SNP Markers Reveal Relationships between Fruit Paternity, Fruit Quality and Distance from a Cross-pollen Source in Persea Americana Orchards

Plant reproductive output is increasingly pollen limited, and cross-pollination can improve fruit yield, fruit size and nutritional quality of many food crops. However, we rarely understand what proportions of the crop result from self- or cross-pollination, how cross-pollination affects crop quality, and how far pollen is transported by pollinators. Management strategies to improve pollination services are consequently not optimal for many crops. We utilised a series of SNP markers, unique for each cultivar of avocado, to quantify proportions of self-and cross-paternity in fruit of Hass avocado at increasing distances from cross-pollen sources. We assessed whether distance from a cross-pollen source determined the proportions of self-pollinated and cross-pollinated fruit, and evaluated how self- and cross-paternity affected fruit size and nutritional quality. Avocado fruit production resulted from both self- and cross-pollination. Cross-pollination levels decreased with increasing distance from a cross-pollen source, from 63% in the row adjacent to another cultivar to 25% in the middle of a single-cultivar block, suggesting that pollen transport was limited across orchard rows. Limited pollen transport did not affect fruit size or quality in Hass avocados as xenia effects of a Shepard polliniser on size and nutritional quality were minor.


Introduction
Pollination is essential for plant reproduction in both natural and agricultural ecosystems [1][2][3] . However, plant reproduction is increasingly limited by the quality or quantity of pollen deposited on the stigmas of owers 4,5 . Roughly 50% of plant species rely on or bene t from cross-pollination for successful reproduction 6,7 . Cross-pollination occurs when pollen from one genotype is transferred to the stigma of another genotype, whereas self-pollination occurs when pollen is transferred within the same genotype 8 . Self-incompatible plants require cross-pollination to reproduce sexually, and some self-compatible plants have increased fruit set when cross-pollinated [9][10][11] . The type of pollen deposited on the stigma can, thus, be important for successful reproduction [12][13][14] .
Tree crop orchards are often established with only a few clonally-propagated cultivars. Each cultivar has one genotype, making trees of a single cultivar a clone 8,15 . Cross-pollination occurs when a stigma of one cultivar receives pollen from owers of another cultivar whereas self-pollination occurs when pollen of the same cultivar is transferred 8 . Cross-pollination increases fruit size, quality and shelf life in many crops, including some tree nuts and berries 14,16−18 . Increased nutritional quality of the fruit can provide health bene ts for consumers; e.g. cross-pollination in almonds increases the oleic to linoleic acid ratio, which has been linked to their cardio-protective effects 17,19 . For most crops, we do not understand to what extent self-v. cross-pollination contribute to the crop at harvest despite plant reproduction being increasingly pollen limited 4,5 . We also do not know how self-v. cross-pollination affect crop quality parameters such as fruit size or nutritional quality.

Results
Paternity and fruit size at different distances from a crosspollen source A total of 52.4% of fruit (N = 190) resulted from self-pollination and 47.6% resulted from cross-pollination. Almost all the cross-pollinated fruit (95%; 86 of 91 fruit) were pollinated by cultivar Shepard. The remaining ve cross-pollinated fruit were pollinated by Lamb Hass (1 fruit) or Sharwil (1 fruit) or the cross-pollen parent could not be assigned de nitively (3 fruit). The percentage of self-pollinated fruit increased with increasing distance (i.e. the number of rows) from a cross-pollen source, from 37-75%, whereas the percentage of cross-pollinated fruit decreased from 63-25% ( Fig. 1; F = 4.00, P = 0.02). The percentage of cross-pollinated fruit declined signi cantly by 11 rows from a cross-pollen source (Fig. 1). Distance from a cross-pollen source did not affect fruit mass (F = 0.39, p = 0.76), esh mass (F = 0.60, p = 0.62), seed mass (F = 0.72, p = 0.55) or seed proportion (F = 2.48, p = 0.09) ( Table 1). Effect of pollen parentage on fruit size, mineral nutrient concentrations and fatty acid composition Hass avocado fruit that were self-pollinated by Hass or cross-pollinated by Shepard did not differ signi cantly in fruit mass, esh mass, seed mass or seed proportion (Table 2). Self-pollinated fruit had 9.1% lower calcium and 10.6% higher phosphorus concentrations than cross-pollinated fruit (Table 3).
Self-and cross-pollinated fruit did not differ in the concentrations of other elements (Table 3). Selfpollinated fruit had a 2.8% higher ratio of unsaturated to saturated fatty acids (UFA:SFA) than crosspollinated fruit (Table 4). Self-and cross-pollinated fruit did not differ signi cantly in the relative contributions of palmitic, palmitoleic, stearic, oleic, elaidic or linoleic acid to the total fatty acid composition (Table 4).

