2.1. Plant Material
The studied plant material for the validation of Turkish molecular markers section in-cluded an Iranian population with 14 very early-and very late-leafing culti-vars/genotypes. For this purpose, a population of 86 walnut individuals located in Horticultural Science Research Institute (HSRI) walnut research orchard in Karaj, Iran was used. The leafing date of 86 cultivars/genotypes was recorded for three consecu-tive years (2017–2019) (Table S1). Finally, among the 86 individuals, 14 very-early and very late-leafing cultivars/genotypes were selected for validation of Turkish markers introduced by Kefayati et al., (2019) (Table S1).
In the other part of the research, the plant material for the implementation of MAS section was an evaluated walnut population including 91 genotypes obtained from a breeding program of Horticultural Science Research Institute (HSRI). This population obtained from the targeted walnut hybridization program of commercial cultivars and superior genotypes, are located in the HSRI walnut research orchard in Karaj, Iran. In this hybridization program, commercial cultivars including ‘Chandler’, ‘Pedro’, ‘Persia’ and ‘Caspian’ as the female parents and cultivars/genotypes including ‘Fran-quette’, ‘Lara’ and the superior genotype of 35T were male parents. For evaluating progenies, the nuts obtained from each cross combination were planted. Leaf samples were gathered from each seedling and dried separately inside the envelopes containing silica gel. Afterward, molecular research was carried out at the Molecular Genetics Laboratory in the Department of Horticulture of Çukurova University, Adana, Turkey.
2.2. Phenotyping of the Populations
Before molecular study, the studied walnut population was phenotypically evaluated for three consecutive years (2017–2019). For this purpose, the leafing date of 86 walnut individuals including superior genotypes and commercial cultivars was recorded based on IPGRI descriptor. leafing date was the date when 50% of terminal buds have enlarged and the bud scales have split exposing the green leaves [11]. The leafing date was reported according to March 1 (Hassani et al., 2020) (Table S1/ Fig. 1). In order to validation of markers and identify the alleles related to early and late-leafing, 7 very late-leafing and 7 very early-leafing cultivars/genotypes were selected.
2.3. DNA Extraction
The genomic DNA of 105 (14 + 91) dry leaf samples was extracted by the modified CTAB method as described by Doyle and Doyle (1987). The Qubit Fluorometer (Ther-moFisher Scientific, Waltham, MA, USA) and 2% agarose gel electrophoresis were used for evaluating the quantity and quality of the isolated DNA. Then, the genomic DNA was diluted to approximately 10 ng/µl for SSR polymerase chain reaction (PCR) ampli-fication. Finally, the samples were stored in the refrigerator at temperature of − 20℃ until the next steps (Kafkas et al. 2005).
2.4. PCR amplification for Validation
The leafing date associated SSR markers “JRHR209732 and CUJRB012” which are de-veloped by Kefayati et al. (2019) were validated and used to evaluate and classify the walnut population of Iran. Sequence information for the two SSR leafing time markers is Shown in Table S2.
According to Scheulke (2000) approach, all SSR-PCR reactions were performed. A total volume of 12.5 µL containing 10 ng DNA, 20 mM (NH4)2SO4, 75 mM Tris–HCl (pH 8.8), 0.01% Tween 20, 2.0 mM MgCl2, 200 µM of each dNTP, 10 nM M13 tailed for-ward primer at the 50 ends, 200 nM reverse primer, 200 nM universal M13 tail primer labeled with FAM, VIC, NED or PET dye, and 0.6 U of Taq DNA polymerase were uti-lized for each reaction (Guney et al., 2021). The PCR amplifications were carried out in two phases. The first step included initial denaturation at 94℃ for 3 min, followed by 28 cycles of 94℃ for 30 s, 58℃ for 45 s, and 72℃ for 60 s. The second step involved 10 cycles of 94℃ for 30 s, 52℃ for 45 s, and 72℃ for 60 s, and a final extension at 72℃ for 5 min (Guney et al., 2021). Then PCR products were separated by capillary electrophoresis on an ABI PRISM 3130xl Genetic Analyzer [Applied Biosystems (ABI) Inc., Tokyo-Japan]. Each well was loaded with 9.7 µL of Hi-Di formamide, 0.3 µL of 500 LIZ size standard, and 1 µL of PCR product. The aliquots were denatured for 5 min at 95℃ and run on the ABI 3130xl automated sequencer using a 36-cm capillary array with POP7 polymer (Kefayati et al., 2019). Finally, fragment size in absolute base pairs was determined by ABI and output charts were used to identify early, mid, and late-leafing genotypes.
2.5. Statistical analysis
In order to identify the association between early and late-leafing, and the presence of each marker allele, chi-square test was performed. The chi-square test with Pearson's method was performed by SPSS (Version 22) software for the alleles obtained for 14 very early-and very late-leafing populations. After validation of turkey leafing date markers, MAS was performed on Iranian walnut population including 91 genotypes. Allele sizes related to early, mid or late-leafing obtained by two markers JRHR209732 and CUJRB012 on 91 genotypes were used for cluster analysis. Cluster analysis and grouping of the studied population were performed based on shared allele genetic dis-tance constructed using the UPGMA (Unweighted Pair Group Method with Arithme-tic Average) method and calculations of distances after standardization of data (Guney et al., 2021). The dendrogram was constructed using the "factoextra" and "dendextend"R Packages.
In this research we compared the kefayati et al (2019) and marrano et al (2019) identified SNP markers associated to leafing date and these markers were located very close to each other on chromosome 1. The study by Kafayati et al. (2019) was performed before the release of the new version of the assembled walnut genome on the chromosome. Therefore, they identified markers related with Leafing Date on the LG4. Thus, they only identified the QTLs on the LGs. Then, we blasted the results of Kafaiti et al. (2019) with chromosome-scale assembly of the walnut reference genome version (Chandler v2.0) published by Marrano et al (2020). The result showed that kefayati's markers were found very closly marrano's markers on the chr 1 (Table S3-S4).