Genetic diversity and phylogenetic analysis in Asian and European Asparagus subgenus species

Garden asparagus (Asparagus officinalis L.) is a diploid (2n = 2x = 20), perennial and dioecious species belonging to Asparagus subgenus and worldwide cultivated as a vegetable crop. A narrow genetic base has been pointed out for the current cultivars. Crop wild related species (CWR) could be a valuable genetic resource in this crop but they have been underused up to now. To investigate the phylogenetic relationships between CWR asparagus species from different origin and A. officinalis L., 12 EST-SSR markers were used to assess the genetic variability of 20 accessions. These accessions belong to 10 Asparagus spp. from Asparagus subgenus including wild and naturalized A. officinalis L. (2x, 4x, 8x, 10x) and CWR species with European (A. tenuifolius Lam. (2x), A. pseudoscaber Grec. (6x), A. macrorrhizus Pedrol & al. (12x), A. prostratus Dumort (4x), A. brachyphyllus Turcz. (6x), A. maritimus (L.) Mill. (6x)) and Asian distribution (A. verticillatus L. (2x), A. persicus Baker (2x), A. breslerianus Schult. & Schult. (8x)). A. albus L. (2x) from the Protasparagus subgenus was used as outgroup. As a result, a total of 248 alleles were obtained and specific alleles of accessions were detected among them. After cluster analysis the accessions did not group by their geographical origin. All wild polyploid accessions with European and Asian distributions were grouped together with A. officinalis L. Hence, that cluster could be considered as the ‘officinalis group’ suggesting a monophyletic origin. The diploid accessions of A. verticillatus L. and A. persicus Baker clustered together and were the most genetically distant respect to ‘officinalis group’. The results obtained in this study may provide useful information to design new crosses among accessions aimed to develop new asparagus germplasm or pre-breeding populations.


Introduction
Asparagus is a large genus comprising about 200 species of herbaceous perennials, tender woody shrubs and vines (Bailey 1942;Bozzini 1959;The plant List 2013). This genus is classified into three subgenera: Asparagus, Protasparagus and Myrsiphyllum (Obermeyer 1983(Obermeyer , 1984Clifford and Conran 1987). The species of the Asparagus subgenus are dioecious and mostly distributed in the temperate climate regions of Europe and Asia whereas the ones belonging to Protasparagus and Myrsiphyllum subgenera are hermaphroditic, and their distribution range encompass different regions of Africa, Oceania and Southern Asia and Europe (Kubota et al. 2012). Garden asparagus (Asparagus officinalis L.) is a dioecious and perennial species cultivated worldwide as a vegetable crop and is considered the most economically important species of the three subgenera. Other species of the Asparagus genus are cultivated or collected from their natural habitats for different uses such as food (A. acutifolius L., A. albus L., A. maritimus Mill.), ornamental (e.g., A. scandens Thunb., A. plumosus Baker, A. densiflorus (Kunth) Jessop, A. falcatus L.) or for their medicinal properties (e.g., A. cochinchinensis (Lour.) Merr., A. racemosus Willd.). The Asparagus genus has a wide range of ploidy levels (2x, 4x, 6x, 8x, 10x and 12x) and a basic chromosome number of x = 10. Also, intraspecific variation in ploidy levels has been detected in some species of this genus (Moreno et al. 2006;Kanno and Yokoyama 2011;Mousavizadeh et al. 2016). Thus, polyploidization seems to have played an important role in the evolution of the Asparagus genus (Castro et al. 2013).