Discussion
Self-and cross-paternity were both common among Hass avocado fruit in our study, but the percentage of cross-pollinated fruit decreased with increasing distance from a cross-pollen source. Self-and crosspollinated fruit did not differ signi cantly in fruit size and they differed little in their nutritional composition. However, self-pollinated fruit had lower calcium concentrations, higher phosphorus concentrations and a slightly elevated UFA:SFA ratio. These results demonstrate that pollen ow is limited across avocado orchards, but that xenia effects of a Shepard polliniser on the size and nutritional quality of Hass fruit quality are minor.
Hass avocado fruit resulted from either self-pollination or cross-pollination, demonstrating some degree of self-compatibility in avocado owers. Between 25% and 63% of fruit were cross-pollinated, with the frequency of outcrossing being dependent on the proximity to owers of another cultivar. The 25% of fruit that were cross-pollinated in the middle of a Hass block had received pollen that was transported at least 130-147 m, even though self-pollen was potentially available from all trees planted at a closer distance.
The orchards in our study were established with Hass, Lamb Hass and Wurtz as type A cultivars and Shepard as the type B cultivar, ensuring that Shepard pollen was available during the female stage of the Hass, Lamb Hass and Wurtz trees. Protogyny can explain why 25% of fruit were cross-pollinated in the middle of the single-cultivar Hass block because only cross pollen was widely released during the female stage of Hass owers. The temporal separation between male and female owers in the owering types is not complete and can be affected by temperature 37 . Other mechanisms such as selective fruitlet abscission might also have occurred 33,38 . Self-pollinated avocado fruitlets can be more prone to selective abortion, leading to an over-representation of cross-pollinated fruit at harvest 38 . However, younger fruitlets resulting from pollination late in owering are also more prone to selective abortion 33 .
A decrease in the percentage of cross-pollinated Hass fruit with increasing distance from a cross-pollen source has been reported previously 33,39 . Our results showed a signi cant decrease in the percentage of cross-pollinated fruit when comparing the middle of a single-cultivar block to the row adjacent to a crosspollen source, but not when comparing 2 or 3 rows from the cross-pollen source to the row adjacent to the cross-pollen source. Other studies have reported a decrease in the percentage of cross-pollinated fruit at smaller distances from a cross-pollen source than we found; e.g. from 0-28 m or from 0-92 m compared with from 0-130 m in our study 33,39 . The high percentages of cross-pollinated fruit at 2 or 3 rows from a cross-pollen source in our study may suggest that Shepard pollen is moved further across orchard rows or it may suggest differences in pollinator movements compared with other studies [40][41][42] .
Self-and cross-pollinated fruit did not differ in fruit mass, esh mass, seed mass or seed proportion.
Cross-pollination increases fruit quality, such as size and nutritional composition, of many crops 5,16−18 .
The effect of different pollen parents on characteristics of the fruit is a phenomenon termed xenia 43 . Fruit mass and seed mass of Fuerte avocado fruit cross-pollinated by Tops-Tops, Teague or Ettinger are higher than of self-pollinated fruit 38 . Likewise, seed mass of Hass fruit cross-pollinated by Ettinger is greater than that of self-pollinated fruit 44 . Potentially, pollination by Ettinger results in larger Hass fruit than does pollination by Shepard.
Nutrient levels and fatty acid composition differed little between self-and cross-pollinated Hass fruit. However, self-pollinated fruit a higher UFA:SFA ratio than cross-pollinated fruit. To our knowledge, no studies have investigated the nutritional quality of self-and cross-pollinated avocado fruit. The slightly higher UFA:SFA ratio of self-pollinated fruit could be bene cial for human health because a diet rich in unsaturated fatty acids decreases LDL-cholesterol levels and other cardiovascular risk factors 22,26,45 . However, the UFA:SFA ratio in avocado is highly variable and the ratio in our study was relatively low at 2.18 and 2.12 in self-and cross-pollinated fruit, respectively 22,46 . The consumption of fruit from cooler growing regions that have higher UFA:SFA ratios will have a much greater effect on health than the small observed difference between self-and cross-pollinated fruit in our study 46 . Self-pollinated fruit had lower calcium and higher phosphorus levels than cross-pollinated fruit. Phosphorus, unlike other micronutrients such as calcium, iron, iodine, magnesium and zinc, whose dietary intakes are often inadequate, is almost never in short supply in the human diet 47 . The calcium nutrient levels of cross-pollinated fruit might be more bene cial for human health.
Self-and cross-pollination both contributed to the harvested avocado crop. The percentage of crosspollinated fruit decreased with increasing distance from a cross-pollen source, indicating limited crosspollen movement to the middle of single-cultivar blocks. Xenia effects of a Shepard polliniser were minimal, because fruit size, the levels of most mineral nutrients, and the contributions of most fatty acids to total fatty acid composition did not differ signi cantly between self-and cross-pollinated fruit.
However, further research should be undertaken to determine whether cross-pollination affects initial fruit set, fruitlet retention and, thus, overall tree yield.

