Gametophyte Factor Research Articles

Risk assessment of pollen‐mediated gene flow from Ga1‐m field corn to dent‐sterile Ga1‐s popcorn

AbstractThe popcorn industry in the United States is at risk of genetic contamination because it utilizes the gametophyte factor 1 gene (Ga1) as a barrier against pollen‐mediated gene flow (PMGF) from field corn (Zea mays L.). Popcorn with the Ga1‐s allele accepts pollen only from Ga1‐s corn, allowing for field corn and popcorn to be nearby without isolation. Germplasm is being introduced to the United States to increase field corn diversity that unknowingly contains the Ga1‐m allele, which can overcome Ga1‐s selectivity and pollinate popcorn. The risk to the popcorn industry has been underassessed. Experiments were conducted to model the frequency of PMGF from Ga1‐m field corn to Ga1‐s popcorn under field conditions and to evaluate the role of wind speed and direction using a concentric donor–receptor design in 2017 and 2018 in Nebraska, USA. The PMGF to white popcorn was detected using a field corn pollen donor with yellow kernel color (dominate) and further confirmed with a molecular test. Popcorn kernels were harvested from cardinal and ordinal directions from 1 to 70 m. More than 7 million kernels were screened to detect the PMGF. Information‐theoretic criteria were used to select the best‐fit model. The greatest PMGF (1.6– 4.1%) was detected at 1 m and declined with distance. The PMGF was detected at 70 m, the maximum distance tested. Amplification of Ga1 followed by restriction enzyme digest confirmed that yellow kernelled progeny were hybrids from the result of PMGF. This is the first assessment of PMGF from field corn to popcorn, and the results are alarming for the popcorn industry and export market.

Expression of the Ga1-s gametophyte factor in shrunken2 sweet corn

Outcrossing is an important problem in specialty maize (Zea mays L.) that can be prevented by using gametophyte factors, such as Ga1-s, which preserve maize plants from pollen contamination. Our objective was to check if the gametophyte factor Ga1-s can protect sweet corn homozygous for sh2 in an efficient and stable way. We combined Ga1-s and sh2 by crossing two popcorn and three sweet corn inbred lines, respectively, in a North Carolina Design II, followed by an ear-to-row breeding program with selection for sh2 phenotype and absence of outcrossing. The released inbred lines homozygous for Ga1-s and sh2 were used for obtaining five hybrids that were evaluated for outcrossing and agronomic performance. Our results show that the gametophyte factor Ga1-s effectively protects the sh2 plants and that this effect was stable across environments. However, the agronomic performance of these inbred lines must be improved. Popcorn donors and sweet corn receptors of Ga1-s were unevenly represented in the released Ga1-s / sh2 inbred lines, suggesting that the viability of sh2 is affected by the genotypes involved. Therefore, breeders should pay attention to the choice of donors of Ga1-s that favors the viability of sh2.

Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize

In maize (Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination (G_GG, G_GB, B_BB, B_BG). A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S.

Identification of M‐Type Gametophyte Factors in Maize Genetic Resources

Maize (Zea mays L.) gametophyte factors are the basis of dent sterile popcorns, where they are used to prevent pollen contamination from dent corns and have been proposed as useful for protection of other specialty types. Current gametophytic systems rely entirely on the strong allele of Gametophyte Factor 1 (Ga1‐s) to impart the selective barrier needed in these hybrids. This allele, however, is naturally overcome by another allele at the same locus (Ga1‐m), which is only detectable by specific evaluation, allowing it to go undetected in breeding lines, thus creating a scenario with substantial risk to Ga1‐s hybrids. Other gametophytic systems exist but have parallel allelic structure with possibly parallel risks, especially from m‐type alleles. By screening a set of maize genetic resources, we assessed the risk posed by the Ga1‐m allele. We identified the allele in several populations readily useful for expanding the genetic base of commercial maize, including several Germplasm Enhancement of Maize lines. To examine the possible distribution of m‐type alleles at other key gametophytic loci, we screened the maize nested association mapping population founder lines for the presence of m‐type gametophytic alleles, identifying 19 previously unreported m‐type gametophytic alleles in these lines. Our results also highlight the frequent concomitancy of gametophytic alleles at different loci, the full interactions of which are ignored by standard phenotyping methods that consider individual loci, confounding allele status determination. We provide a method for determining allele status at multiple gametophytic loci and highlight the implications of concomitant alleles, especially Ga1‐m and Tcb1‐m, on the possible deployment of new barrier systems.

