Whole‐genome resequencing‐based QTL‐seq identified AhTc1 gene encoding a R2R3‐MYB transcription factor controlling peanut purple testa colour

Abstract

SummaryPeanut (Arachis hypogaea. L) is an important oil crop worldwide. The common testa colours of peanut varieties are pink or red. But the peanut varieties with dark purple testa have been focused in recent years due to the potential high levels of anthocyanin, an added nutritional value of antioxidant. However, the genetic mechanism regulating testa colour of peanut is unknown. In this study, we found that the purple testa was decided by the female parent and controlled by a single major gene named AhTc1. To identify the candidate gene controlling peanut purple testa, whole‐genome resequencing‐based approach (QTL‐seq) was applied, and a total of 260.9 Gb of data were generated from the parental and bulked lines. SNP index analysis indicated that AhTc1 located in a 4.7 Mb region in chromosome A10, which was confirmed by bulked segregant RNA sequencing (BSR) analysis in three segregation populations derived from the crosses between pink and purple testa varieties. Allele‐specific markers were developed and demonstrated that the marker pTesta1089 was closely linked with purple testa. Further, AhTc1 encoding a R2R3‐MYB gene was positional cloned. The expression of AhTc1 was significantly up‐regulated in the purple testa parent YH29. Overexpression of AhTc1 in transgenic tobacco plants led to purple colour of leaves, flowers, pods and seeds. In conclusion, AhTc1, encoding a R2R3‐MYB transcription factor and conferring peanut purple testa, was identified, which will be useful for peanut molecular breeding selection for cultivars with purple testa colour for potential increased nutritional value to consumers.

Translational genomics for achieving higher genetic gains in groundnut

“…The availability of quality reference genomes for both the subspecies of cultivated groundnut (Bertioli et al 2019;Chen et al 2019;Zhuang et al 2019) will further enhance the precision and accuracy of sequence alignment and SNP calling which will eventually facilitate precise discovery of genomic regions and candidate genes (Zhuang et al 2019). Among all the pooled sequencing-based approaches, namely QTL-Seq, MutMap, Seq-BSA InDel-Seq and BSR-Seq (see Pandey et al 2016), the 'QTL-Seq' has been successfully applied for discovery of genomic regions and candidate genes in groundnut (Pandey et al 2017b;Clevenger et al 2018;Luo et al 2019a, b;Zhuang et al 2019;Kumar et al 2020;Zhao et al 2020). The first QTL-seq study using diploid reference genomes identified a colocalized genomic region on chromosome A03 for rust and LLS resistance in TAG 24 × GPBD 4 followed by development and validation of allele-specific diagnostic markers for rust and LLS resistance (Pandey et al 2017b).…”

“…The fifth QTL-seq study was performed for seed size (one bulk with 54 big seeded RILs and second bulk with 54 small seeded RILs) in the population (Yueyou92 × Xinhuixiaoli) getting two QTL-conformed regions leading to isolation of candidate genes between two co-segregated SNPs (Zhuang et al 2019). The sixth QTL-Seq study determined that the purple testa color is controlled by female parent and identified a single major gene, AhTc1 encoding a R2R3-MYB transcription factor, followed by successful development of allele-specific markers (Zhao et al 2020). Most recently, the seventh QTL-seq analysis in RIL population (ICGV 00350 × ICGV 97045) for fresh seed dormancy identified two candidate genes-RING-H2 finger protein and zeaxanthin epoxidase which significantly express during seed development and control abscisic acid (ABA) accumulation (Kumar et al 2020).…”

“…As far as using RNA-Seq analysis on pooled samples is concerned, the Zhao et al (2020) performed bulked segregant RNA sequencing (BSR-Seq) for purple testa color and confirmed the results achieved through QTL-Seq analysis by achieving similar results. It is worth mentioning that the BSR-Seq approach provides a cheaper option for candidate gene discovery as compared to QTL-Seq approach; nevertheless, the QTL-Seq generated more SNPs across the genome than BSR-Seq which helps in narrowing down the candidate genomic regions and genes (Zhao et al 2020).…”

Fine mapping and gene cloning in the post-NGS era: advances and prospects

“…Based on the non-synonymous SNPs found between the extreme bulks, allele-specific diagnostic markers were reported for three SNPs for rust and one SNP for LLS (Pandey et al 2017b). More recently, QTL-Seq by Zhao et al (2019) localizes AhTc1 gene in peanut controlling purple testa to a 4.7 Mb region and the underlying J3K16L gene was confirmed through bulked segregant RNA sequencing (BSRseq) and gene overexpression analyses. A similar QTL-Seq approach in groundnut was associated with 2.4 Mb and 0.74 Mb genomic regions on the pseudomolecules B05 and A09, respectively, with fresh seed dormancy trait (Kumar et al 2019).…”

Whole‐genome resequencing‐based QTL‐seq identified candidate genes and molecular markers for fresh seed dormancy in groundnut

“…Of the available sequencing-based approaches, QTL-seq approach offers great benefits by identifying genomic region(s) and candidate genes leading to development of diagnostic markers. This approach has already been deployed successfully in some legume crops including groundnut for foliar disease (rust and LLS) resistance (Pandey et al, 2017), shelling percentage (Luo et al, 2019a), bacterial wilt (Luo et al, 2019b) and test a colour (Zhao et al, 2019). QTL-seq approach has also been successfully deployed in discovery of genomic regions and candidate genes with high accuracy and precision in some other crops such as cucumber (Lu et al, 2014), tomato (Illa-Berenguer et al, 2015), pigeonpea (Singh et al, 2016a) and chickpea (Das et al, 2015;Singh et al, 2016b).…”