RNA-Seq analysis and development of SSR and KASP markers in lentil (Lens culinaris Medikus subsp. culinaris)

Abstract Seed weight is a major target of peanut breeding as an important component of seed yield. However, relatively little is known about QTLs and candidate genes associated with seed weight in peanut. In this study, three major QTLs on chromosomes A05, B02 and B06 were determined by applying NGS-based QTL-seq approach for a RIL population. These three QTL regions have been successfully narrowed down through newly developed SNP and SSR markers based on traditional QTL mapping. Among these three QTL regions, qSWB06.3 exhibited stable expression with large contribution to phenotypic variance across all environments. Furthermore, RNA-seq were applied for early, middle and late stages of seed development, and differentially expression genes (DEGs) were identified in ubiquitin-proteasome pathway, serine/threonine protein pathway and signal transduction of hormones and transcription factors. Notably, DEGs at early stage were majorly related to regulating cell division, whereas DEGs at middle and late stages were mainly associated with cell expansion during seed development. Through integrating SNP variation, gene expression and functional annotation, candidate genes related to seed weight in qSWB06.3 were predicted and distinct expression pattern of those genes were exhibited using qRT-PCR. In addition, KASP-markers in qSWB06.3 were successfully validated in diverse peanut varieties and the alleles of parent Zhonghua16 in qSWB06.3 was associated with high seed weight. This suggested that qSWB06.3 was reliable and the markers in qSWB06.3 could be deployed in marker-assisted breeding to enhance seed weight. This study provided insights into the understanding of genetic and molecular mechanisms of seed weight in peanut.

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Genome-Wide Development and Validation of Cost-Effective KASP Marker Assays for Genetic Dissection of Heat Stress Tolerance in Maize

International Journal of Molecular Sciences ◽

10.3390/ijms21197386 ◽

2020 ◽

Vol 21 (19) ◽

pp. 7386

Author(s):

Ashok Babadev Jagtap ◽

Yogesh Vikal ◽

Gurmukh Singh Johal

Keyword(s):

Heat Stress ◽

Stress Tolerance ◽

Cost Effective ◽

Genetic Dissection ◽

Rna Seq ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Genome Wide ◽

Heat Stress Tolerance ◽

Kasp Markers

Maize is the third most important cereal crop worldwide. However, its production is vulnerable to heat stress, which is expected to become more and more severe in coming years. Germplasm resilient to heat stress has been identified, but its underlying genetic basis remains poorly understood. Genomic mapping technologies can fill the void, provided robust markers are available to tease apart the genotype-phenotype relationship. In the present investigation, we used data from an RNA-seq experiment to identify single nucleotide polymorphisms (SNPs) between two contrasting lines, LM11 and CML25, sensitive and tolerant to heat stress, respectively. The libraries for RNA-seq were made following heat stress treatment from three separate tissues/organs, comprising the top leaf, ovule, and pollen, all of which are highly vulnerable to damage by heat stress. The single nucleotide variants (SNVs) calling used STAR mapper and GATK caller pipelines in a combined approach to identify highly accurate SNPs between the two lines. A total of 554,423, 410,698, and 596,868 SNVs were discovered between LM11 and CML25 after comparing the transcript sequence reads from the leaf, pollen, and ovule libraries, respectively. Hundreds of these SNPs were then selected to develop into genome-wide Kompetitive Allele-Specific PCR (KASP) markers, which were validated to be robust with a successful SNP conversion rate of 71%. Subsequently, these KASP markers were used to effectively genotype an F2 mapping population derived from a cross of LM11 and CML25. Being highly cost-effective, these KASP markers provide a reliable molecular marker toolkit to not only facilitate the genetic dissection of the trait of heat stress tolerance but also to accelerate the breeding of heat-resilient maize by marker-assisted selection (MAS).

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Development of KASP Markers and Identification of a QTL Underlying Powdery Mildew Resistance in Melon (Cucumis melo L.) by Bulked Segregant Analysis and RNA-Seq

Frontiers in Plant Science ◽

10.3389/fpls.2020.593207 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yanyan Cao ◽

Qiannan Diao ◽

Youyuan Chen ◽

Haijun Jin ◽

Yongping Zhang ◽

...