Nearly all current cultivars of garden asparagus are diploid and derive from the Dutch population 'Violet Dutch' (eighteenth century) (Knaflewski 1996). Different studies employing molecular markers have revealed the narrow genetic base of this crop (Brettin and Sink 1992;Geoffriau et al. 1992;Khandka et al. 1996;Moreno et al. 2006) supporting its common origin. In the current climate change scenario, the development of new cultivars adapted to new environmental conditions and increasing the genetic diversity available in breeding programs are issues of the utmost importance to meet future environmental challenges (Galluzzi et al. 2020). Landraces and wild species may play a key role in broadening the genetic base of cultivated species (Zeven 1998;Dempewolf et al. 2014). Recently, a new approach called 'introgressiomics' has been proposed (Prohens et al. 2017). It consists of mass scale development of plant materials and populations with introgressions from crop wild relatives (CWR) into the genetic background of crops. The development of new germplasm is a major goal in asparagus breeding, and several studies have been performed to that end in the last years. For instance, Castro et al. (2013) developed a diploid population carrying introgressions from 'Morado de Huetor', which is a tetraploid landrace with an interspecific (A. officinalis L. x A. maritimus (L.) Mill.) origin (Moreno et al. 2008). Riccardi et al. (2011) used tetraploid plants derived from interspecific crosses between two wild species (A. acutifolius, A. maritimus) and one landrace ('Violetto d'Albenga') to obtain diphaploid plants by in vitro anther culture. However, the use of CWR in the development of new asparagus germplasm has largely remained unexploited. In this sense, wild relative species of the Asparagus subgenus could be a source of new alleles for the genetic improvement of yield, nutritional quality, adaptability, and resistance or tolerance to biotic or abiotic stresses affecting the asparagus crop (Kanno and Yokoyama 2011;Nothnagel et al. 2017;Jaramillo-Carmona et al. 2017). Extensive knowledge of the phylogenetic relationships between the wild species related to A. officinalis can be useful to guide the choice of the most suitable species to include in a breeding program. To date, the phylogenetic studies employing genetic resources of this crop have been mainly focused on landraces cultivated in the south of Europe ('Morado de Huétor', 'Violetto d'Albenga') and CWR species with European or Eurasian distribution such as A. prostratus Dumort, A. maritimus Mill., A. pseudoscaber Grec., A. tenuifolius Lam., A. macrorrhizus Pedrol and al. or A. brachyphyllus Turcz. (Stajner et al. 2002;Moreno et al. 2006Moreno et al. , 2008Castro et al. 2013).
The proposed centre of origin of garden asparagus includes eastern Europe, Caucasus, and Siberia (Sturtevant 1919;Ellison 1986). In these regions, other CWR species such as A. persicus Baker, A. breslerianus Schult. & Schult. or A. verticillatus L. and wild populations of A. officinalis L (Mousavizadeh et al. 2015) have been also described. Some Iranian populations of these species have been employed in genetic variability studies based on morphological traits and molecular markers (Sarabi et al. 2010;Mousavizadeh et al. 2015Mousavizadeh et al. , 2018. Phylogenetic studies using wild populations of A. officinalis and European and Asian wild species closely related to A. officinalis will provide new information useful for asparagus breeding and knowledge of the evolutionary pathway of Asparagus genus.
Simple sequence repeats (SSR) markers derived from genomic DNA are widely used for assessing genetic diversity and inferring phylogenetic relationships between closely related species or different populations from the same species (Simpson 2006). In Asparagus genus, SSR markers have been employed in different studies to assess the genetic variability and phylogenetic relationships of diverse genetic stocks such as current cultivars, landraces, wild species and introgressed populations (Caruso et al. 2008;Li et al. 2016;Castro et al. 2013Castro et al. , 2014. In previous studies, we analyzed the genetic variability of Asparagus spp. with European distribution (Castro et al. 2013) and Asparagus spp. from Iran (Mousavizadeh et al. 2018) using EST-SSR markers (Expressed Sequence Tag-derived Simple Sequence Repeats). To have a more comprehensive picture of the genetic variability and the relationships between the wild asparagus species, in this study, we have analyzed together the whole collection of wild asparagus species (European plus Near East accessions) using a higher number of EST-SSR markers than in previous studies.

