Self-Incompatibility in Apricot: Identifying Pollination Requirements to Optimize Fruit Production.

In recent years, an important renewal of apricot cultivars is taking place worldwide, with the introduction of many new releases. Self-incompatible genotypes tolerant to the sharka disease caused by the plum pox virus (PPV), which can severely reduce fruit production and quality, are being used as parents in most breeding programs. As a result, the self-incompatibility trait present in most of those accessions can be transmitted to the offspring, leading to the release of new self-incompatible cultivars. This situation can considerably affect apricot management, since pollination requirements were traditionally not considered in this crop and information is lacking for many cultivars. Thus, the objective of this work was to determine the pollination requirements of a group of new apricot cultivars by molecular identification of the S-alleles through PCR amplification of RNase and SFB regions with different primer combinations. The S-genotype of 66 apricot cultivars is reported, 41 for the first time. Forty-nine cultivars were considered self-compatible and 12 self-incompatible, which were allocated in their corresponding incompatibility groups. Additionally, the available information was reviewed and added to the new results obtained, resulting in a compilation of the pollination requirements of 235 apricot cultivars. This information will allow an efficient selection of parents in apricot breeding programs, the proper design of new orchards, and the identification and solution of production problems associated with a lack of fruit set in established orchards. The diversity at the S-locus observed in the cultivars developed in breeding programs indicates a possible genetic bottleneck due to the use of a reduced number of parents.

Pollen Development and Viability in Diploid and Doubled Diploid Citrus Species.

Seedlessness is one of the most important agronomic traits in mandarins on the fresh fruit market. Creation of triploid plants is an important breeding strategy for development of new commercial varieties of seedless citrus. To this end, one strategy is to perform sexual hybridizations, with tetraploid genotypes as male parents. However, while seed development has been widely studied in citrus, knowledge of key steps such as microsporogenesis and microgametogenesis, is scarce, especially in polyploids. Therefore, we performed a study on the effect of ploidy level on pollen development by including diploid and tetraploid (double diploid) genotypes with different degrees of pollen performance. A comprehensive study on the pollen ontogeny of diploid and doubled diploid "Sanguinelli" blood orange and "Clemenules" clementine was performed, with focus on pollen grain germination in vitro and in planta, morphology of mature pollen grains by scanning electron microscopy (SEM), cytochemical characterization of carbohydrates by periodic acid-Shiff staining, and specific cell wall components revealed by immunolocalization. During microsporogenesis, the main difference between diploid and doubled diploid genotypes was cell area, which was larger in doubled diploid genotypes. However, after increase in size and vacuolization of microspores, but before mitosis I, doubled diploid "Clemenules" clementine showed drastic differences in shape, cell area, and starch hydrolysis, which resulted in shrinkage of pollen grains. The loss of fertility in doubled diploid "Clemenules" clementine is mainly due to lack of carbohydrate accumulation in pollen during microgametogenesis, especially starch content, which led to pollen grain abortion. All these changes make the pollen of this genotype unviable and very difficult to use as a male parent in sexual hybridization with the objective of recovering large progenies of triploid hybrids.

Male Meiosis as a Biomarker for Endo- to Ecodormancy Transition in Apricot.

Dormancy is an adaptive strategy in plants to survive under unfavorable climatic conditions during winter. In temperate regions, most fruit trees need exposure to a certain period of low temperatures to overcome endodormancy. After endodormancy release, exposure to warm temperatures is needed to flower (ecodormancy). Chilling and heat requirements are genetically determined and, therefore, are specific for each species and cultivar. The lack of sufficient winter chilling can cause failures in flowering and fruiting, thereby compromising yield. Thus, the knowledge of the chilling and heat requirements is essential to optimize cultivar selection for different edaphoclimatic conditions. However, the lack of phenological or biological markers linked to the dormant and forcing periods makes it difficult to establish the end of endodormancy. This has led to indirect estimates that are usually not valid in different agroclimatic conditions. The increasing number of milder winters caused by climatic change and the continuous release of new cultivars emphasize the necessity of a proper biological marker linked to the endo- to ecodormancy transition for an accurate estimation of the agroclimatic requirements (AR) of each cultivar. In this work, male meiosis is evaluated as a biomarker to determine endodormancy release and to estimate both chilling and heat requirements in apricot. For this purpose, pollen development was characterized histochemically in 20 cultivars over 8 years, and the developmental stages were related to dormancy. Results were compared to three approaches that indirectly estimate the breaking of dormancy: an experimental methodology by evaluating bud growth in shoots collected periodically throughout the winter months and transferred to forcing chambers over 3 years, and two statistical approaches that relate seasonal temperatures and blooming dates in a series of 11-20 years by correlation and partial least square regression. The results disclose that male meiosis is a possible biomarker to determine the end of endodormancy and estimate AR in apricot.