Unilateral incompatibility is linked to reduced pollen expression of a farnesyl pyrophosphate synthase.

Unilateral incompatibility is linked to reduced pollen expression of a farnesyl pyrophosphate synthase.

Interaction between RNA helicase ROOT INITIATION DEFECTIVE 1 and GAMETOPHYTIC FACTOR 1 is involved in female gametophyte development in Arabidopsis.

ROOT INITIATION DEFECTIVE 1 (RID1) is an Arabidopsis DEAH/RHA RNA helicase. It functions in hypocotyl de-differentiation, de novo meristem formation, and cell specification of the mature female gametophyte (FG). However, it is unclear how RID1 regulates FG development. In this study, we observed that mutations to RID1 disrupted the developmental synchrony and retarded the progression of FG development. RID1 exhibited RNA helicase activity, with a preference for unwinding double-stranded RNA in the 3' to 5' direction. Furthermore, we found that RID1 interacts with GAMETOPHYTIC FACTOR 1 (GFA1), which is an integral protein of the spliceosome component U5 small nuclear ribonucleoprotein (snRNP) particle. Substitution of specific RID1 amino acids (Y266F and T267I) inhibited the interaction with GFA1. In addition, the mutated RID1 could not complement the seed-abortion phenotype of the rid1 mutant. The rid1 and gfa1 mutants exhibited similar abnormalities in pre-mRNA splicing and down-regulated expression of some genes involved in FG development. Our results suggest that an interaction between RID1 and the U5 snRNP complex regulates essential pre-mRNA splicing of the genes required for FG development. This study provides new information regarding the mechanism underlying the FG developmental process.

Live and let die: a REM complex promotes fertilization through synergid cell death in Arabidopsis.

Fertilization in flowering plants requires a complex series of coordinated events involving interaction between the male and female gametophyte. We report here molecular data on one of the key events underpinning this process - the death of the receptive synergid cell and the coincident bursting of the pollen tube inside the ovule to release the sperm. We show that two REM transcription factors, VALKYRIE (VAL) and VERDANDI (VDD), both targets of the ovule identity MADS-box complex SEEDSTICK-SEPALLATA3, interact to control the death of the receptive synergid cell. In vdd-1/+ mutants and VAL_RNAi lines, we find that GAMETOPHYTIC FACTOR 2 (GFA2), which is required for synergid degeneration, is downregulated, whereas expression of FERONIA (FER) and MYB98, which are necessary for pollen tube attraction and perception, remain unaffected. We also demonstrate that the vdd-1/+ phenotype can be rescued by expressing VDD or GFA2 in the synergid cells. Taken together, our findings reveal that the death of the receptive synergid cell is essential for maintenance of the following generations, and that a complex comprising VDD and VAL regulates this event.

