C. chinense as genetic bridge species.

Despite a high crossability between the species of the annuum complex has been widely assumed [6, 8, 9], many crosses of C. annuum accessions with both C. chinense or C. frutescens were unsuccessful (Fig 2). Thus, in the C. annuum (♀) × C. chinense (♂) cross scheme, fruit set occurred in 10 of the 24 possible combinations and only three C. annuum accessions (California Wr. red, Bola and Guindilla) were able to set fruit with any of the C. chinense accessions used (Fig 2). Furthermore, only the seeds of four combinations germinated and developed in normal hybrids, which also showed pollen viability estimates comprised between 17–31% (Table 3), considerably lower than those from their parent accessions, always higher than 80% (Table 1). Capsicum annuum accession Bola was the only one which produced hybrid plants with both C. chinense accessions (Table 3). Moreover, one of these (Bola × P15) was the hybrid with the highest pollen viability (31%). Therefore, this accession appears as suitable germplasm for the GB strategy, as well as for the transfer of genes from C. chinense to commercial peppers. Although Zijlstra et al. [11] discarded prezygotic barriers between species belonging to the annuum complex, the low or even nil fruit set rates obtained in our experiment suggest strong prezygotic incompatibility between C. annuum and C. chinense. Even more, as observed in some cases by Inai et al. [39], our low germination rates could be due to embryo abortion, which also suggest postzygotic barriers.

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larger image TIFF original image Download: Fig 2. Cross diagram showing the results of the different crosses performed within the genetic bridge approach starting with crosses between C. annuum and the bridge species. (A) First cross table indicates the crossability degree of C. annuum with C. chinense or C. frutescens, (B) Second cross table indicates the crossability degree of hybrids obtained in A with C. baccatum. Numbers over the cells indicate percentage of fruit set over an average of 25 artificial pollinations. Green indicates non-viable crosses, where no fruits were obtained (0% of fruits set, not indicated as number); blue indicates fruit set but non-viable seeds; red indicates fertile hybrids with normal development; and grey indicates hybrid inviable plants. https://doi.org/10.1371/journal.pone.0144142.g002

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larger image TIFF original image Download: Table 3. Descriptive results of the genetic bridge (GB) strategy using the alternative of obtaining hybrids between C. annuum (Ca) and C. chinense (Cch) for being subsequently crossed with C. baccatum to obtain three-way hybrids. Cross combinations that did not set fruit or set fruit but seeds did not germinate are not included in the table. https://doi.org/10.1371/journal.pone.0144142.t003

The reciprocal cross C. chinense (♀) × C. annuum (♂) enabled a higher number of successful combinations in terms of fruit set, with 16 out of the 24 possible combinations (Fig 2). However, only hybrid seeds from four combinations germinated (Table 3) and, moreover, hybrids showed stunted growth, filiform leaves, short internodes and did not enter into the reproductive phase (Fig 3). These symptoms were not observed in other plants grown in the same trial and all samples were negative to ELISA tests for Tobamoviruses or tomato spotted wilt virus (TSWV). Thus, the most likely reason for this abnormal development was the virus-like syndrome (VLS), which might be due to the interaction between cytoplasm and nuclear genes of C. chinense and C. annuum respectively [6, 39]. Therefore, the results suggest that C. chinense should always be utilised as male parent to achieve viable hybrids with C. annuum.

The four viable C. annuum (♀) × C. chinense (♂) hybrids available were then utilized to complete the bridge cross with C. baccatum. Crosses involving C. baccatum Aji amarillo did not set any fruit (Fig 2), suggesting a low cross compatibility of this accession. Then, regarding the rest of combinations, the best results were obtained using hybrids as female parents, with seven out of the eight possible combinations setting fruit, and viable seeds being obtained from six combinations (Fig 2 and Table 3). All these materials showed a normal appearance, although a wide range of pollen viability was observed, which was comprised between 4% and 83% in (Pas × PI15) (♀) × BrP (♀) and (Bola × AjíP) (♀) × AjíR (♂) respectively (Table 3). On the contrary, when C. annuum (♀) × C. chinense (♂) hybrids were utilized as pollen donors, fruit set and successful hybrid plants were only achieved in AjíR (♀) × (Bola × AjíP) (♂) (Fig 2). Surprisingly, this hybrid showed the highest pollen viability (95%) among all hybrid materials of the GB approach involving C. chinense as bridge species, which suggests that the involved genotypes Bola (C. annuum), AjíP (C. chinense), and AjíR (C. baccatum) have a high compatibility. Consequently, given the number of successful combinations (6/8), the following scheme [C. annuum (♀) × C. chinense (♂)] (♀) × C. baccatum (♂) is recommended when using the GB approach.

