High cross-pollination rate of Greek oregano (O. vulgare ssp. hirtum) with Common oregano (O. vulgare ssp. vulgare) under open field conditions as revealed by microsatellite marker analysis

Abstract We studied the mode of pollination in Greek oregano (Origanum vulgare ssp. hirtum) under both controlled and open pollination conditions. When grown indoors without the presence of insects, Greek oregano plants did not develop any seeds, indicating a low level of spontaneous self-pollination. Applying manual self-pollination under the same conditions resulted in only 16 seeds, of which only five were able to germinate. At the same time, a clonally propagated Greek oregano plant of the same genotype produced a rich set of over 300 seeds in open field conditions when the flowers were visited by insects in an area where no other Origanum species were observed. Analysis with SSR markers showed that over 70% of the seeds likely resulted from self-pollination, indicating that insect-mediated pollination is essential for the seed development. We further analyzed the cross-pollination of Greek oregano with Common oregano (O. vulgare ssp. vulgare) in open field conditions where the two subspecies were grown in close proximity. Applying SSR markers, we analyzed 83 plants obtained from seeds of three vegetatively propagated Greek oregano mother plants. Surprisingly, the results showed that all analyzed seedlings resulted from cross-pollination of Greek oregano with Common oregano, indicating that cross-pollination between the two subspecies can completely take over the self-pollination or cross-pollination between the Greek oregano plants. The possible impact of the observed high cross-pollination rate on the genetic origin of seeds of selected Greek oregano lines and varieties, as well as on the genetic diversity and structure of natural populations, is discussed.


Introduction
Origanum is one of the widely known genera in the Lamiaceae family (subfamily Nepetoideae, tribe Mentheae, subtribe Menthineae).The genus consists of 44 species and 21 hybrids, among which are medicinal, fragrant, culinary and ornamental plants [1][2][3].Origanum vulgare L. is one of the most popular species of the genus Origanum, which has been used in traditional medicine since ancient times and is also an important spice in the Mediterranean cuisine.It is widely accepted that Origanum vulgare L. comprises six subspecies, among which are O. vulgare ssp.vulgare and O. vulgare ssp.hirtum.O. vulgare ssp.vulgare, or Common oregano, is widely distributed in Europe and Asia and has relatively low essential oil content, whereas O. vulgare ssp.hirtum, also known as Greek oregano, grows on the Balkan Peninsula and in Turkey and produces high quality essential oil [3][4][5].In Bulgaria, Greek oregano grows only in the southernmost parts of the country: in the Eastern part of the Rhodope Mountains and in the Kresna Gorge in the Struma River Valley [6,7].
Hybridization is considered an important driver of diversity in plants.It has played a role in plant domestication and has also been used in plant breeding.Hybridization can have immediate phenotypic consequences, or over longer evolutionary timescales, hybridization can lead to local adaption through the introgression of novel alleles and transgressive segregation, and in some cases result in the formation of new hybrid taxa [8,9].Interspecific hybridization under natural conditions was observed for several species in the genus Origanum.The studies of natural interspecific hybrids of O. vulgare ssp.hirtum demonstrate that hybridization occurs relatively often when the species distribution overlaps, and also that hybridization has played a major role in the process of speciation of the genus [10][11][12][13].The intraspecific hybridization between subspecies of oregano species is not well studied, but is thought to occur frequently and play a significant role in the genetic diversity and population structure of different Origanum species [1].
In the present study, we evaluate the rate of intraspecific cross-pollination under natural open field conditions between the two of the most popular representatives of the Origanum vulgare species, the essential oil-rich O. vulgare ssp.hirtum (Greek oregano) and the more widespread O. vulgare ssp.vulgare (Common oregano) using highly polymorphic SSR markers recently developed for the genus Origanum [14].Furthermore, the study presents information on spontaneous and open field self-pollination of Greek oregano plants.