Study sites and design
Hass avocado fruit were harvested from two commercial orchards near Childers, Queensland, Australia (25°08'17"S 152°22'40"E and 25°13'32"S 152°17'53"E). The soil at both orchards was red clay-loam. The Fruit were stored at 4 °C for 10 or 20 d, before being moved to room temperature (21 °C) to allow onset of ripening. Fruit were ripe after 10.6 ± 1.0 days (mean ± SE) at room temperature. Ripeness was con rmed by measuring skin and esh rmness with a handheld sclerometer (8 mm head; Lutron Electronic Model: FR-5120, Coopersburg, PA). Fruit were considered ripe when the maximum force required to impress the sclerometer tip 1 mm deep was < 15 N for the skin and < 5 N for the esh 50,51 . Flesh rmness was measured after removing small patches of skin at two locations along the equator of the fruit, with the two measurements taken at 90° from each other. Fruit and seed fresh mass were recorded. Subsamples of esh were then taken to measure the: (i) relative contribution of six fatty acids to the total fatty acid composition; (ii) ratios of saturated and unsaturated fatty acids; and (iii) concentrations of mineral nutrients. A ~ 50 mg subsample of the seed was also taken for genotyping.

Mineral nutrients
We determined the concentrations of 14 nutrients from esh taken from two locations, near the apex and along the equator of each fruit. We used combustion analysis ( Double-digest RADseq is commonly performed using 75 bp reads. We opted for longer reads (150 bp) to support downstream assay development for MassARRAY genotyping assays. Sequences extracted for private alleles from each cultivar were imported into Agena Assay Designer software (AgenaCX). All proximal variants identi ed by Stacks were annotated onto the sequences, and preference was given to sequences with low degrees of variation. Standard design parameters were used except for the following changes to improve multiplexing: false priming threshold (0.8), primer dimer threshold (0.8), amplicon length variation (0.9), PCR primer T m variation (0.9), maximum pass iteration base (200). The design produced a single multiplex containing primer pairs and extension primers for 28 assays (Supplementary Material S1 and S2).
High-throughput genotyping was performed using the Agena MassARRAY platform (Agena Bioscience, San Diego, CA, USA) to assign paternity of avocado seeds. Brie y, the extracted avocado seed DNA (2 uL; 10 ng/ul) was ampli ed in 5 uL multiplex PCR reactions containing 1 U of Taq, 2.5 pmol of each PCR primer, and 500 µM of each dNTP (PCR Accessory and Enzyme Kit, Agena). Thermocycling was performed at 94 °C for 4 min followed by 45 cycles of 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min, and a nal extension at 72°C for 3 min. Unincorporated dNTPs were deactivated using 0.5 U of shrimp alkaline phosphatase (37 °C for 4 min, 85 °C for 5 min). Primer extension was initiated by adding 1.3 U of iPLEX GOLD, dideoxy nucleotide terminators and extension primers. The reaction conditions consisted of 95 °C for 30 s, 40 cycles of 95 °C for 5 s plus ve inner cycles of 52 °C for 5 s and 80 °C for 5 s, and a nal extension at 72 °C for 3 min. A cation exchange resin was added to remove residual salt, and 7 nL of the puri ed primer extension product was loaded onto the matrix pad of a SpectroCHIP (Agena) using an RS1000 nanodispenser. The extension products were analysed by matrix assisted laser desorption ionization-time of ight mass spectrometry (MALDI-TOF MS) using a MassARRAY Analyser 4 (Agena). Mass spectra (4300 to 9000 Daltons) were interpreted with TYPER 4.0 software (Agena) to identify the alleles and to genotype the samples. A total of 98% of samples could be assigned by mass array.