Plant materials
Nineteen accessions from 10 Asparagus species of Asparagus subgenus and one accession (A. albus L.) from the Protasparagus subgenus were employed in this study. Those species include A. officinalis L.  (Table 1). A. albus L. was included as outgroup. The origin, ploidy level and number of plants studied per accession are detailed in Table 1. All the accessions except A. albus L. (ABB) and A. tenuifolius Lam. (ATU) are growing in an experimental field trial which contains the collection of genetic resources of the asparagus breeding program developed at the University of Cordoba.

EST-SSR markers analysis
Total genomic DNA of individual plants was isolated from 1 g of tips from young spears following a modified CTAB extraction protocol (Torres et al. 1993). The DNA was quantified by spectrophotometric absorbance at 260 nm (NanoDrop ND-1000; Thermo SCIENTIFIC, Waltham, MA). Two DNA bulks each composed of 4-5 individuals (depending on the number of plants available per accession) were made for all accessions but A. tenuifolius Lam. and A. officinalis (AOB). Each bulk was generated by pooling equal amount of DNA from each individual. There were 37 bulks total. Bulks were used for PCR amplification. A set of 12 EST-SSR markers developed in A. officinalis (Caruso et al. 2008;Mercati et al. 2013) were employed to analyze the genetic variability and relationships between accessions. The markers are distributed in 9 out of the 10 chromosomes of the basic chromosome number of this species . The forward primers of the markers (AAT1, AG2, AG7, AG8, TC1, TC3, TC7, TC9) developed by Caruso et al. (2008) were synthesized with fluorescent dyes (6-FAM or HEX) at the 5'-ends whereas the forward primers of the markers (asp_c12534, asp_c1367, asp_c17476 and asp_c5587) developed by Mercati et al. (2013) were synthesized with a 19 bp long M13 tail (5 0 -CACGACGTTG-TAAAACGAC-3 0 ) at the 5' ends for fluorescent labeling PCR fragments following Schuelke (2000). The PCR amplifications carried out with the markers developed by Mercati et al. (2013) were performed in a 10-ll reaction mixture containing 20 ng of bulked genomic DNA, 5X polymerase buffer, 2 mM MgCl 2 , 0.8 mM of dNTP, 0.125 lM of forward primer, 0.25 lM of reverse primer, 0.25 lM of the fluorescent labeled M13 primer (HEX or 6-FAM) and 1U of Taq polymerase (Promega). No fluorochrome M13 and equal amounts of both forward and reverse primers was added in the PCR reaction mix performed with the SSR markers developed by Caruso et al. (2008).
Amplification cycles were performed in a Perkin Elmer Cetus DNA Thermal Cycler (9600) programmed as follows: (A) 1 min at 94 8C for DNA denaturation, (B) 35 cycles with the following temperature profile: 94 8C for 1 min, 55 8C for 1 min and 72 8C for 1.5 min; (C) followed by a final extension at 72 8C for 10 min.
The PCR products were separated using an automated capillary sequencer (ABI 3130 Genetic Analyzer: Applied Biosystems/HITACHI, Madrid, Spain) in the Unit of Genomics of the Central Research Support Service at the University of Córdoba. The size of the amplified bands was calculated based on an internal DNA standard (400HD-ROX) with GeneScan software (version 3.x) and the results were interpreted using the Genotyper program (version 3.7) all from Applied Biosystems.

Phylogenetic and genetic diversity analyses
A large number of the accessions used in this study are polyploid, hence the difficulty to infer allele frequency. Therefore, for each marker the bands (putative alleles) obtained in each accession were scored as 1 (presence) or 0 (absence). The number of different alleles, specific accession alleles and polymorphic information content (PIC) were also computed. The PIC value of each marker was calculated as PIC = 1 -P _p j 2 , where p j is the band frequency of the jth allele in the examined bulk of plants. The marker data were also used to create a binary data matrix for the phylogenetic analysis. The Dice similarity index was used to calculate the genetic distance (1-Dice) among accessions. All the species analyzed are members of the Asparagus subgenus. Therefore, the assumption of a molecular clock was acceptable and the unweighted pair group method with arithmetic mean (UPGMA) was used to group the accessions. A dendrogram was obtained using the software NTSYS (Rohlf 2000). The correlation coefficient between the similarity matrix and the cophenetic value matrix was computed to test the goodness of fit of the cluster analysis.

