Abstract
We developed novel miRNA-based markers based on salt responsive miRNA sequences to detect polymorphisms in miRNA sequences and locations. The validation of 76 combined miRNA + miRNA and miRNA + ISSR markers in the three extreme pistachio populations led to the identification of three selected markers that could link salt tolerance phenotype to genotype and divided pistachio genotypes and Pistacia species into three clusters. This novel functional marker system, in addition to more efficient performance, has higher polymorphisms than previous miRNA-based marker systems. The functional importance of the target gene of five miRNAs in the structure of the three selected markers in regulation of different genes such as ECA2, ALA10, PFK, PHT1;4, PTR3, KUP2, GRAS, TCP, bHLH, PHD finger, PLATZ and genes involved in developmental, signaling and biosynthetic processes shows that the polymorphism associated with these selected miRNAs can make a significant phenotypic difference between salt sensitive and tolerant pistachio genotypes. The sequencing results of selected bands showed the presence of conserved miRNAs in the structure of the mitochondrial genome. Further notable findings of this study are that the sequences of PCR products of two selected markers were annotated as Gypsy and Copia retrotransposable elements. The transposition of retrotransposons with related miRNAs by increasing the number of miRNA copies and changing their location between nuclear and organellar genomes can affect the regulatory activity of these molecules. These findings show the crucial role of retrotransposon-derived miRNAs as mobile epigenetic regulators between intracellular genomes in regulating salt stress responses as well as creating new and tolerant phenotypes for adaptation to environmental conditions.
Highlights
Genotypes requires sufficient knowledge about their gene sequences and functions
Molecular marker studies on pistachio are often focused on genetic relationships and diversity, as well as sex determination by using different markers such as Simple Sequence Repeat (SSR)[19,20,21], Amplified Fragment Length Polymorphism (AFLP)[22,23], Inter-Simple Sequence Repeats (ISSR)[24,25,26,27], Randomly Amplified Polymorphic DNA (RAPD)[28,29,30], Retrotransposon Microsatellite Amplified Polymorphism (REMAP)[31], Selective Amplification of Microsatellite Polymorphic Loci (SAMPL)[32] and Single Nucleotide Polymorphism (SNP)[33]
Plant miRNA-based DNA markers can be divided into two types: one type is miRNAbased simple sequence repeat (SSR) markers reported in several plants such as Legume species45, rice[40,43,46] and Punica granatum 47 and another type is designed based on conserved regions of pre-miRNAs reported in Gossypium species[48], Brassica s pecies[44], and Setaria italica 49
Summary
Genotypes requires sufficient knowledge about their gene sequences and functions. the sequencing and molecular data on plants have increased rapidly, only a few studies have been done on P. vera. The aims of this study were (1) the generation of novel miRNA-based FMs and validation of these markers in the three extreme pistachio populations, (2) development of a new type of semi-random ISSR markers and validation of new combination of miRNA + ISSR markers in the three extreme pistachio populations, (3) application of polymorphic candidate markers for grouping pistachio genotypes and Pistacia species, and (4) sequencing PCR products of selected markers to confirm the performance of primers and locate PCR products related to selected markers This is the first report of salt responsive novel FMs development designed based on high-throughput sequencing data and validation in P. vera for selecting salt tolerant genotypes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
