Discovery of miRNAs and Development of Heat-Responsive miRNA-SSR Markers for Characterization of Wheat Germplasm for Terminal Heat Tolerance Breeding.

Pooja Sihag,Vijeta Sagwal,Upendra Kumar,Priyanka Balyan,Anuj Kumar,Reyazul Rouf Mir,Om Parkash Dhankher

doi:10.3389/fgene.2021.699420

Abstract

A large proportion of the Asian population fulfills their energy requirements from wheat (Triticum aestivum L.). Wheat quality and yield are critically affected by the terminal heat stress across the globe. It affects approximately 40% of the wheat-cultivating regions of the world. Therefore, there is a critical need to develop improved terminal heat-tolerant wheat varieties. Marker-assisted breeding with genic simple sequence repeats (SSR) markers have been used for developing terminal heat-tolerant wheat varieties; however, only few studies involved the use of microRNA (miRNA)-based SSR markers (miRNA-SSRs) in wheat, which were found as key players in various abiotic stresses. In the present study, we identified 104 heat-stress-responsive miRNAs reported in various crops. Out of these, 70 miRNA-SSR markers have been validated on a set of 20 terminal heat-tolerant and heat-susceptible wheat genotypes. Among these, only 19 miRNA-SSR markers were found to be polymorphic, which were further used to study the genetic diversity and population structure. The polymorphic miRNA-SSRs amplified 61 SSR loci with an average of 2.9 alleles per locus. The polymorphic information content (PIC) value of polymorphic miRNA-SSRs ranged from 0.10 to 0.87 with a mean value of 0.48. The dendrogram constructed using unweighted neighbor-joining method and population structure analysis clustered these 20 wheat genotypes into 3 clusters. The target genes of these miRNAs are involved either directly or indirectly in providing tolerance to heat stress. Furthermore, two polymorphic markers miR159c and miR165b were declared as very promising diagnostic markers, since these markers showed specific alleles and discriminated terminal heat-tolerant genotypes from the susceptible genotypes. Thus, these identified miRNA-SSR markers will prove useful in the characterization of wheat germplasm through the study of genetic diversity and population structural analysis and in wheat molecular breeding programs aimed at terminal heat tolerance of wheat varieties.

Highlights

Triticum aestivum L. is the most commonly used hexaploid bread wheat (AABBDD; 6X = 2n = 42) with genome size of approximately 17 GB
80 heat-responsive miRNA families consisting of 104 members were recognized in Arabidopsis, wheat, rice, maize, and sorghum crops (Supplementary Table 1)
Primer designing using BatchPrimer3 v1.0 resulted in 70 significant miRNA-simple sequence repeats (SSRs) primers pair, whereas no SSR flanking primers were obtained for two miRNAs, and the absence of SSRs were noticed in 27 pre-miRNA sequences

Summary

Introduction

Triticum aestivum L. is the most commonly used hexaploid bread wheat (AABBDD; 6X = 2n = 42) with genome size of approximately 17 GB. It is derived via crossing among tetraploid Triticum turgidum (AABB) and diploid Aegilops tauschii (DD), whereas T. turgidum (AABB) was derived via crossing between Triticum urartu (AA) and Aegilops speltoides (BB) (Petersen et al, 2006). Heat stress at the time of anthesis and grain filling adversely affects wheat quality and yield in terms of grain number and grain weight (Ferris et al, 1998; Kumar et al, 2013). In comparison to the heat-susceptible lines, the genotypes maintaining yields under higher temperatures must contain genes that prevent degradation of chlorophyll molecules, maintain low canopy temperature, and encode enzymes that can maintain activity under elevated temperatures

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Conclusion