Morphological And Molecular Identication of Double Flowered Stock (Matthiola Incana R. Br) Cultivars With High Fertility

Garden stock (Matthiola incana R. Br.) is a commercially important horticultural crop owing to its ornamental effect. There are different stock cultivars varied in color and shape, especially owered phenotype is an essential index evaluating its commercial value, because double owered cultivars have more brilliant owers compared to single owered one. The present work aimed: (1) to make superior cultivars with different colors, high fertility, being capable of early selecting only double owered seedlings by leaf color and to investigate morphological characteristics and (2) to select RAPD and ISSR primers for the cultivar certication and identication to culture and produce good commercial stock cultivars. Here we obtained new double owered stock cultivars with different colors including pink, pale pink and white, through outcrossing between “white” cultivar (high fertile but unable to select double ower phenotype) and “pink” cultivar (vice. versa). Among newly obtained stock cultivars, single and double ower seedlings are distinguishable from each other by leaf color, having about 70% of fertility. Moreover RAPD and ISSR markers selected in this study can be applied to identify different stock cultivars in seed production, culture and to establish cultivar certication system. for phenotypic traits such as leaf color, leaf shape and ower color. The following plant measurements were recorded: plant height, stem length, number of leaves per plant, leaf length and width, ower diameter, inorescence length; numbers of owers in inorescence and pod (silique) length. The data obtained from the research study was statistically analyzed by using ANOVA in Statistica ver. 6.0 and means were compared using Tukey’ s tests at the 5% level of signicance. All means are presented with standard error.

There are single and double owered M. incana cultivars, and latter is more widely used for oriculture because it has more beautiful owers. Seed production is only possible in single owered individuals. It is impossible in double owered plants due to degenerative pistils and stamens. Therefore many researchers and breeders have aimed to produce of more double owered plants than single owered ones in M. incana and to develop markers to select them.
To date, several morphological traits such as cotyledon shape, serrate leaf, seed color, and leaf color have been used in early selection of double owered stock individuals, because these traits were linked to double owers (Saunders 1911;1915;1921;1928;Ecker et al. 1993).
Takashi and Kanae (2018) identi ed the gene related to double owers and developed a molecular marker to select double owered plants e ciently, based on the study of double ower-linked molecular markers in Japanese gentian (Tasaki et al. 2017).
In general, the double ower trait is inherited by a single locus, s and it is linked to leaf color. In some stock cultivars, leaf color is a simple morphological marker, that is, green color indicates single owered individual while pale green color represents double owered individual (Ecker et al. 1993).
And also the fertility of plant is an important trait in cultivation and breeding of stock as well as other plants. Consequently, it is necessary to make a good stock cultivar with high fertility, different colors, and being capable of early discrimination of double owered phenotype, in culture and production for commercial use. Moreover, RAPD method has also been used to assess genetic diversity on the level of variety or cultivar in following plants, for example, common beans And also ISSR method has been widely used in genetic diversity analysis because of its high reproducibility than RAPD method (Domenyuk et al. 2002;Galvan et al. 2003;Dogan et al. 2007). The present work aimed to: (1) make superior cultivars with different colors, high fertility, being capable of early selecting only double owered seedlings by leaf color and examine morphological characteristics and (2) select RAPD and ISSR primers for cultivar certi cation and identi cation to culture and maintain good commercial stock cultivars.
The white cultivar is highly fertile, but it is not possible to distinguish between single owered individuals and double owered ones. Otherwise, in case of the pink cultivar, although it shows very low fertility, it is possible to select double owered seedlings according to leaf color; single ower phenotype with green color and double ower phenotype with pale green color.
Here we evaluated genetic diversity among new double owered stock cultivars obtained in cross "white" cultivar × "pink" cultivar by using morphological traits including leaf color, leaf shape, ower color and double ower phenotype, and RAPD and ISSR markers.
In this study we made some good stock cultivars with different colors including pink, pale pink and white (high fertile and capable of distinguishing between single and double owered seedlings) through outcrossing between white cultivar (high fertile but impossible to select double ower phenotype) and pink cultivar (vis. versa). In newly obtained stock cultivars, it is possible to distinguish between single and double ower seedlings by leaf color, having about 70 ~ 75% of fertility, thereby they are e cient good cultivars than both parent cultivars in seed production and culture.
Additionally, our cultivars were classi ed into two main groups, including the pink and the white cultivars respectively, the genetic distances among them varied from 0.03 to 0.24, with similarity to the previous research results in apricot and cowpea (Chroboková et al. 2011;Masvodza et al. 2014).
In the future, by using our selected RAPD and ISSR primers, the present method might be contributed to establish the cultivar certi cation, identi cation, and conservation system in culture and management of double owered stock cultivars with high fertility and different colors.