Identification of maize-derived dominant gametophyte factors

The use of gametophyte factors to protect specialty-type maize has long been advocated, but as of yet, they have made very little impact on preventing pollen contamination due to the complications associated with breeding with these materials, mainly the additive nature of the alleles. A dominant gametophyte factor (DGF) overcomes this problem, allowing for less time consuming production of gametophytic hybrids, but effectively utilized sources do not exist. Tcb1-s, a known DGF, is a teosinte introgression into maize and the leading candidate for utilization, however, it has several issues that limit its effective use in expediting the breeding process for gametophytic hybrids. The use of maize for a source of DGFs may overcome this problem; with the idea years of selection by farmers would likely have minimized any segregation for yield associated with these alleles, making their use for production of gametophytic hybrids an appealing option for modern breeders. Through screening and backcrossing selected maize accessions, we identified DGFs in seven accessions from race Maiz Dulce, which we document here as a starting point for identification of additional maize-derived DGFs. These accessions did not appear to segregate for yield, a marked improvement over existing DGFs. Additionally, we assessed the compatibility of identified maize-derived DGFs from one accession, and showed that, while lines are generally compatible, they are not obligately so since a single accession may segregate for multiple gametophyte factors. There is, therefore, a need to consider the compatibility of pairs of DGFs early in the inbreeding process. Maize-derived DGFs provide a more effective method of producing gametophytic hybrids, making their production economical enough to be brought to market. The use of DGFs has wider potential to benefit any producers interested in preventing pollen contamination with gametophytic hybrids through the same benefits provided to breeders for organic and other specialty systems. In combination with Ga1-m resistance, maize-derived DGFs provide a long-term gametophytic solution to pollen contamination, in a more expeditious way.

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.

Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1) in Maize.

Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L.) and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1); which did not carry a gametophytic factor and W22 (Ga1), a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1) and W22 (Ga1). A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1) and W22 (Ga1) whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1) and W22 (Ga1). However, in pollen, 2 proteins were identified only in the W22 (ga1) and 12 proteins only in the W22 (Ga1) whereas 10 proteins were confirmed from the both of W22 (ga1) and W22 (Ga1). In contrary, 10 proteins were appeared only in the pistil of W22 (ga1) and 7 proteins from W22 (Ga1) while 3 proteins confirmed in the both of W22 (ga1) and W22 (Ga1). Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

Fine Mapping of the Maize Cross‐Incompatibility Locus Gametophytic Factor 1 (ga1) Using a Homogeneous Population

ABSTRACTPhenotyping of a mapping population is usually the bottleneck that limits the size of the mapping population and mapping resolution. A homogeneous population mapping approach was used for mapping maize (Zea mays L.) gametophytic factor 1 (ga1) that could completely eliminate phenotyping during the mapping process. The strong allele of maize ga1 (Ga1‐S), from popcorn inbred line SDGa25, showed a 100% cross‐incompatibility with the majority of Chinese dent and flint maize. A homogeneous mapping population was developed by crossing an (SDGa25/Jing66) F1 male back to an SDGa25 female. During the pollination process, pollen grains of ga1 were completely excluded from fertilization, and a homogeneous BC1F1 (SDGa25//SDGa25/Jing66) population was created in which only the Ga1‐S/Ga1‐S genotype existed, making phenotyping unnecessary. A total of 2245 individuals of this population were genotyped with SD9 and SD12 markers and 20 recombinants were identified. The Ga1‐S locus was quickly delineated to a 100 Kb region between markers dCS1 and insertion deletion ID7 at position 9,491,422 and 9,591,946 bp based on the B73 RefGen_v2 sequence. By marker‐assisted selection, Ga1‐S was introgressed into parental lines of an elite white waxy maize hybrid by six generations of backcrossing and one generation of selfing. The homozygous Ga1‐S/Ga1‐S hybrid showed full cross‐incompatibility to ga1 maize. The Ga1‐S allele could be used as a biological reproductive barrier in reducing cross‐pollination between different types of maize such as waxy and non‐waxy, genetically modified (GM) and non‐GM maize.

