The following plant materials were used in this study: A. officinalis ‘Mary Washington 500W’; purple asparagus cultivar ‘Pacific Purple’, ‘Purple Passion’, NJ1064 (an all-male strain), and ‘Erasmus’ (an all-male cultivar, provided by Bejo Japan KK); and A. maritimus. The list of these samples were shown in Table 1 . These plants were cultivated in a greenhouse at the Graduate School of Life Sciences, Tohoku University, Japan.

Total DNA was extracted from asparagus cladodes as described previously (Honda and Hirai, 1990). For PCR analysis, two sets of male-specific primers were used: AspMSD-fw and AspMSD-rv, and MspHd-fw and MspHd-rv published in Mitoma et al. (2018). These primer sets amplify 0.55 kb and 1.1 kb fragments, respectively. AODEFint4fw and AODEFint4rv, which amplify 0.5 kb of the intron of AODEF gene, was used for positive control as described previously (Kubota et al., 2012). For indel specific PCR, we used MSE1-InDel-fw and MSE1-InDel-rv primers. The primers used in this study were listed in Table S1 .

PCR was performed in a volume of 25 μl reaction mixture containing 50 ng total DNA and 50 pmol each primer using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) with a TaKaRa PCR Thermal Cycler Dice (TaKaRa, Shiga, Japan). The PCR reactions were denatured for 2 min at 94°C, followed by 30 cycles of 30 sec at 94°C, 30 sec at 58°C, and 1 min at 72°C, with a final cycle of 72°C for 10 min. Subsquently, gel electrophoresis for the amplified DNA fragments were performed with 0.9% agarose gels in 1×TAE buffer. After stained with ethidium bromide, the gels were observed and photographed under UV light.

Total DNA was extracted from cladodes as described above (Honda and Hirai, 1990). MSE1-fw and MSE1-rv were used for cloning the full-length of genomic clone of AspTDF1/MSE1 gene. The primers used in this study were listed in Table S1 . For genomic PCR, TaKaRa ExTaq polymerase (TaKaRa, Japan) was used in a volume of 25 µl reaction mixture containing 50 ng of total DNA, 0.2 mM of each dNTPs, 1 × Ex Taq buffer, 0.5 units of TaKaRa ExTaq polymerase, and 0.5 µM of each primer with a TaKaRa PCR Thermal Cycler Dice (TaKaRa). The PCR consisted of an denaturation step for 2 min at 96°C, followed by 30 cycles of 30 sec at 96°C, 30 sec at 60°C, and 2.5 min at 72°C, with a final cycle of 72°C for 10 min. The PCR products were cloned into pGEM-T Easy vector (Promega, USA) and sequenced using an Applied Biosystems Big Dye Terminator V3.1 (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Phylogenetic analysis was conducted using MEGA11 software (Tamura et al., 2021). Full-length predicted amino acid sequences and nucleotide sequences were aligned by MEGA. The phylogenetic tree was constructed using Neighbor joining method and a bootstrap consensus tree was inferred from 1,000 replicates (Felsenstein, 1985).

To date, several dominant sex identification markers, such as AspMSD, MSSTS710, Asp1-T7sp and MspHd, have been developed and all of these markers were applicable to identify the sex of many cultivars in A. officinalis ( Figure 1A ; Nakayama et al., 2006; Kanno et al., 2014; Mitoma et al., 2018). Among these markers, the applicability of AspMSD, MSSTS710 and Asp1-T7sp markers were analyzed in other Asparagus species (Kubota et al., 2012; Mitoma et al., 2018; Kanno et al., 2020). Here we analyzed the applicability of MspHd marker for the sex identification in PP-m and PP-m* of ‘Pacific Purple’, including A. officinalis and A. maritimus. Although the applicability of MspHd marker in ‘Pacific Purple’ was previously reported (Mitoma et al., 2018), we added A. maritimus for this analysis because RAPD analysis has revealed that VA might originated from cross hybridization between A. officinalis and related species such as A. maritimus (Moreno et al., 2008a).