Plant material and cross
Matthiola incana R. Br. cultivar "white" (DPR Korea) as a maternal parent and "pink" (DPR Korea) as a paternal parent were grown in a greenhouse at Pyongyang oriculture institute. In the cultivar "white", plant height is high with no hairy leaves, white owers, and high ratio of seed production (approximately 95%) otherwise, in the cultivar "pink", plant height is lower than "white" with hairy leaves, pink owers, and very low ratio of seed production (about 2.2%), besides with different leaf color between single and double owered individuals.
We fertilized a "white" plant's ovules using pollen produced by "pink" plant.
We obtained F 1 offspring by growing 320 seeds resulted from a hybridized plant, and mixed the seeds by self-fertilizing F 1 heterozygous plants. In the F 2 progeny produced from 10 plants of them, the selection was performed according to oral and leaf's traits among 1757 plants. Subsequently, we conducted a family selection in the F 3 generation and selection of xed line in the F 4 generation, nally obtained 4 pink, 2 pale pink and 2 white stock cultivars xed in targeting traits in the F 5 generation.

Morphological analysis
Observations were made on each plant for phenotypic traits such as leaf color, leaf shape and ower color.
The following plant measurements were recorded: plant height, stem length, number of leaves per plant, leaf length and width, ower diameter, in orescence length; numbers of owers in in orescence and pod (silique) length.
The data obtained from the research study was statistically analyzed by using ANOVA in Statistica ver. 6.0 and means were compared using Tukey' s tests at the 5% level of signi cance. All means are presented with standard error. Molecular analysis Genomic DNA isolation Fresh leaf samples were collected from individuals of all cultivars according to ower type (single or double) and leaf shape (hairy and no hairy). Genomic DNA were isolated from 500 mg of fresh leaf material using CTAB method (Cetyl Trimethyl Ammonium Bromide) developed by Doyle and Doyle (1990).
Qualitative and Quantitative estimation of DNA DNA quality was assessed by using Nanodrop Spectrophotometer (Thermoscienti c Nanodrop 1000, USA) at the absorbance ratio of 260 and 280 nm providing a value of 1.8 which determines pure DNA preparation. Quality of DNA fragment was electrophoretically analyzed through 0.8% agarose gel using 1X TAE buffer at 50 V for 45 mins.
RAPD PCR ampli cation RAPD PCR ampli cation was performed to amplify randomly unknown target sequences by using arbitary random primers according to the protocol described by Williams et al. (1990). PCR was carried out in a 25µl reaction volume containing 2.5µl of 10X Taq buffer, 2.5µl of dNTPs, 2.5µL of MgCl 2, 1µl of primer, 0.1µL 0.1U/µL Taq polymerase, 1µl of temple DNA (100 ng/ µl) and 15.4 µl of ddH 2 O for each sample in a Mastercycler (nexus gradient).
We initially tested 25 primers from RAPD primer set (Opéron, sets D, H, I, N and P) on two individuals for each of the eight cultivars. We selected 10 primers (OPD3, OPH18, OPI14, OPN4, OPP19, OPH8, OPH19, OPP9, OPP17 and OPP20) with both reproducible and polymorphic variation with well de ned and darkly staining bands.
Mastercycler (nexus gradient) programmed for an initial denaturation step of 94°C for 5 min, followed by 40 cycles of 30 s at 94°C, 60 s at 37°C and 90 s at 72°C, a nal extension step of 72°C for 2 min.
Ampli cation products were separated on 1.5% agarose gels in 1×TAE buffer at 80 V for 1h 30 mins.
Gels were stained with ethidium bromide and photographed under UV light by using Geldoc-It™ (USA).
ISSR PCR ampli cation ISSR PCR ampli cation was performed by using 7 primers with high GC content according to the protocol described by Bekir et al. (2016). Data analysis constructed. DNA fragments of identical size ampli ed with the same primer were considered to be the same DNA marker.
To examine the genetic relationship among populations, a dendrogram was constructed using the unweighted paired group method of cluster analysis using arithmetic averages (UPGMA) and principal coordinate analysis (PCA) was performed with NTSYSpc version 2.20 (Rohlf 2008).
Based on RAPD and ISSR data, the POPGENE ver. 1.32 (Yeh et al. 2000) was used to estimate values meaning genetic diversity: number of polymorphic band, observed number of alleles, effective number of alleles (Kimura and Crow 1964), Nei's (1973) gene diversity (h), Shannon's information index (I) (Lewontin 1972) and genetic distances among populations (Nei 1978).
A dendrogram of the populations was constructed which has been based on the unweighed pair group method (UPGMA) analysis of Nei's (1978) genetic distances. And also, G ST representing population differentiation and gene ow (Nm) among populations were estimated.
Using the Arlequin ver. 3.5.2.2 (Exco er and Lischer 2010), an analysis of molecular variance (AMOVA) was performed to evaluate the distribution of genetic variation within and among stock cultivars, and the Fixation index (Fst) was estimated too.