Results and discussion
A total of 248 alleles were detected across all 20 asparagus accessions analyzed in this study using 12 EST-SSR markers. The allele sizes ranged from 118 (Asp_c5587) to 256 bp (AG8). The number of different alleles per marker varied from 4 (Asp_c5587) to 41 (TC9), with an average of 20.7 alleles per marker ( Table 2). The total number of alleles per accession varied from 17 (A. verticillatus L. (AVP)) to 88 (A. maritimus (L.) Mill.). The markers TC9, TC1 and AG7 did not show amplification in A. persicus Baker, A. verticillatus L. (AVP, AVH, AVP) and the wild A. officinalis accession (AOK), respectively. Two markers (TC7 and Asp_c12534) did not amplify in A. albus L. These results suggest that the transferability of the markers is high between Asparagus species. Sixtythree of the 248 alleles obtained were specific from accession. The number of specific alleles per marker ranged from 1 (Asp_c17476) to 16 (TC9). Specific alleles were found in 13 of the 20 accessions ranging from 1 to 8 alleles. A. albus L. and A. maritimus (L.) Mill. had the highest number of specific alleles whereas A. officinalis (AOT) had the lowest number ( Table 2). The specific alleles could be useful to verify the hybrid origin of plants derived from crosses among accessions included in this study. All EST-SSR except Asp_c5587, were considered informative markers (PIC [ 0.8) ( Table 2). The phylogenetic relationship among the species was investigated by cluster analysis. A cophenetic correlation of 0.89 was obtained, indicating a good fit between the tree and the similarity matrix. According to the dendrogram results, there are five different groups (Fig. 1). The first group is separated from all the others and contains just A. albus L. (2x), which belongs to Protasparagus subgenus and is considered as an outgroup. The remaining groups cluster together and are formed by the species of the Asparagus subgenus, showing a monophyletic origin like it has been reported by other authors (Fukuda et al. 2005; Kubota el at. 2012). All wild polyploid accessions with European or Asian distribution are grouped together with A. officinalis in the same cluster (group V), indicating that they are genetically related. Hence, that cluster could be considered as the 'officinalis group' previously reported by Castro et al. (2013). Within the 'officinalis group' the accessions did not group according to their geographical origin and seem to have a monophyletic origin within Asparagus subgenus. High crossability among hexaploid and tetraploid European accessions of this 'officinalis group', together with a regular meiotic behavior and high pollen and seed fertility was reported by these authors. The dodecaploid A. macrorrhizus Pedrol & al., an endemic species from the southeast of Spain growing near to the seaside, was previously classified as A. maritimus (L.) Mill. species (Sánchez-Gómez et al. 2007). Recent botanical studies catalogued this accession as a new species within the Asparagus subgenus (Pedrol et al. 2013;Regalado et al. 2017 (Amian et al. 2018), proving its close phylogenetic relationship with A. officinalis. Another polyploid species included in this group, the octoploid A. breslerianus Schult. & Schult. from Iran, grows in a warm and dry climate with a high salinity soil (Mousavizadeh et al. 2016). This species has been also successfully crossed with the landrace 'Morado de Huétor' (unpublished data). All these results support the hypothesis that the species clustered in group V could be in a recent process of speciation. As it has been suggested in previous work, polyploidization could be playing an important role in the evolutionary process of Asparagus subgenus (Castro et al. 2013). Unreduced gametes could explain the different polyploidy levels. This type of gametes has been described in A. officinalis (2x) (Camadro et al. 1992) and in the landrace 'Morado de Huétor' (4x) (Regalado et al. 2015). 'Morado de Huétor' has an interspecific origin and derives from diploid A. officinalis x hexaploid A. maritimus (L.) Mill. (Moreno et al. 2008). Also, introgression of this landrace into current diploid cultivars has been reported (Castro et al. 2014).  The number of specific alleles is shown in brackets The group formed by A. verticillatus L. and A. persicus Baker (group III) was the most genetically distant from the 'officinalis group'. This result could explain the unsuccessful crosses between A. verticillatus L. and A. officinalis reported up to now (Ito et al. 2008). To our knowledge, there is no information regarding the crossability between A. persicus Baker and A. officinalis. Resistance to Stemphilium vesicarium Wallr. has been reported in A. verticillatus L. (Kanno and Yokoyama 2011). Hence, a crossability study including A. persicus, A. verticillatus and diploid A. officinalis could be interesting in order to find out whether it is possible to transfer the resistance genes into the diploid current cultivars.