Total DNA was extracted from male and female individuals of A. officinalis, A. maritimus and ‘Pacific Purple’ including two types of males (PP-m and PP-m*). PCR analysis with these total DNAs as template was performed using AspMSD and MspHd markers, as well as AODEFint4 primer set for positive control (Kubota et al., 2012). Using AspMSD marker, PCR products were observed in all male individuals of A. officinalis, A. maritimus and ‘Pacific Purple’, while no amplification was detected in female individuals of these species ( Figure 1B ). Meanwhile, PCR fragment was detected only in male individuals of A. officinalis and PP-m and there was no amplification in PP-m*, male of A. maritimus and all female individuals by MspHd marker ( Figure 1B ). The applicability of other sex identification markers, MSSTS710 and Asp1-T7sp, in Asparagus species have already been reported (Kanno et al., 2014; Mitoma et al., 2018; Kanno et al., 2020), and these data were summarized in Table 2 .

In order to analyze the genetic polymorphism in the non-recombining Y region of PP-m and PP-m*, AspMSD marker region can be used to compare the sequences among A. officinalis, A. maritimus and ‘Pacific Purple’. Since AspMSD marker was created based on the sequence of MSE1/AoMYB35/AspTDF1 gene, the genomic sequence of this gene was analyzed among several purple asparagus cultivars as well as A. officinalis and A. maritimus. Total DNAs were extracted from two male individuals of each purple asparagus cultivars (PP-m and PP-m* of ‘Pacific Purple’, ‘Purple Passion’, ‘Erasmus’ and NJ1064), A. officinalis and A. maritimus. We also used A. cochinchinensis for the outgroup of phylogenetic analysis and these samples were listed in Table 1 . After PCR amplification with gene specific primers, amplified fragments were cloned into plasmid vector and sequenced. To understand the phylogenetic relationship of MSE1/AoMYB35/AspTDF1 gene among asparagus cultivars and related species shown above, deduced amino acid sequences were used to construct the phylogenetic tree. As shown in Figure 2A , PP-m* of ‘Pacific Purple’ belongs to the same clade of A. maritimus, and the other purple asparagus cultivars such as PP-m of ‘Pacific Purple’, ‘Purple Passion’, ‘Erasmus’ and NJ1064 were in the same clade of A. officinalis. This indicates that the non-recombining Y region of PP-m and PP-m* from ‘Pacific Purple’ is likely to have a high homology with that from A. officinalis and A. maritimus, respectively. Furthermore, comparison of deduced amino acid sequences showed that the MSE1/AoMYB35/AspTDF1 sequence from PP-m* of ‘Pacific Purple’ was very similar to that from A. maritimus (or its closely related species) and that gene from the other purple asparagus cultivars was very similar to that from A. officinalis ( Figure 2B ).

In addition, the insertion and deletion (indel) was observed in the intron region of the MSE1/AoMYB35/AspTDF1 gene ( Figure 3A ). The size of indel was 55bp and the intron size of PP-m* of ‘Pacific Purple’ and A. maritimus was shorter than that of PP-m and A. officinalis. The size and the position of indel was completely the same in PP-m* of ‘Pacific Purple’ and A. maritimus, and also in PP-m and A. officinalis ( Figure 3A ). To analyze this indel polymorphism among ‘Pacific Purple’, we isolated total DNAs from 13 PP-m male individuals, 26 PP-m* individuals and 6 female individuals of ‘Pacific Purple’. PCR amplification was performed with the primers which amplify the indel region. As shown in Figure 3B , one PCR fragment was detected in PP-m and PP-m* and the size of the fragment of PP-m* is shorter than that of PP-m, while no PCR fragment was detected in female.

In order to detect the sex ratio of ‘Pacific Purple’, male individuals of PP-m (MM029M) and PP-m* (MM060M) were selected to cross with female individuals (MM001F and MM018F). The sex of the progeny generated these crosses were analyzed with AspMSD marker. As shown in Table 3 , we got 92 progeny from MM018F x MM060M (PP-m*) and the number of male and female was 52 and 40, respectively. From MM001F x MM029M (PP-m), we got 105 progeny and the number of male and female was 62 and 43, respectively. This result showed that the sex ratio of ‘Pacific Purple’ was male:female = 1:1.