We cross-validated the mass array results with pre-published microsatellite markers 60,61 . Details on the microsatellite markers and laboratory protocols are presented in Supplementary Material S2. The microsatellite markers could only assign 74% of the samples, but in those cases, 98% agreed with the mass array assignment.

Statistical analyses
We calculated the proportions of cross-pollinated and self-pollinated fruit per tree. We used two-way ANOVA to test whether distance from a cross-pollen source (measured as number of rows) and orchard affected the proportion of cross-pollinated and self-pollinated fruit. The interaction between distance and orchard was not signi cant. We used linear mixed models with tree number, transect and orchard as random effects to test whether distance from a cross-pollen source, as a xed and categorical variable, affected fruit mass, esh mass, seed mass and seed proportion. Flesh mass was calculated by subtracting seed mass from the fruit mass, and seed proportion was calculated as the amount of fruit mass that consisted of seed mass. Tukey's all-pair comparisons tests were performed when differences were detected.
We also used linear mixed models with tree number, transect and orchard as random effects to compare size and nutritional quality between self-pollinated fruit and fruit that were cross-pollinated by the predominant polliniser, Shepard. We compared (i) fruit mass, (ii) esh mass, (iii) seed mass, (iv) seed proportion, (v) concentrations of each of 14 mineral nutrients, (vi) relative contributions of six fatty acids to the total fatty acid composition, (vii) relative contributions of saturated and unsaturated fatty acids to the total fatty acid composition, and (viii) ratio of unsaturated to saturated fatty acids. Data was logtransformed before analysis when necessary to achieve normal data distribution. Tukey's all-pair comparisons tests were performed when differences were detected.
Statistical analyses were performed using R version 3.1.1 for Macintosh OS X 62 . Mixed models were performed with the package 'lmerTest' and 'multcomp' in R 63 . Figure 1 Percentage of cross-pollinated and self-pollinated Hass avocado fruits at different numbers of rows from a cross-pollen source. Fruits were sampled along transects starting at trees adjacent to another cultivar (Row 1) and ending in the middle row of the Hass block ( Row 11,12 or 14). Means (+ SE) for crossparentage and self-parentage with different letters are signi cantly different (two-way ANOVA and Tukey's HSD; P < 0.05; n = 8).

Figure 1
Percentage of cross-pollinated and self-pollinated Hass avocado fruits at different numbers of rows from a cross-pollen source. Fruits were sampled along transects starting at trees adjacent to another cultivar (Row 1) and ending in the middle row of the Hass block ( Row 11,12 or 14). Means (+ SE) for crossparentage and self-parentage with different letters are signi cantly different (two-way ANOVA and Tukey's HSD; P < 0.05; n = 8).

Supplementary Files
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