Plant material
Vegetatively propagated plants of O. vulgare ssp.hirtum line BR19 were used in the study.Line BR19 originated from а plant from a natural population of the species located in the Eastern Rhodopes region, Bulgaria.The original plant was collected in 2019 and its taxonomic affiliation was determined following DNA barcoding with sequences from the chloroplast genome including the rubisco large subunit (rbcL), the gene for maturase K (matK), the intergenic spacer trnH-psbA as well as by visual assessment of the plant appearance.The BR19 plants were propagated vegetatively by rhizome division, grown in soil containers and further used in the study.Seeds of O. vulgare ssp.vulgare plants were purchased from Florian Ltd. (Bulgaria) and were used to grow a 3 × 0.5 m bed of Common oregano in the experimental field of the AgroBioInstitute (ABI) near the town of Kostinbrod (Bulgaria) during the 2022 growing season.

Self-pollination in insect-free environment
Two BR19 plants were grown in a soil container placed in an insect-proof room under natural light and growing conditions with periodic irrigation.The experiment was carried out at the experimental station of ABI (Kostinbrod, Bulgaria) during the 2021 growing season.One of the plants was left to self-pollinate spontaneously.Тhe flowers of the second plant were periodically hand-pollinated using fine tweezers.

Self-pollination test under open field conditions
A single BR19 plant was grown in a garden near the village of Zagore (Bulgaria) during the 2022 growing season, with no other Origanum plants growing in the same area.The plant was irrigated periodically and the flowers were allowed to be visited by insects.

Cross-pollination under open field conditions
Three vegetatively propagated BR19 plants grown in soil containers were placed next to the bed of O. vulgare ssp.vulgare plants, grown in the experimental field of ABI, (Kostinbrod, Bulgaria) during the 2022 growing season.The plants were irrigated periodically and allowed to pollinate under natural conditions.

Seed collection and germination
All flowers produced by the BR19 plants grown within the three pollination experiments were collected at the end of the season and the formation of seeds was evaluated and seeds were collected for further germination.The obtained seeds were germinated on a cotton gauze moistened with aqueous solution of 500 ppm gibberellic acid (GA3) in Petri dishes at room temperature.After approximately 1 week, the seedlings were transplanted into peat pellets and further grown in a Sanyo MLR-351 plant growth chamber under the following conditions: 16 h of light at 22 °C and 8 h of darkness at 18 °C.After reaching 1.5-2 cm height, the plantlets were transferred to seedling trays filled with a 2:1 mixture of soil and perlite.The plants were grown in а greenhouse to an approximate height of 10 cm, when one leaf from each plant was collected and immediately frozen in a plastic container for isolation of genomic DNA.

DNA isolation
The frozen leaf samples were ground to a fine powder using liquid nitrogen and a TissueLyser (Qiagen) laboratory mill.Genomic DNA was purified according to the CTAB protocol [15].Genomic DNA concentration was measured spectrophotometrically using Nanodrop 2000 (Thermo Scientific) and diluted to a final concentration of 25 ng/µL with Type I ultrapure water.

PCR amplification and analysis of SSR markers
Six primer pairs shown in Table 1 (R-6M, R-38C, R-40C, R-103C, R-105C and R-115C) were used for PCR amplification of SSR markers as previously described [14].Two different types of tails were added at the 5′ end to each forward primer based on the calculated melting temperature of the respective reverse primer.The tails used were Tail C (5′-CAGGACCAGGCTACCGTG-3′) and Tail M13 (5′-GTAAAACGACGGCCAGT-3′) [16,17].Tail C was used when the Tm of the reverse primer was > 58 °C.Tail M13 was used when the Tm of the reverse primer was ≤ 58 °C.Based on the respective tail used, the annealing temperatures of the PCR reactions were 57 °C for Tail C and 54 °C for Tail M13.The PCR reactions were performed in a volume of 16 µL, containing 0.8 µL of forward primer (3 pmol/µL), 1 µL of Tail primer (10 pmol/µL) labelled with FAM, 1 µL of reverse primer (10 pmol/µL), 8 µL 2× MyTaqTM Mix (Bioline), 4 µL ultra-pure water and 1.3 µL genomic DNA (total amount of 32.5 ng) using the following PCR conditions: 95 °C for 3 min followed by 33 cycles of 95 °C for 15 s, T annealing (Table 1) for 30 s, 72 °C for 30 s and final elongation at 72 °C for 10 min, as described [14].Fragment analysis was performed on an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, uSA) using 36-cm long capillaries (Thermo Fisher Scientific, Waltham, MA, uSA), POP-7 polymer (Thermo Fisher Scientific, Waltham, MA, uSA) and GeneScan TM 500 LIZ TM as a size standard (Thermo Fisher Scientific, Waltham, MA, uSA).GeneMapper 4.0 (Thermo Fisher Scientific, Waltham, MA, uSA) was used for fragment sizing and alleles were reported as base pairs.IDENTITy 1.0 [16] was used for parental analysis based on allele configurations.