We analyzed the applicability of sex identification markers among A. officinalis, A. maritimus, and ‘Pacific Purple’ ( Figure 1B ; Table 2 ). AspMSD marker is applicable for all of these species, while MspHd and MSSTS710 were applicable in A. officinalis and PP-m of ‘Pacific Purple’, and not in PP-m* and A. maritimus. These results indicated that the sequence homology of these marker regions is different, although these markers are located on the non-recombining Y region ( Figure 1A ). AspMSD marker was generated using the exon sequence of MSE1/AoMYB35/AspTDF1 gene ( Figure 1A ; Mitoma et al., 2018). Therefore, the sequence within the MSE1/AoMYB35/AspTDF1 gene is probably conserved among A. officinalis, A. maritimus and ‘Pacific Purple’. On the other hand, the sequence around MspHd and MSSTS710 markers, which are located on the intergenetic regions, do not appear to be conserved.

It is interesting to note that the applicability of Asp1-T7sp marker is different in PP-m* of ‘Pacific Purple’ and A. maritimus: Asp1-T7sp marker is applicable in A. maritimus but not in PP-m* ( Table 2 ). Asp1-T7sp marker can be used to identify the sex of various cultivars of A. officinalis and several dioecious Asparagus species, such as A. kiusianus, A. maritimus, A. pseudoscaber and A. schoberioides (Nakayama et al., 2006; Kubota et al., 2012; Kanno et al., 2020). Furthermore, this marker is applicable in ‘Morado de Huetor’ (MH), which is tetraploid landrace in Spain (Regalado et al., 2014; Regalado et al., 2016). ‘Pacific Purple’ was bred from VA, tetraploid landrace in Italy, and VA and MH are likely an interspecific hybrid between A. officinalis and A. maritimus (Falloon and Andersen, 1999; Moreno et al., 2008a). In addition, Asp1-T7sp marker region is located on just downstream of SOFF gene ( Figure 1A ). This indicated that the sequence around Asp1-T7sp marker is well conserved among the cultivars of A. officinalis and related species including A. maritimus. Since Asp1-T7sp marker cannot amplify the male-specific fragment in PP-m*, there might be some mutation and/or rearrangement occurred at the primer sequence of Asp1-T7sp marker in PP-m* of ‘Pacific Purple’. Difference of the applicability of Asp1-T7sp and MspHd markers for A. maritimus is also interesting because the position of Asp1-T7sp marker is located within MspHd marker region ( Figure 1A ; Mitoma et al., 2018). Further analysis comparing the sequence around MspHd marker region is necessary to clarify the genetic variation of the non-recombining Y region among ‘Pacific Purple’, A. officinalis and A. maritimus.

Recently, we developed SSM01 marker, which is able to distinguish between X and Y sex genotypes among A. officinalis, A. maritimus and ‘Pacific Purple’, including PP-m and PP-m* (Akahori and Kanno, 2022). The sequence of SSM01 marker region have previously been determined in these Asparagus species (Akahori and Kanno, 2022). However, due to the high similarity of their sequences, their phylogenetic relationship could not be clarified. SSM01 marker is located at the end of the non-recombining Y region although this marker is far from two sex determination genes ( Figure 1A ; Akahori and Kanno, 2022). Highly conserved SSM01 region may have some function in asparagus, but further analysis is needed.

In diploid asparagus, the genotype of male and female is known as [Mm] and [mm], respectively (Rick and Hanna, 1943; Sneep, 1953). On the other hand, the report for the genotype of tetraploid asparagus is limited. The tetraploid Spanish landrace MH is well analyzed and the male and female genotypes of this population are [Mmmm] and [mmmm], respectively (Moreno et al., 2008b). The Italian landrace VA and purple asparagus cultivar derived from VA are known as tetraploid (Falloon and Andersen, 1999), however, the genotype of these population is not known well. Here we analyzed the sex ratio of ‘Pacific Purple’, and this cultivar showed male:female=1:1 ( Table 3 ). Furthermore, the indel polymorphism observed in the MSE1/AoMYB35/AspTDF1 gene showed that a single type of PCR fragment of different size is amplified when using PP-m and PP-m* DNAs as templates ( Figure 3B ). Our results lead us to assume that the male and female genotypes of ‘Pacific Purple’ is likely to be [Mmmm] and [mmmm], respectively.