Genetic analysis and fine mapping of the Ga1-S gene region conferring cross-incompatibility in maize

Cross-incompatibility genes known as gametophyte factors (ga) are numerous in maize. Many popcorn strains carry these genes and cannot be fertilized by pollen of dent and flint maize strains although the reciprocal crosses are successful. A Chinese popcorn strain SDGa25 carries the strongest allele of Ga1 (Ga1-S) and the majority of Chinese dent and flint maize germplasm are incompatible with SDGa25. The incompatibility is due to pollen tube growth obstruction 2 h after pollination. The pollen tube is arrested in the silk segment 5.5 cm distal to the pollination area and never reaches the ovule. The Ga1-S carried by SDGa25 behaves as a single dominant gene. This gene was mapped between markers SD3 on BAC AC200747 0.827 cM apart on the telomere side and SD12 on BAC AC204382 0.709 cM apart on the centromere side. The genetic region mapped spanning the Ga1-S locus was estimated to be 1.5 cM in length and the physical distance is 2,056,343 bp on ctg156 based on the B73 RefGen_v2 sequence. Gametophyte factors influence gene flow direction and the strongest Ga1-S allele is useful for isolating one category of commercial varieties from another. The eight tightly linked markers to Ga1-S developed in this study would greatly improve marker-assisted introgression efficiency and the fine mapping would facilitate the isolation of the Ga1-S.

GAMETOPHYTIC FACTOR 1, Involved in Pre‐mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homologs of gene GFA1 in yeast and human encode putative U5 snRNP-specific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogenesis and embryogenesis in plant.

VAJ/GFA1/CLO is Involved in the Directional Control of Floral Organ Growth

Flowers assume variant forms of reproductive structures, a phenomenon which may be partially due to the diversity among species in the shape and size of floral organs. However, the organ size and shape of flowers usually remain constant within a species when grown under the same environmental conditions. The molecular and genetic mechanisms that control organ size and shape are largely unknown. We isolated an Arabidopsis mutant, vajra-1 (vaj-1), exhibiting defects in the regulation of floral organ size and shape. In vaj-1, alterations in the size and shape of floral organs were caused by changes in both cell size and cell number. The vaj-1 mutation also affected the number of floral organs. In vaj-1, a mutation was found in GAMETOPHYTIC FACTOR 1 (GFA1)/CLOTHO (CLO), recently shown to be required for female gametophyte development. The VAJ/GFA1/CLO gene encodes a translational elongation factor-2 (EF-2) family protein, of which the human U5-116 kD and yeast Snu114p counterparts are U5 small nuclear ribonucleoprotein (snRNP)-specific proteins. A transient expression assay using Arabidopsis protoplasts revealed that VAJ protein co-localized with SC35, a serine/arginine-rich (SR) protein involved in pre-mRNA splicing. Our results showed that VAJ/GFA1/CLO has a novel role in the directional control of floral organ growth in Arabidopsis, possibly acting through pre-mRNA splicing.

Segregation distortion in Arabidopsis gametophytic factor 1 (gfa1) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle.

Genes in conflict: the biology of selfish genetic elements

Genes in conflict: the biology of selfish genetic elements

Mapping QTL for popping expansion volume in popcorn with simple sequence repeat markers.

Popping expansion volume is the most important quality trait in popcorn ( Zea mays L.), but its genetics is not well understood. The objectives of this study were to map quantitative trait loci (QTLs) responsible for popping expansion volume in a popcorn x dent corn cross, and to compare the predicted efficiencies of phenotypic selection, marker-based selection, and marker-assisted selection for popping expansion volume. Of 259 simple sequence repeat (SSR) primer pairs screened, 83 pairs were polymorphic between the H123 (dent corn) and AG19 (popcorn) parental inbreds. Popping test data were obtained for 160 S(1) families developed from the [AG19(H123 x AG19)] BC(1) population. The heritability ( h(2)) for popping expansion volume on an S(1) family mean basis was 0.73. The presence of the gametophyte factor Ga1(s) in popcorn complicates the analysis of popcorn x dent corn crosses. But, from a practical perspective, the linkage between a favorable QTL allele and Ga1(s) in popcorn will lead to selection for the favorable QTL allele. Four QTLs, on chromosomes 1S, 3S, 5S and 5L, jointly explained 45% of the phenotypic variation. Marker-based selection for popping expansion volume would require less time and work than phenotypic selection. But due to the high h(2) of popping expansion volume, marker-based selection was predicted to be only 92% as efficient as phenotypic selection. Marker-assisted selection, which comprises index selection on phenotypic and marker scores, was predicted to be 106% as efficient as phenotypic selection. Overall, our results suggest that phenotypic selection will remain the preferred method for selection in popcorn x dent corn crosses.