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses.

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Ovary Signals for Pollen Tube Guidance in Chalazogamous Mangifera indica L.

Most flowering plants show porogamy in which the pollen tubes reach the egg apparatus through the micropyle. However, several species show chalazogamy, an unusual pollen tube growth, in which the pollen tubes reach the embryo sac through the chalaza. While ovary signals for pollen tube growth and guidance have been extensively studied in porogamous species, few studies have addressed the process in chalazogamous species such as mango (Mangifera indica L.), one of the five most important fruit crops worldwide in terms of production. In this study, we characterize pollen-pistil interaction in mango, paying special attention to three key players known to be involved in the directional pollen tube growth of porogamous species such as starch, arabinogalactan proteins (AGPs), and γ-aminobutyric acid (GABA). Starch grains were observed in the style and in the ponticulus at anthesis, but their number decreased 1 day after anthesis. AGPs, revealed by JIM8 and JIM13 antibodies, were homogenously observed in the style and ovary, but were more conspicuous in the nucellus around the egg apparatus. GABA, revealed by anti-GABA antibodies, was specifically observed in the transmitting tissue, including the ponticulus. Moreover, GABA was shown to stimulate in vitro mango pollen tube elongation. The results support the heterotrophic growth of mango pollen tubes in the style at the expense of starch, similarly to the observations in porogamous species. However, unlike porogamous species, the micropyle of mango does not show high levels of GABA and starch, although they were observed in the ponticulus and could play a role in supporting the unusual pollen tube growth in chalazogamous species.

Establishing a framework for female germline initiation in the plant ovule.

Female gametogenesis in flowering plants initiates in the ovule, where a single germline progenitor differentiates from a pool of somatic cells. Germline initiation is a fundamental prerequisite for seed development but is poorly understood at the molecular level due to the location of the cells deep within the flower. Studies in Arabidopsis have shown that regulators of germline development include transcription factors such as NOZZLE/SPOROCYTELESS and WUSCHEL, components of the RNA-dependent DNA methylation pathway such as ARGONAUTE9 and RNA-DEPENDENT RNA POLYMERASE 6, and phytohormones such as auxin and cytokinin. These factors accumulate in a range of cell types from where they establish an environment to support germline differentiation. Recent studies provide fresh insight into the transition from somatic to germline identity, linking chromatin regulators, cell cycle genes, and novel mobile signals, capitalizing on cell type-specific methodologies in both dicot and monocot models. These findings are providing unique molecular and compositional insight into the mechanistic basis and evolutionary conservation of female germline development in plants.

Polyploidy in Fruit Tree Crops of the Genus Annona (Annonaceae).