In this study, we determined the MSE1/AoMYB35/AspTDF1 sequence from A. officinalis, A. maritimus and ‘Pacific Purple’. Phylogenetic analysis based on the amino acid sequences of this gene showed that PP-m* was closely related to A. maritimus and PP-m grouped in the same clade of A. officinalis ( Figure 2A ). The comparison of the amino acid sequence and the indel found in the intron of the MSE1/AoMYB35/AspTDF1 gene among these species showed that the MSE1/AoMYB35/AspTDF1 sequence from PP-m and PP-m* was very similar to that from A. officinalis and A. maritimus, respectively ( Figures 2B , 3A ). These results revealed that MSE1/AoMYB35/AspTDF1 gene from PP-m and PP-m*of ‘Pacific Purple’ was derived from A. officinalis and A. maritimus, respectively. This nicely fit with the results of the applicability of sex determination markers ( Table 2 ). MSSTS710 and MspHd markers were available for A. officinalis and PP-m of ‘Pacific Purple’, but not for A. maritimus and PP-m* of ‘Pacific Purple’, suggesting that the non-recombining Y region of PP-m and PP-m* have high homology with that of A. officinalis and A. maritimus, respectively.

The purple asparagus cultivar ‘Pacific Purple’ was developed using the Italian landrace VA. This cultivar is a polycross hybrid and its offspring from the polycross block showed a uniform purple color (Falloon and Andersen, 1999). This indicates that multiple parents were used for the breeding of ‘Pacific Purple’. Based on the result of MSE1/AoMYB35/AspTDF1 sequence, PP-m has the same MSE1/AoMYB35/AspTDF1 gene sequence as A. officinalis, and PP-m* has the same as A. maritimus. In addition, the male genotype of ‘Pacific Purple’ is likely to be [Mmmm] as shown above. These results indicate that two types of pollen parents with different MSE1/AoMYB35/AspTDF1 genes were included in the polycross block used for the breeding of ‘Pacific Purple’. This is consistent with the speculation that VA is likely an interspecific hybrid between A. officinalis and A. maritimus (Moreno et al., 2008a).

Based on our results, the putative breeding process of ‘Pacific Purple’ is shown in Figure 4 . Since ‘Pacific Purple’ is a cultivar developed from VA (Falloon and Andersen, 1999), it is expected that ancestral population of VA had maintained the interspecific crossing between A. officinalis and A. maritimus. During the selection process of VA from ancestral population, two types of pollen parents with different non-recombining Y region might have been maintained in their population. At the final step of pollen parent selection of ‘Pacific Purple’, the breeder selected two types of males with different MSE1/AoMYB35/AspTDF1 sequence, resulting that two types of male, i.e. PP-m and PP-m*, were included in ‘Pacific Purple’.

On the other hand, other purple asparagus cultivars/lines of ‘Purple Passion’, ‘Erasmus’, NJ1064, have one type of MSE1/AoMYB35/AspTDF1 sequence of A. officinalis/PP-m type ( Figures 2 , 3 ). Among these purple asparagus cultivars, at least ‘Purple Passion’ has been shown to be derived from VA, like ‘Pacific Purple’ (Benson et al., 1996). ‘Purple Passion’ was bred from single plant selection, while ‘Pacific Purple’ bred from polycross hybrid (Benson et al., 1996; Falloon and Andersen, 1999). This is consistent with the result that only the A. officinalis/PP-m type of MSE1/AoMYB35/AspTDF1 sequence was obtained from ‘Purple Passion’.

In conclusion, PP-m and PP-m* of ‘Pacific Purple’ has the similar sequence of MSE1/AoMYB35/AspTDF1 gene from A. officinalis and A. maritimus, respectively. ‘Pacific Purple’ was bred from VA, which is likely to be developed from an interspecific crossing between A. officinalis and A. maritimus. Since ‘Pacific Purple’ was a polycross hybrid, two types of pollen parents with different MSE1/AoMYB35/AspTDF1 sequence is likely to be used for the breeding of this cultivar. Since several purple asparagus cultivars derived from VA are known to date, it is interesting to analyze the MSE1/AoMYB35/AspTDF1 sequence of those cultivars.