Regarding the bridge cross strategy that involved C. baccatum to obtain the first set of interspecific hybrids, it was possible to achieve normal C. baccatum (♀) × C. chinense (♂) hybrids from all combinations, with the only exception of those combinations involving the Ají Amarillo accession (C. baccatum), which did not to set any fruit (Fig 4) and confirmed its low aptitude for interspecific crosses. Despite these hybrids had low pollen viability (19–33%) (Table 4), similarly to C. annuum (♀) × C. chinense (♂) materials, it was possible to obtain subsequent crosses with C. annuum.

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larger image TIFF original image Download: Fig 4. Cross diagram showing the results of the different crosses performed within the genetic bridge approach starting with crosses between C. baccatum and the bridge species. (A) First cross tables indicate the crossability degree of C. baccatum with C. chinense and C. frutescens, (B) Second cross tables indicate the crossability degree of the hybrids obtained with C. annuum. Numbers over the cells indicate percentage of fruit set over an average of 25 artificial pollinations. Green indicates non-viable crosses, where no fruits were obtained (0% of fruits set, not indicated as number); blue indicates fruit set but non-viable seeds; red indicates fertile hybrids with normal development; and grey indicates hybrid inviable plants. https://doi.org/10.1371/journal.pone.0144142.g004

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larger image TIFF original image Download: Table 4. Descriptive results of the genetic bridge (GB) strategy using the alternative of obtaining hybrids between C. baccatum (Cb) and C. chinense (Cch) for being subsequently crossed with C. annuum to obtain three-way hybrids. Cross combinations that did not set fruit or set fruit but seeds did not germinate are not included in the table. https://doi.org/10.1371/journal.pone.0144142.t004

By contrast, within the reciprocal crosses C. chinense (♀) × C. baccatum (♂), fruit set and viable seeds were observed in all possible combinations (Fig 4). Unfortunately, as observed in C. chinense (♀) × C. annuum (♂) plants, all these hybrids also showed VLS, indicating the presence of detrimental genes in C. chinense cytoplasm that may also induces detrimental effects in hybrids with C. baccatum (Table 4). This is consistent with other works related to interspecific hybridization in pepper [6, 7, 39, 40]. Therefore, as suggested for crosses with C. annuum, C. chinense should be also utilized as pollen donor in crosses with C. baccatum to prevent detrimental effects in the hybrid offspring.

Finally, the use of C. baccatum (♀) × C. chinense (♂) hybrids as male parents for finishing the GB approach only succeed in 1 out of 48 possible combinations (and in one single plant), which also showed a 3% pollen viability (Fig 4, Table 4). Such findings are in agreement with those observed in the C. baccatum (♀) × [C. annuum (♀) × C. chinense (♂)](♂) scheme, confirming that due to the frequently low pollen viability of both interspecific hybrids, C. annuum (♀) × C. chinense (♂) and C. baccatum (♀) × C. chinense (♂), they should not be used as male parents. On the contrary, the best results were achieved utilizing C. baccatum (♀) × C. chinense (♂) hybrids as pistillate parent, which allowed finishing the GB in 15 out of the 48 possible combinations (Fig 4 and Table 4). In addition, many of these hybrids showed high levels of pollen viability (>50%). Therefore, this alternative is more efficient for the GB when starting the cross scheme crossing C. baccatum with C. chinense.

As a whole, the comparison between the different alternatives for GB showed that the use of [C. annuum (♀) × C. chinense (♂)] (♀) × C. baccatum (♂) crossing schemes allowed obtaining plants with a range of pollen viability (4–83%) in six cross combinations. The use of the [C. baccatum (♀) × C. chinense (♂)] (♀) × C. annuum (♂) scheme provided 13 fertile combinations with up to 94% pollen viability. Therefore, according to these findings, we recommend to utilize the latter alternative to introgress genes from C. baccatum to C. annuum. Moreover, this strategy provides 50% C. annuum genome, while three-way hybrids from the other scheme will only carry 25% of C. annuum genome. Nevertheless, it should be also considered that this strategy, depending on the character to introgress, may require checking the resulting phenotypes at the first step from crosses between C. baccatum and C. chinense before performing the second cross involved in the bridge cross. In fact, Yoon and Park (2005) [12] utilized the other alternative, despite they also found low pollen viability.