Prosize 2 0 version 1

In the present study, a total of 21 microsatellite loci, 14 of which are
recommended by FAO (2011) to determine genetic diversity in sheep, were
used. PCR amplifications were carried out in a total volume of 25  $\mathrm{\mu }$ L
PCR mixture. PCR mixture consisted of 2–2.5  $\mathrm{\mu }$ L genomic DNA (50 ng  $\mathrm{\mu }$ L ${}^{-\mathrm{1}}$ ), 1.2  $\mathrm{\mu }$ L HQ buffer (Geneall), 2  $\mathrm{\mu }$ L dNTPs (2.5 mM  $\mathrm{\mu }$ L ${}^{-\mathrm{1}}$ ),
0.25  $\mathrm{\mu }$ L of each primer (10 pmol  $\mathrm{\mu }$ L ${}^{-\mathrm{1}}$ ), 0.4  $\mathrm{\mu }$ L (2.5 U  $\mathrm{\mu }$ L) Taq polymerase
(Geneall), and distilled deionized water. PCR reaction conditions were
performed as follows: initial denaturation at 95  ${}^{\circ }$ C for 5 min,
followed by 30 cycles of denaturation at 94  ${}^{\circ }$ C for 45 s,
annealing (at 50–60  ${}^{\circ }$ C for different loci) for 45 s,
extension at 72  ${}^{\circ }$ C for 45 s, and final extension at
72  ${}^{\circ }$ C for 5 min.
In this study, 96 automated capillary electrophoresis systems
(Advanced Analytical Technologies, Iowa, USA) were used for fragment
analysis. The capillary conditioning solution, inlet buffer, separation gel,
and 35–500 bp marker were prepared according to the user manual provided by
the manufacturer. After capillary electrophoresis separation, the raw data
were recorded, and band sizes were calculated using PROSize^®2.0 version 1.3.1.1 (Advanced Analytical Technologies, Iowa, USA).

For the RNAseq experiment, pooled pedicel samples of individual plants were obtained 7 days after the last GA₃ application (corresponding to EL31 stage according to the Einhoch-Lorenz phenology system [42 (link)], plus 7 days—defined in this study as 7DAT). Two levels were used in the treatment factor, control and GA; and two levels were considered in the genotype factor, cv. Thompson Seedless and L23. Four biological replicates were used for each condition, summing to 16 libraries which were synthesized and sequenced.
Prior to library synthesis, total RNA was assessed by fragment analyzer PROSize® 2.0 version 1.3.1.1 (Advanced Analytical Technologies, Inc., Ames, IA, USA). Mean RNA quality in all samples was 7.35 (SD: 0.56), confirming the integrity of extractions. Then 2.5 μg aliquots were used to isolate poly(A) mRNA for preparation of libraries using TruSeq RNA Sample Prep Kit v2, following the manufacturer’s instructions described in the TruSeq RNA Sample Preparation v2 Guide, Part #15026495 Rev. F (Illumina, Inc.). Libraries were sequenced using the HiSeq 4000 platform (Illumina). Libraries were sequenced as paired-end data (2 × 100 bp).