Self-pollination of Greek oregano plants
The self-pollination of the Greek oregano line BR19 was tested under two different conditions.First, a BR19 plant was allowed to self-pollinate spontaneously after growing in an insect-proof room, and in the second experiment, a BR19 plant was grown in open field conditions with no other plants of oregano species detected in the nearby area.The closer observation of the flowers collected from the BR19 plant grown in the insect-proof room did not show any seed formation, suggesting an absence of spontaneous self-pollination.Furthermore, the repeated hand pollination of the flowers of a second BR19 plant grown in parallel in the insect-proof room, showed the formation of only 16 seeds, of which only 5 germinated further.
In a second type of pollination condition experiment, a single BR19 plant was grown under open field conditions with no other Origanum species detected in the nearby area.Periodic observations showed that this plant attracted a large number of different insect pollinators to land on the open flowers during the flowering period.This resulted in the formation of a large seed set with over 300 seeds and germination efficiency of over 60%.The seedlings produced from the germinated seeds were used to grow a total of 183 plants, potentially resulting from insect-mediated self-pollination of the BR19 plant.The further genotyping of these plants with six highly polymorphic SSR markers and the comparison of the obtained data with the corresponding SSR profile of the BR19 plant suggested that the large majority, 129 out of 183 (70.5%) of these plants, were the result of self-pollination.The SSR profiles of the remaining 54 plants showed SSR alleles that were not present in the BR19 line and most likely resulted from cross-pollination with unknown Table 1.SSR primer pairs used in the present study.Bold letters indicate tails added to the forward primer (tail m13 -5′-gtaaaacgacggccagt-3′ and tail c -5′-caggaccaggctaccgtg-3′), ta -annealing temperature.

SSR locus
Repeat motif primer Origanum plants, through long-distance pollen transfer carried by insect pollinators.
Sarrou et al. [17] managed to produce seeds by self-pollination of Greek oregano plants following application of paper bag insulators on the flowers in open field conditions [17].However, the authors did not report the number of seeds produced by self-pollination in comparison to the seeds produced by open pollination from the same plants, which hampers the comparison of data between the two studies.The results from the present study suggest that the presence of insect pollinators is essential for the efficient self-pollination of Greek oregano plants.A similar absence of spontaneous self-pollination as a result of pollinator exclusion by insect insulators placed on flowers has been reported for other Lamiaceae species, including Lavandula angustifolia [18], Sideritis scardica [19].The results further confirm the belonging of Origanum vulgare plants to the vast group of angiosperms whose pollination is mainly mediated by insect pollinators [20].