Morpological diversity of new double owered stock cultivars
We compared and analysed the morphological traits in new double owered stock cultivars obtained from outcrossing including 4 pink, 2 pale pink and 2 white cultivars (Fig. 1). In the present study the genetic relationship among and within cultivars of M. incana were analysed by ten informative RAPD and ISSR primers ( Table 2). Based on above obtained RAPD and ISSR pro les (Fig. 3), genetic relationship among 21 stock samples was estimated using NTSYSpc2.11.
We obtained data matrix based on Nei (1973)'s genetic distance and constructed a dendrogram using UPGMA method.
As shown in Fig. 4, at genetic distance of 0.15, dendrogram grouped all individuals in two clusters, and principal coordinate analysis result (Fig. 5) was agreed with cluster analysis (Fig. 4). Nei's genetic identity (above diagonal) and genetic distance (below diagonal).
As shown in Fig. 6, eleven stock cultivars were classi ed into two main groups; pink and white cultiivar group.
In the group of white cultivars, new cultivar "white 2" was genetically closest to control cultivar "white" (genetic distance-0.0498), and new cultivar "white 1" was the most distant to maternal cultivar "white" (genetic distance-0.1062). Otherwise, in the group of pink cultivars, new cultivar "pink 2" was the genetically closest to paternal cultivar "pink" (genetic distance-0.0534), and new cultivar "pink 1" was the most distant to new cultivar "pale pink 1" (genetic distance-0.0965).
The values of genetic diversity indices for studied stock cultivars are given in Table 4. The highest n a was observed for "pale pink 2" (1.26) and the least for "pink 3" (1.07), showing the lowest Shannon's index (0.04) and Nei's gene diversity (0.03) as well.
Nei's gene diversity were estimated to be 0.03 and 0.11 and the Shannon indices of the M. incana cultivars ranged from 0.04 to 0.16. Genetic variation among cultivars was more than that within cultivars, as indicated by the sum of squares values calculated by AMOVA using Arlequin ver. Table 5). Of the total genetic diversity, 67.27 % was attributable to differences among culivars and 32.73% was to differences within culivars, showing a signi cant varietal differentiation in newly obtained stock cultivars. The xation index (Fst) was 0.673.

Discussion
Because double owered cultivars have superior ornamental value to single owered ones, stock breeders have attempted to make double owered cultivars with different colors.
Additionally, in breeding of M. incana, the studies also have been reported to identify the morphological and molecular markers which could be used to discriminate the double owered individuals. Ecker et al. (1993) founded that the c allele for sinuate leaf shape might be recessive to the C allele for normal entire leaf and it was tightly linked to the s recessive allele for double owering, suggesting its role as a double owering marker in the cultivation and the breeding of M. incana.
Takashi and Kanae (2018) revealed that co-dominant marker, MiAG could be used for early discrimination of double owered individuals among seedlings not showing phenotypic differences. He examined that the single owered individuals of cultivars being distinguished double owered ones according to cotyledon shape ('Kiss me white', 'Iron white', 'Quartet cherry'), serrated leaf ('Pygmy white'), seed color ('White wonder no.2′) were heterozygotes MiAG/miag1, whereas the individuals correlated with leaf color (in 'Aida' and 'Opera') were heterozygotes MiAG/miag2.
In this study we made the garden stock varieties with different colors (pink, pale pink and white) and high fertility which were capable of discriminating double owered individuals in cross "white" cultivar (high fertile but impossible in selection of double owered plants) × "pink" cultivar (very low fertile but possible in selection of double owered plants  (Fig. 2), and harboring high fertility, therefore they might be superior to both parents cultivars in the cultivation and seed production for commercial use.
To date, random ampli ed polymorphic DNA (RAPD) method have been widely applied to assess the intra-and inter-speci c genetic diversity in many plants.

Conclusion
We bred new double owered stock cultivars with different colors and high fertility in cross white cultivar that was high fertile but impossible to select double ower phenotype × pink cultivar (vice. versa). RAPD and ISSR markers selected in this study can be used to identify different stock cultivars according to ower color and owered phenotype, therefore in addition to morphological traits, they will be applied to cultivar certi cation, identi cation, and establishing conservation system in their culture and management.

Declarations
Con ict of interest The authors declare that there are no con icts of interest. Figure 1 Phenotypes of newly obtained stock plants and both parent plants "-s" means single owered phenotype and "-d" means double owered phenotype.

Figure 2
Leaf color in young seedlings in newly obtained stock plants (green: double owered seeding, pale green: single owered seeding)   UPGMA dendrogram illustrating the genetic relationships between eleven cultivars of M. incana.