Keyword(s):

Powdery Mildew ◽

Candidate Gene ◽

Powdery Mildew Resistance ◽

Bulked Segregant Analysis ◽

Target Area ◽

Rna Seq ◽

Mildew Resistance ◽

Specific Pcr ◽

Polymorphic Snps ◽

Kasp Markers

Powdery mildew (PM), caused by Podosphaera xanthii (Px), is one of the most devastating fungal diseases of melon worldwide. The use of resistant cultivars is considered to be the best and most effective approach to control this disease. In this study, an F2 segregating population derived from a cross between a resistant (wm-6) and a susceptible cultivar (12D-1) of melon was used to map major powdery mildew resistance genes using bulked segregant analysis (BSA), in combination with next-generation sequencing (NGS). A novel quantitative trait locus (QTL) named qCmPMR-12 for resistance to PM on chromosome 12 was identified, which ranged from 22.0 Mb to 22.9 Mb. RNA-Seq analysis indicated that the MELO3C002434 gene encoding an ankyrin repeat-containing protein was considered to be the most likely candidate gene that was associated with resistance to PM. Moreover, 15 polymorphic SNPs around the target area were successfully converted to Kompetitive Allele-Specific PCR (KASP) markers (P < 0.0001). The novel QTL and candidate gene identified from this study provide insights into the genetic mechanism of PM resistance in melon, and the tightly linked KASP markers developed in this research can be used for marker-assisted selection (MAS) to improve powdery mildew resistance in melon breeding programs.

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Comparative RNA-seq analysis of the drought-sensitive lentil (Lens culinaris) root and leaf under short- and long-term water deficits

Functional & Integrative Genomics ◽

10.1007/s10142-019-00675-2 ◽

2019 ◽

Vol 19 (5) ◽

pp. 715-727 ◽

Cited By ~ 4

Author(s):

Hande Morgil ◽

Mehmet Tardu ◽

Gül Cevahir ◽

İbrahim Halil Kavakli

Keyword(s):

Lens Culinaris ◽

Rna Seq ◽

Water Deficits ◽

Short And Long Term

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Insights into the Host-Pathogen Interaction Pathways through RNA-Seq Analysis of Lens culinaris Medik. in Response to Rhizoctonia bataticola Infection

Genes ◽

10.3390/genes13010090 ◽

2021 ◽

Vol 13 (1) ◽

pp. 90

Author(s):

Gyan P. Mishra ◽

Muraleedhar S. Aski ◽

Tejas Bosamia ◽

Shiksha Chaurasia ◽

Dwijesh Chandra Mishra ◽

...

Keyword(s):

Lens Culinaris ◽

Molecular Mechanisms ◽

Vital Role ◽

Rna Seq ◽

Cell Wall Modification ◽

Rhizoctonia Bataticola ◽

Host Pathogen Interaction ◽

Host Pathogen ◽

Molecular Aspects ◽

Related Proteins

Dry root rot (Rhizoctonia bataticola) is an important disease of lentils (Lens culinaris Medik.).To gain an insight into the molecular aspects of host-pathogen interactions, the RNA-seq approach was used in lentils following inoculation with R.bataticola. The RNA-Seq has generated >450 million high-quality reads (HQRs) and nearly 96.97% were properly aligned to the reference genome. Very high similarity in FPKM (fragments per kilobase of exon per million mapped fragments) values (R > 0.9) among biological replicates showed the consistency of the RNA-Seq results. The study revealed various DEGs (differentially expressed genes) that were associated with changes in phenolic compounds, transcription factors (TFs), antioxidants, receptor kinases, hormone signals which corresponded to the cell wall modification enzymes, defense-related metabolites, and jasmonic acid (JA)/ethylene (ET) pathways. Gene ontology (GO) categorization also showed similar kinds of significantly enriched similar GO terms. Interestingly, of the total unigenes (42,606), 12,648 got assembled and showed significant hit with Rhizoctonia species. String analysis also revealed the role of various disease responsive proteins viz., LRR family proteins, LRR-RLKs, protein kinases, etc. in the host-pathogen interaction. Insilico validation analysis was performed using Genevestigator® and DEGs belonging to six major defense-response groups viz., defense-related enzymes, disease responsive genes, hormones, kinases, PR (pathogenesis related) proteins, and TFs were validated. For the first time some key miRNA targets viz. miR156, miR159, miR167, miR169, and miR482 were identified from the studied transcriptome, which may have some vital role in Rhizoctonia-based responses in lentils. The study has revealed the molecular mechanisms of the lentil/R.bataticola interactions and also provided a theoretical approach for the development of lentil genotypes resistant to R.bataticola.

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