According to our results, A. tenuifolius Lam. (group III) seems to be closer to the 'officinalis group' in terms of evolution. Successful crosses between A. tenuifolius Lam. and A. officinalis were early reported by Bozzini (1963). Group IV is the closest to the 'officinalis group' and is formed by A. officinalis wild population (AOK). This accession shows a vining growth habit and has a different morphology compared with the rest of the group V (Mousavizadeh et al. 2018). Successful crosses between wild A. officinalis (AOK) and cultivated asparagus have been obtained by our research group (unpublished data).
The genetic relationships among accessions obtained in this study agree with previous genetic variability studies that used rDNA or cpDNA markers to analyze A. officinalis, A. verticillatus L., A. maritimus (L.) Mill. or A. pseudoscaber Grec. species, among other Asparagus spp. (Stajner et al. 2002;Ito et al. 2008;Fukuda et al. 2005;Kubota el at. 2012).
Several studies have pointed out the existence of wild and naturalized populations of A. officinalis (Stutervant 1919;Geoffriau et al. 1992;Mousavizadeh et al. 2015;Sarabi et al. 2010;Melyan et al. 2016). Besides, different species closely related to the crop distributed across different parts of Europe or Asia have been also described (Valdes et al. 1980;Kay et al. 2001;Komarov et al. 1935;Xinqi and Tamanian 2000). To date, there is no general agreement in the taxonomic classifications of the CWR species of A. officinalis. Some of them have been considered like subspecies of A. officinalis. In breeding, it is important to know the possibility of transferring genes from CWR to the crop. In this sense, Harlan and de Wet (1971) proposed a classification system for cultivated plants and its related species assigning taxa to primary, secondary, and tertiary pools. Because the polyploid nature of some accessions in the 'officinalis group', these could be classified in the secondary pools. Our findings support the results from previous studies performed by our group in which the European and Asian accessions were analyzed separately (Castro et al. 2013;Mousavizadeh et al. 2018). In this study, we have used both groups of accessions and a higher number of markers providing a clearest insight into the genetic relationships within the Asparagus subgenus. Most accessions are currently growing in a field ex-situ collection of asparagus genetic resources that is being used in our asparagus breeding program at the University of Cordoba (Spain). To create highly diverse introgressed populations, as proposed by Prohens et al. (2017), the specific alleles found in our study could be helpful to verify the hybrid origin of plants derived from crosses between wild relatives and the crop. The results obtained in the current study may provide useful information to design new crosses aimed to develop new asparagus germplasm or prebreeding populations.
Author contributions SJM, JG and RM conceived and designed the study; SJM and RM developed the plant material; SJM, PC and RM performed the DNA extraction, analyzed the EST-SSR data; SJM, JG and RM statistical analysis; SJM, JG and RM drafted the manuscript; PC and MRH revised the manuscript. All authors agree and approved the final version of the manuscript.
Funding This research was cofunded by two projects of Ministry of Economy and Competitiveness of Spain Kingdom, project number AGL2014-57575R and RFP2015-00005-00-00 both cofinanced by the EU through ERDF.

Declarations
Conflict of interest The authors declare no conflict of interest.
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