A pair of non-Mendelian genes at the Ga2 locus confer unilateral cross-incompatibility in maize

Maize unilateral cross-incompatibility (UCI) that causes non-Mendelian segregation ratios has been documented for more than a century. Ga1, Ga2, and Tcb1 are three major UCI systems, described but not fully understood. Here, we report comprehensive genetic studies on the Ga2 locus and map-based cloning of the tightly linked male determinant ZmGa2P and female determinant ZmGa2F that govern pollen-silk compatibility among different maize genotypes. Both determinants encode putative pectin methylesterases (PME). A significantly higher degree of methyl esterification is detected in the apical region of pollen tubes growing in incompatible silks. No direct interaction between ZmGa2P and ZmGa2F is detected in the yeast two-hybrid system implying a distinct mechanism from that of self-incompatibility (SI). We also demonstrate the feasibility of Ga2 as a reproductive barrier in commercial breeding programs and stacking Ga2 with Ga1 could strengthen the UCI market potentials.


Minor revisions:
Title: "the Ga2 locus" P1 L21: "A significantly higher degree" P8 L18: cite reference for Mo17 being Ga2-M P8 L23: both purple and yellow kernels (currently implies a color mixture within one kernel) P9 L13: first time using the term incompatible, make clear that the pollen is ga2 and silks are Ga2 P11 L20: determinant and be self-incompatible P14 L8: are under way P14-15 L27-1: correct grammar "If the male determinant was gametophytic, only one genotype (Ga2/Ga2) would be present in the 511L (♀)×(W22×511L) (♂) BC1F1 population, otherwise if it were sporophytic then two genotypes (Ga2/Ga2 and Ga2/ga2) would be present." P15 L20: correct grammar "If the female determinant was gametophytic in nature, the BC1F1 population should have only one genotype ga2/ga2, otherwise if were sporophytic, there would be two genotypes Ga2/ga2 and ga2/ga2." P18 L18: self-incompatible not self-incompatibility P18 L23-24: change to "while heterozygous plants set both purple and yellow kernels" P18 L25: change "mix-colored" to "a mix of colored" P19 L3-4: grammar corrections "and the other is a single pollination with a mixture of both pollen genotypes. Besides the full yellow and the mixed color ears, barren ears were also observed due to some individuals which lack the male determinant" Figure   Reviewer #2: Remarks to the Author: This is an excellent manuscript, and contributes valuable knowledge concerning the Unilateral Cross-Incompatibility systems in maize. They are important because of the value to the popcorn industry, as well as organic/non-gmo markets.
There are several significant contributions: a) confirming that there are separable male and female determinants in the Ga2 locus & finding recombinants separating male and female funcional determinates, b)determining potential functional genes by mapping & following with expression data matching the phenotypic observations to identify the gene, c)that the female function is conditioned by a single recessive gene and that it is of sporophytic operation, d) map-based cloning and identification of the likely female gene, and e)proving that no direct interaction between the silk function determinate and the male function determinate is involved in their phenotypic operation.
The materials, methods, and the data are of good quality and clearly support the conclusions reached.
I am enclosing some language/form suggestions that reflect standard English usage, however, the paper is very well written, and is much better than I could do in a language which isn't my first.
Page 2, line 4 ...has been "known" for more... Page 2, line 10 ...a pair of female... (leave out second "a" Page 2, line 28 ... of UCI, "exploration" , not exploitation Page 2, line 29 ...major "progress has"... Reviewer #3: Remarks to the Author: The manuscript of Chen and coworkers reports a very interesting study of the Ga2 locus, one of the locus involved in the unilateral cross incompatibility (UCI) system of maize and one of the lesser known. The authors perform a robust genetic and molecular study of the male and female determinants of Ga2, both of them encode pectin methylesterases (PME), responsible of deesterification of pectins. It is known that PME activity regulates the level of pectin esterification in cell walls and that the dymanyc cell wall of the tip of growing pollen tube is enriched in highly methyl esterified pectins, while other parts of the tube wall contain major proportion of de-esterified pectins. On the other hand, in maize it has been postulated that PME activity in the growing pollen tube tip has a critical role in controlling maize UCI, by regulating the degree of methylesterification of the pollen tube cell wall, i.e. low esterification involves no progression of the tube and no fertilization.
Functional analyses of the manuscript are based in the study of the level of pectin esterification in pollen tube tips from different genetic crosses, growing in compatible and incompatible silks. Results indicate that pollen tube tips contain less esterified pectins in incompatible silks than in compatible ones. These findings together with other results of the manuscript lead authors to conclude that ZmGa2P determinant may not possess in vivo PME activity and to hypothesize that it may interact with other active PMEs that have both PME and PMEI domains to finely tune PME activities in the apical region of pollen tube.
The main support for the conclusion of the manuscript regarding the function of the Ga2 determinants is the different esterification level of pectins of the pollen tube tips in compatible versus incompatible silks. This analysis is based in a unique experimental approach: immunofluorescence with LM20 antibody which detects highly esterified pectins, and quantification of signal intensities. Since no other approach is performed to estimate changes in pectin esterification, the methodology and results of these assays should be clearly explained. Some concerns in this sense: Immunofluorescence experiments with LM20 in pollen tubes growing in vitro would be interesting to compare with in vivo situation. This comparison would help to understand the function of female determinant and to give addittional support to the results in vivo.
Page 9, line 10: Title is confuse, the genes are PMEs, and pectins are synthesized as highly esterified forms that can be de-esterified later.
Page 9, lines 22-24: a clear definition of the region considered as "tip", "apical region" of the pollen tube should be included (length, shape). Images at lower magnification, showing pollen tube regions with no LM20 signal would support the results.
For an appropriate comparison among immunofluorescence signal intensities methodology should be clear. In M&M or Results section, a short description on the method used for quantification of signals is required (software used, number of images, type of images (optical sections, projections, z-stack collection...) also, definition of ROI (region of interest) to quantify the signal, and if confocal settings were the same for acquisition of every image, etc.