Chromosomal regions associated with segregation distortion in maize.

Segregation distortion skews the genotypic frequencies from their Mendelian expectations. Our objectives in this study were to assess the frequency of occurrence of segregation distortion in maize, identify chromosomal regions consistently associated with segregation distortion, and examine the effects of gametophytic factors on linkage mapping. We constructed a simple sequence repeat (SSR) linkage map for a LH200/LH216 F(2)Syn3 (i.e., random-mated three times) population, and compared the segregation distortion in this map with the segregation distortion in three published linkage maps. Among 1,820 codominant markers across the four mapping populations, 301 (17%) showed segregation distortion ( P < 0.05). The frequency of markers showing segregation distortion ranged from 19% in the Tx303/CO159 mapping population to 36% in the B73/Mo17 mapping population. A positive relationship was found between the number of meioses and the frequency of segregation distortion detected in a population. On a given chromosome, nearly all of the markers showing segregation distortion favored the allele from the same parent. A total of 18 chromosomal regions on the ten maize chromosomes were associated with segregation distortion. The consistent location of these chromosomal regions in four populations suggested the presence of segregation distortion regions (SDRs). Three known gametophytic factors are possible genetic causes of these SDRs. As shown in previous research, segregation distortion does not affect the estimate of map distance when only one gametophytic factor is present in an SDR.

A mitochondrial DNA sequence is associated with abnormal pollen development in cytoplasmic male sterile bean plants.

Cytoplasmic male sterility (CMS) in common bean is associated with the presence of a 3-kb unique mitochondrial sequence designated pvs. The pvs sequence encodes at least two open reading frames (297 and 720 bp in length) with portions derived from the chloroplast genome. Fertility restoration by the nuclear restorer gene Fr results in the loss of this transcriptionally active unique region. We examined the effect of CMS (pvs present) and fertility restoration by Fr (pvs absent) on the pattern of pollen development in bean. In the CMS line, pollen aborted in the tetrad stage late in microgametogenesis. Microspores maintained cytoplasmic connections throughout pollen development, indicating aberrant or incomplete cytokinesis. Pollen-specific events associated with pollen abortion and fertility restoration imply that a gametophytic factor or event may be involved in CMS. In situ hybridization experiments suggested that significant reduction or complete loss of the mitochondrial sterility-associated sequence occurred in fertile pollen of F2 populations segregating for fertility. These observations support a model of fertility restoration by the loss of a mitochondrial DNA sequence prior to or during microsporogenesis/gametogenesis.

Flower Colors in Common Bean Produced by Interactions of the Sal and V Loci and a Gametophyte Factor Ga Linked to Sal

Inheritance of red flower color was investigated in crosses using Lamprecht's lines M0169 and M0056, which are derived from Phaseolus coccineus L., and Univ. of Florida P. vulgaris L. breeding line 5-593. Based on segregation in the F2populations from 5-593 × M0169 and 5-593 × M0056, we hypothesize that the genotypes for flower colors are sal/sal V/V for 5-593 and Sal/Sal v/v for M0169 and M0056. The backcross 5-593 × F, (5-593 × M0056) segregated for four flower colors in about equal frequencies, and F2, F3, and F4progeny tests of the backcross plants provided confirmation of all the genotypes in the digenic model. The two recombinant true-breeding colors/genotypes were white (sal/sal v/v) and china rose (Sal/Sal V/V). We hypothesize that the large deficiency of plants carrying the Sal allele in segregating populations is due to a gametophyte factor linked to Sal. We propose the gene symbol Ga for the gametophyte factor locus, which achieves complete selection for pollen carrying Ga on female plants carrying Ga, i.e., no pollen carrying ga achieves fertilization. The linkage between Ga and the marker locus Sal is 17 CM (centiMorgan).