Open field cross-pollination of Greek oregano with Common oregano plants
In the second part of the study, the efficiency of open field cross-pollination of Greek and Common oregano plants was evaluated by growing the two subspecies in close proximity, placing three soil containers with vegetatively propagated BR19 (O.vulgare ssp.hirtum) plants next to a bed of Common oregano (O.vulgare ssp.vulgare) plants.The observation of the flower fruit set of the BR19 plants after the end of the flowering season showed that each plant produced a substantial seed set of over 500 seeds per plant.Some of the seeds collected from each plant were germinated and used to grow plants.In order to understand the origin of these plants, 83 of them were genotyped at the six highly polymorphic SSR loci.The allele sizes of all six SSR markers were scored manually for each plant and compared with the allele profile of the BR19 mother plant.Surprisingly, all 83 analyzed plants possessed alleles that differed and did not originate from the BR19 mother plant.This suggested that all analyzed plants did not originate from self-pollination or cross-pollination between the three BR19 plants (Supplemental Table S1), and most likely resulted from cross-pollination with neighboring Common oregano plants.To test the latter, we genotyped seven randomly chosen Common oregano plants that were in close proximity to the Greek oregano mother plants at the six SSR loci and compared their alleles with those of the 83 plants.Analysis of the obtained data using the Identity 1.0 software showed that the allele profiles of 32 of the progeny plants could be explained as a result of hybridization between the BR19 mother plants and one of the seven randomly chosen Common oregano plants.This is most likely a consequence of the insect-mediated pattern of flower pollination observed above for the BR19 line (O.vulgare ssp.hirtum), suggesting a high probability that pollinators transfer pollen between neighboring plants.Considering that no other flowering Origanum sp.plants were present in the nearby area, it is possible to speculate that all analyzed plants from the BR19 seed progeny were the result of insect-mediated pollination with pollen from Common oregano plants grown in the plant bed next to the three containers of BR19 Greek oregano plants.Thus, the obtained results clearly showed that, under open field conditions, the cross-pollination of Greek oregano with pollen of Common oregano occurs at a very high rate and can completely take over the self-pollination or cross-pollination between vegetatively propagated Greek oregano plants.The results further support the earlier suggestion that the intraspecific hybridization between subspecies of Origanum species occurs frequently and plays a significant role for the genetic diversity and population structure of different Origanum species [1].
Considering the increased interest in breeding and semi-industrial scale cultivation of Greek oregano, the results of the present study could be considered in several practical applications related to oregano breeding, cultivation, genetic resources management and biodiversity of natural populations, as follows: • The results of self-pollination tests without the presence of insects suggest that spontaneous or mechanical self-pollination is unlikely to be successfully applied to generate large seed sets and populations of self-pollinated breeding lines required for use in oregano breeding.

•
The efficient insect-mediated self-pollination of the BR19 line suggests that such pollination could be used to generate large seed sets and segregating populations of selected breeding lines if they are grown under open field conditions without other oregano plants present in the nearby areas or when grown indoors using dedicated greenhouse pollinators.

•
The observed high rate of cross-pollination, taken together with the possible long-distance pollen transfer by insects, suggests that seed propagation of elite breeding lines and varieties should be done carefully or better avoided and their propagation should be carried out vegetatively in order to preserve their important agronomic traits.

•
The above is particularly important for the production of planting material from seeds, which, due to the high rate of cross-pollination observed in oregano, could lead to deterioration in the quality of the new plantations due to segregation of traits as a result of cross-subspecies hybridization.Accordingly, vegetative propagation of superior O. vulgare ssp.hirtum breeding lines and varieties should preferably be used in the production of planting material for the development of new plantations.

•
Growing larger plantations of oregano varieties close to natural oregano populations should be avoided in order to prevent large-scale pollen transfer and cross-pollination of naturally grown plants with the cultivated varieties, which could lead to significant reduction in the genetic diversity and changes in the structure of natural populations.

Conclusions
The results of our study showed that the presence of insect pollinators is essential for the course of both self-and cross-pollination in Greek oregano.The generation of large seed sets in breeding programs should be assisted by the use of insect pollinators in dedicated closed greenhouses.The observed high rate of cross-pollination of Greek oregano with Common oregano in our study points towards several important considerations which should be taken into account in breeding programs of Greek oregano.The utilization of seeds for the propagation of planting material of elite varieties should be done carefully or better avoided and the planting material should preferably be produced by vegetative propagation due to the high rate of cross-subspecies hybridization.Growing large oregano plantations next to natural populations could result in changes in the genetic structure of natural populations due to the high rate of exchange of genetic material.