genotypic classes could be distinguished by the percentages of dwarf seedlings in their test cross progeny. No differences were found in the genotypic distributions of the reciprocal families, which indicates gametophytic selection at this locus is not the cause of reciprocal yield differences. Superior-Adg 11.1 individuals classified by ga1 locus genotype were evaluated for tuber yield. Tuber yields were highly correlated with genotype in both reciprocal families. Yields followed the pattern of simplex>duplex>triplex, indicating that the recessive mutant ga1 allele exhibited a dose effect stimulating yield.

Cloning the Arabidopsis GA1 Locus by Genomic Subtraction.

Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.

Cloning the Arabidopsis GA1 Locus by Genomic Subtraction

Arabidopsis thaliana ga l mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive g a l deletion mutant, gal-3. The cloned sequences correspond to a 5.0-kb deletion in the gal-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the gal-3 mutant is located at the GAl locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GAl locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in gal-3 complemented the dwarf phenotype when integrated into the gal-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga l alleles within the 5.0-kb region deleted in mutant gal-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GAl locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GAl locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GAl region of the Arabidopsis genome.

The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) heynh.

By selecting for germinating seeds in the progeny of mutagen-treated non-germinating gibberellin responsive dwarf mutants of the ga-1 locus in Arabidopsis thaliana, germinating lines (revertants) could be isolated. About half of the revertants were homozygous recessive for a gene (aba), which probably regulates the presence of abscisic acid (ABA). Arguments for the function of this gene were obtained from lines homozygous recessive for this locus only, obtained by selection from the F2 progeny of revertant X wild-type crosses. These lines are characterized by a reduced seed dormancy, symptoms of withering, increased transpiration and a lowered ABA content in developing and ripe seeds and leaves.