What do dotted lines mean in figure 5?
Ideally, immunofluorescence assays with other antibody which detects pectins with low degree of esterification (e.g. LM19) could be added to the manuscript, to support the results with LM20 (or any other experiment that can provide addittional support).

Reviewer #1:
Q1: Since this paper has the opportunity to name these genes, I wonder if the authors would consider calling the female determinant something other than ZmGa2S? The male determinant as ZmGa2P makes sense and is distinct from the phenotype (Ga2-M). It also is consistent with ZmGa1P. Perhaps the female determinants could have an -F designation, similar to Tcb-f? This becomes relevant when talking to breeders who are not well versed in genetics and making distinctions between a phenotype or haplotype (Ga2-Strong) and a gene.  RE: This is a good point and we totally agree that an in vitro assay would give additional support. However, in vivo assay would reflect the actual in-plant situation.
In fact, we have tried to express ZmGa2P and ZmGa2F in vitro using both Escherichia coli and Pichia methanolica expression systems. Unfortunately, we failed to detect any PME activity in the assay reaction, thus no active proteins were obtained for the in vitro immunofluorescence experiments. Similar situation happened for ZmGa1P. We proposed the possible reason as the lack of post-translational modification in these expression systems. This provides an important topic for future studies.
Q2: Page 9, lines 10: Title is confused, the genes are PMEs, and pectins are synthesized as highly esterified forms that can be de-esterified later.

RE:
We revised the title as "ZmGa2P and ZmGa2F are putative PMEs mediating cross-incompatibility" (Page 9, Lines 9). Q3: Page 9, lines 22-24: a clear definition of the region considered as "tip", "apical region" of the pollen tube should be included (length, shape). Images at lower magnification, showing pollen tube regions with no LM20 signal would support the results.

RE:
The "tip" and "apical region" both indicate the region shown as the figure below. Q6: Ideally, immunofluorescence assays with other antibody which detects pectins with low degree of esterification (e.g. LM19) could be added to the manuscript, to support the results with LM20 (or any other experiment that can provide additional support).

RE:
We fully agree that immunofluorescence assays with additional antibody could provide additional support for LM20 results. This study mainly focused on the genetic study, map-based cloning and functional validation of the Ga2 locus, which laid foundation for subsequent molecular, genetic and biochemical studies. Additional immunofluorescence assays with other antibody are necessary for future study.