Genome duplication or polyploidy is one of the main factors of speciation in plants. It is especially frequent in hybrids and very valuable in many crops. The genus Annona belongs to the Annonaceae, a family that includes several fruit tree crops, such as cherimoya (Annona cherimola), sugar apple (Annona squamosa), their hybrid atemoya (A. cherimola × A. squamosa) or pawpaw (Asimina triloba). In this work, genome content was evaluated in several Annona species, A. triloba and atemoya. Surprisingly, while the hybrid atemoya has been reported as diploid, flow cytometry analysis of a progeny obtained from an interspecific cross between A. cherimola and A. squamosa showed an unusual ploidy variability that was also confirmed karyotype analysis. While the progeny from intraspecific crosses of A. cherimola showed polyploid genotypes that ranged from 2.5 to 33%, the hybrid atemoyas from the interspecific cross showed 35% of triploids from a total of 186 genotypes analyzed. With the aim of understanding the possible implications of the production of non-reduced gametes, pollen performance, pollen size and frequency distribution of pollen grains was quantified in the progeny of this cross and the parents. A large polymorphism in pollen grain size was found within the interspecific progeny with higher production of unreduced pollen in triploids (38%) than in diploids (29%). Moreover, using PCR amplification of selected microsatellite loci, while 13.7% of the pollen grains from the diploids showed two alleles, 41.28% of the grains from the triploids amplified two alleles and 5.63% showed up to three alleles. This suggests that the larger pollen grains could correspond to diploid and, in a lower frequency, to triploid pollen. Pollen performance was also affected with lower pollen germination in the hybrid triploids than in both diploid parents. The results confirm a higher percentage of polyploids in the interspecific cross, affecting pollen grain size and pollen performance. The occurrence of unreduced gametes in A. cherimola, A. squamosa and their interspecific progeny that may result in abnormalities of ploidy such as the triploids and tetraploids observed in this study, opens an interesting opportunity to study polyploidy in Annonaceae.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A. cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the cereals.

Grain production in cereal crops depends on the stable formation of male and female gametes in the flower. In most angiosperms, the female gamete is produced from a germline located deep within the ovary, protected by several layers of maternal tissue, including the ovary wall, ovule integuments and nucellus. In the field, germline formation and floret fertility are major determinants of yield potential, contributing to traits such as seed number, weight and size. As such, stimuli affecting the timing and duration of reproductive phases, as well as the viability, size and number of cells within reproductive organs can significantly impact yield. One key stimulant is the phytohormone auxin, which influences growth and morphogenesis of female tissues during gynoecium development, gametophyte formation, and endosperm cellularization. In this review we consider the role of the auxin signaling pathway during ovule and seed development, first in the context of Arabidopsis and then in the cereals. We summarize the gene families involved and highlight distinct expression patterns that suggest a range of roles in reproductive cell specification and fate. This is discussed in terms of seed production and how targeted modification of different tissues might facilitate improvements.

Identification of Self-Incompatibility Alleles by Specific PCR Analysis and S-RNase Sequencing in Apricot.

Self-incompatibility (SI) is one of the most efficient mechanisms to promote out-crossing in plants. However, SI could be a problem for fruit production. An example is apricot (Prunus armeniaca), in which, as in other species of the Rosaceae, SI is determined by an S-RNase-based-Gametophytic Self-Incompatibility (GSI) system. Incompatibility relationships between cultivars can be established by an S-allele genotyping PCR strategy. Until recently, most of the traditional European apricot cultivars were self-compatible but several breeding programs have introduced an increasing number of new cultivars whose pollination requirements are unknown. To fill this gap, we have identified the S-allele of 44 apricot genotypes, of which 43 are reported here for the first time. The identification of Sc in 15 genotypes suggests that those cultivars are self-compatible. In five genotypes, self-(in)compatibility was established by the observation of pollen tube growth in self-pollinated flowers, since PCR analysis could not allowed distinguishing between the Sc and S8 alleles. Self-incompatible genotypes were assigned to their corresponding self-incompatibility groups. The knowledge of incompatibility relationships between apricot cultivars can be a highly valuable tool for the development of future breeding programs by selecting the appropriate parents and for efficient orchard design by planting self-compatible and inter-compatible cultivars.

Pollen wall development in mango (Mangifera indica L., Anacardiaceae).

The mango (Mangifera indica) is a woody perennial crop currently cultivated worldwide in regions with tropical and subtropical climates. Despite its importance, an essential process such as pollen development, and, specifically, cell wall composition that influences crosstalk between somatic cells and the male germline, is still poorly understood in this species and in the Anacardiaceae as a whole. A detailed understanding of this process is particularly important to know the effect of low temperatures during flowering on pollen development that can be a limiting factor for fertilization and fruit set. To fill this gap, we performed a thorough study on the cell wall composition during pollen development in mango. The results obtained reveal a clear differentiation of the cell wall composition of the male germline by pectins, AGPs and extensins from the early developmental stages during microsporogenesis and microgametogenesis reflecting a restricted communication between the male germline and the surrounding somatic cells that is very sensitive to low temperatures. The combination of the results obtained provides an integrated study on cell wall composition of the male germline in mango that reveals the crucial role of the sporophyte and the gametophyte and the vulnerability of the process to low temperatures.

Optimizing Production in the New Generation of Apricot Cultivars: Self-incompatibility, S-RNase Allele Identification, and Incompatibility Group Assignment.

Apricot (Prunus armeniaca L.) is a species of the Rosaceae that was originated in Central Asia, from where it entered Europe through Armenia. The release of an increasing number of new cultivars from different breeding programs is resulting in an important renewal of plant material worldwide. Although most traditional apricot cultivars in Europe are self-compatible, the use of self-incompatible cultivars as parental genotypes for breeding purposes is leading to the introduction of a number of new cultivars that behave as self-incompatible. As a consequence, there is an increasing need to interplant those new cultivars with cross-compatible cultivars to ensure fruit set in commercial orchards. However, the pollination requirements of many of these new cultivars are unknown. In this work, we analyze the pollination requirements of a group of 92 apricot cultivars, including traditional and newly-released cultivars from different breeding programs and countries. Self-compatibility was established by the observation of pollen tube behavior in self-pollinated flowers under the microscope. Incompatibility relationships between cultivars were established by the identification of S-alleles by PCR analysis. The self-(in)compatibility of 68 cultivars and the S-RNase genotype of 74 cultivars are reported herein for the first time. Approximately half of the cultivars (47) behaved as self-compatible and the other 45 as self-incompatible. Identification of S-alleles in self-incompatible cultivars allowed allocating them in 11 incompatibility groups, six of them reported here for the first time. The determination of pollination requirements and the incompatibility relationships between cultivars is highly valuable for the appropriate selection of apricot cultivars in commercial orchards and of parental genotypes in breeding programs. The approach described can be transferred to other woody perennial crops with similar problems.

Pollen-pistil interaction in pawpaw (Asimina triloba), the northernmost species of the mainly tropical family Annonaceae.

PREMISE OF THE STUDY: The pawpaw, Asimina triloba, is an underutilized fruit crop native to North America that belongs to the mainly tropical, early-divergent family Annonaceae. Asimina is the only genus within the Annonaceae with species adapted to cold climates. A thorough analysis of its reproductive biology, specifically pollen-pistil interaction during the progamic phase, is essential to understand both its adaptation to cold climates and how to optimize its fertilization and fruit set. METHODS: We characterized pollen-pistil interaction in Asimina triloba, including the floral cycle and anatomy, stigmatic receptivity, and the pollen tube pathway. We used a combination of histological, cytological, and immunolocalization approaches. KEY RESULTS: Asimina triloba has a gynoecium formed by plicate carpels with a short stylar canal. Unicellular papillae form a continuous tissue covered by a copious secretion from the stigma to the ovary, which is most prominent on the stigma surface where it forms an extragynoecial compitum. Compared to the stigmas of other species in the Annonaceae, the stigmas of A. triloba show a long stigmatic receptivity associated with a long flowering cycle. Stigmatic receptivity is concomitant with the secretion of cell-wall-related arabinogalactan proteins (AGPs). CONCLUSIONS: A long female phase with a long period of stigmatic receptivity is unusual among protogynous flowers of the magnoliid clade, suggesting a derived condition of A. triloba within the Annonaceae. This phase further correlates with the presence of cell-wall-related arabinogalactan proteins in the secretion, which may indicate the conservation of these glycoproteins during stigmatic receptivity and pollen tube growth in angiosperms.

The transition from somatic to germline identity shows conserved and specialized features during angiosperm evolution.

How and why specific plant cells adopt germline identity during ovule development has proved challenging to address, and the pathways that are active in the ovules of basal/early-divergent angiosperms possessing a multilayered nucellus are still unclear. Here, we compare megasporogenesis between two early-divergent angiosperms (Annona cherimola and Persea americana) and the evolutionarily derived Arabidopsis thaliana, studying the three-dimensional spatial position of the megaspore mother cell (MMC), the compositional details of the MMC wall and the location of PIN1 expression. Specific wall polymers distinguished the central position of the MMC and its meiotic products from surrounding tissues in early-divergent angiosperms, whereas, in A. thaliana, only callose (in mature MMCs) and arabinogalactan proteins (AGPs) (in megaspores) distinguished the germline. However, PIN1 expression, which regulates polar auxin transport, was observed around the MMC in the single-layer nucellus of A. thaliana and in the multilayered nucellus of A. cherimola, or close to the MMC in P. americana. The data reveal a similar microenvironment in relation to auxin during megasporogenesis in all three species. However, the different wall polymers that mark MMC fate in early-divergent angiosperms may reflect a specific response to mechanical stress during differentiation, or the specific recruitment of polymers to sustain MMC growth.

The Diversity of the Pollen Tube Pathway in Plants: Toward an Increasing Control by the Sporophyte.

Plants, unlike animals, alternate multicellular diploid, and haploid generations in their life cycle. While this is widespread all along the plant kingdom, the size and autonomy of the diploid sporophyte and the haploid gametophyte generations vary along evolution. Vascular plants show an evolutionary trend toward a reduction of the gametophyte, reflected both in size and lifespan, together with an increasing dependence from the sporophyte. This has resulted in an overlooking of the importance of the gametophytic phase in the evolution of higher plants. This reliance on the sporophyte is most notorious along the pollen tube journey, where the male gametophytes have to travel a long way inside the sporophyte to reach the female gametophyte. Along evolution, there is a change in the scenery of the pollen tube pathway that favors pollen competition and selection. This trend, toward apparently making complicated what could be simple, appears to be related to an increasing control of the sporophyte over the gametophyte with implications for understanding plant evolution.

Transition from two to one integument in Prunus species: expression pattern of INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT).

While gymnosperm ovules have one integument, in most angiosperms two integuments surround the ovules. Unitegmic ovules have arisen independently several times during the evolution of angiosperms, but the ultimate genetic cause of the presence of a single integument remains elusive. We compared species of the genus Prunus that have different numbers of integuments: bitegmic species, such as Prunus armeniaca (apricot) and Prunus persica (peach), and unitegmic species, such as Prunus incisa, analyzing the expression pattern of genes that are involved in integument development in Arabidopsis thaliana: INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT). Bitegmic and unitegmic species showed similar INO expression patterns, indicative of the conservation of an outer integument. However, expression of ETT, which occurs in the boundary of the outer and inner integuments, was altered in unitegmic ovules, which showed lack of ETT expression. These results strongly suggest that the presence of a single integument could be attributable to the amalgamation of two integuments and support the role of ETT in the fusion of the outer and inner integuments in unitegmic ovules, a situation that could be widespread in other unitegmic species of angiosperms.

Microspore development in Annona (Annonaceae): differences between monad and tetrad pollen.

UNLABELLED: • PREMISE OF THE STUDY: Permanent tetrads are the most common form of pollen aggregation in flowering plants. The production of pollen in monads is plesiomorphic in angiosperms, but the aggregation into tetrads has arisen independently different times during the evolution of flowering plants. The causes behind the recurrent evolution of pollen aggregation from monads remain elusive. Permanent tetrad pollen is quite common in the Annonaceae, the largest family in the early-divergent order Magnoliales. In some genera, such as Annona, both tetrad- and monad-producing species can be found.• METHODS: In this comparative study of pollen development, we use immunolocalization, cytological characterization, and enzymatic assays of four species in the genus Annona and one species in its closely related genus Asimina that release pollen in tetrads and two species in the genus Annona that release pollen in monads.• KEY RESULTS: The main difference between species with tetrad and monad pollen is a delayed digestion of callose and cellulose at the pollen aperture sites that resulted in nonlayering of the exine in these areas, followed by a rotation and binding of the young microspores at the aperture sites.• CONCLUSIONS: Small changes in development resulted in clear morphological changes on pollen dispersal time and open a window on the possible selective advantage of the production of aggregated pollen.

Arabidopsis WIH1 and WIH2 genes act in the transition from somatic to reproductive cell fate.

BACKGROUND: Unlike animals, higher plants do not establish a germ line in embryo development but form haploid germ cells from diploid somatic cells late in their life cycle. However, despite its prime importance, little is known about how this transition is regulated. RESULTS: Here, we show that the WUSCHEL (WUS) gene, initially identified as a stem cell regulator in the shoot meristem, is required for megasporogenesis and thus ultimately for the formation of female generative cells. WUS functions in this process by indirectly activating the expression of the WINDHOSE1 (WIH1) and WIH2 genes that encode small peptides found in plants and fungi, but not in animals. WIH genes function together with the tetraspanin-type protein TORNADO2 (TRN2)/EKEKO in promoting megasporogenesis. CONCLUSIONS: Together, our studies identify a pathway promoting germ cell formation from somatic precursor cells.

Stigmatic receptivity in a dichogamous early-divergent angiosperm species, Annona cherimola (Annonaceae): influence of temperature and humidity.

PREMISE OF THE STUDY: A variety of mechanisms to prevent inbreeding have arisen in different angiosperm taxa during plant evolution. In early-divergent angiosperms, a widespread system is dichogamy, in which female and male structures do not mature simultaneously, thus encouraging cross pollination. While this system is common in early-divergent angiosperms, it is less widespread in more recently evolved clades. An evaluation of the consequences of this system on outbreeding may provide clues on this change, but this subject has been little explored. METHODS: In this work, we characterized the cycle and anatomy of the flower and studied the influence of temperature and humidity on stigmatic receptivity in Annona cherimola, a member of an early-divergent angiosperm clade with protogynous dichogamy. KEY RESULTS: Paternity analysis reveals a high proportion of seeds resulting from self-fertilization, indicating that self-pollination can occur in spite of the dichogamous system. Stigmatic receptivity is environmentally modulated--shortened by high temperatures and prolonged by high humidity. CONCLUSIONS: Although spatial and temporal sexual separation in this system seems to effectively decrease selfing, the system is modulated by environmental conditions and may allow high levels of selfing that can guarantee reproductive assurance.

Seedless fruits and the disruption of a conserved genetic pathway in angiosperm ovule development.

Although the biological function of fruiting is the production and dissemination of seeds, humans have developed seedless fruits in a number of plant species to facilitate consumption. Here we describe a unique spontaneous seedless mutant (Thai seedless; Ts) of Annona squamosa (sugar apple), a member of the early-divergent magnoliid angiosperm clade. Ovules (seed precursors) of the mutant lack the outer of two normal integuments, a phenocopy of the inner no outer (ino) mutant of Arabidopsis thaliana. Cloning of the INO ortholog from A. squamosa confirmed conservation of the outer integument-specific expression pattern of this gene between the two species. All regions of the gene were detectable in wild-type A. squamosa and in other members of this genus. However, no region of the INO gene could be detected in Ts plants, indicating apparent deletion of the INO locus. These results provide a case of a candidate gene approach revealing the apparent molecular basis of a useful agronomic trait (seedless fruit) in a crop species, and indicate conservation of the role of a critical regulator of ovule development between eudicots and more ancient lineages of angiosperms. The outer integument is one synapomorphy of angiosperms separating them from other extant seed plants, and the results suggest that the evolution of this structure was contemporaneous with the derivation of INO from ancestral YABBY genes. Thus, a unique lateral structure appears to have coevolved with a novel gene family member essential for the structure's formation.