Conjunctive Analyses of BSA-Seq and BSR-Seq to Identify Candidate Genes Controlling the Black Lemma and Pericarp Trait in Barley

Abstract

Black barley seeds are a health-beneficial diet resource because of their special chemical composition and antioxidant properties. The black lemma and pericarp (BLP) locus was mapped in a genetic interval of 0.807 Mb on chromosome 1H, but its genetic basis remains unknown. In this study, targeted metabolomics and conjunctive analyses of BSA-seq and BSR-seq were used to identify candidate genes of BLP and the precursors of black pigments. The results revealed that five candidate genes, purple acid phosphatase, 3-ketoacyl-CoA synthase 11, coiled-coil domain-containing protein 167, subtilisin-like protease, and caffeic acid-O-methyltransferase, of the BLP locus were identified in the 10.12 Mb location region on the 1H chromosome after differential expression analysis, and 17 differential metabolites, including the precursor and repeating unit of allomelanin, were accumulated in the late mike stage of black barley. Phenol nitrogen-free precursors such as catechol (protocatechuic aldehyde) or catecholic acids (caffeic, protocatechuic, and gallic acids) may promote black pigmentation. BLP can manipulate the accumulation of benzoic acid derivatives (salicylic acid, 2,4-dihydroxybenzoic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid, protocatechuic aldehyde, and syringaldehyde) through the shikimate/chorismite pathway other than the phenylalanine pathway and alter the metabolism of the phenylpropanoid-monolignol branch. Collectively, it is reasonable to infer that black pigmentation in barley is due to allomelanin biosynthesis in the lemma and pericarp, and BLP regulates melanogenesis by manipulating the biosynthesis of its precursors.

Transcriptional convergence after repeated duplication of an amino acid transporter gene leads to the independent emergence of the black husk/pericarp trait in barley and rice

“…We screened 3200 inbred lines from our BC 2 F 6 and TH1-7 populations using previous markers (Long et al, 2019), plus 10 newly designed indel markers (Table S1), which enabled us to position HvBlp between markers BLZK98 and MC_1570156, spanning 0.484 Mb using Morex (V3) as reference (Figure 1d). Within this genetic interval, Morex (yellow husk) contains only seven annotated genes, none of which seem to be related to melanin or anthocyanin production.…”

“…The candidate gene controlling the black husk/pericarp trait in barley has puzzled barley researchers for a long time. Several previous studies (Bungartz et al ., 2016 ; Glagoleva et al ., 2017 , 2022 ; Jia et al ., 2017 ; Liu et al ., 2023 ; Long et al ., 2019 ; Shoeva et al ., 2016 ) have attempted to uncover its molecular basis but have all failed to date. In the present study, we identified the corresponding candidate gene HvBlp by screening an exceptionally large collection of 3200 inbred lines.…”

“…L: likelihood value. Amino acid sites were numbered according to HvBlp 2022; Jia et al, 2017;Liu et al, 2023;Long et al, 2019;Shoeva et al, 2016) have attempted to uncover its molecular basis but have all failed to date. In the present study, we identified the corresponding candidate gene HvBlp by screening an exceptionally large collection of 3200 inbred lines.…”

“…In nature, mature grains of rice, maize, wheat and barley plants have evolved different colours such as yellow, purple, red, blue and black due to different pigmentations (Abdel-Aal et al, 2006;Paulaneyer et al, 2017), potentially as a result of environmental adaptation because common pigments such as anthocyanins and flavonoids are strong antioxidants and can help plants address various stress conditions (Jia et al, 2020;Kaur et al, 2022;Shomali et al, 2022). Unlike other colours, the black grain trait, prominently observed in wild rice (Zhu et al, 2011) and wild barley (Long et al, 2019;Shoeva et al, 2020), was attributed to the accumulation of plant melanins instead of anthocyanins in the lemma and/or pericarp (Fei et al, 2021;Shoeva et al, 2020;Varga et al, 2016). Noteworthy, the black hull trait in rice needs to be differentiated from the deep purple colouration trait (often mistaken as black), which is mainly due to anthocyanin accumulation and is controlled by candidate genes in the anthocyanin biosynthesis pathway (Mackon et al, 2021;Oikawa et al, 2015;Upadhyaya et al, 2021).…”

“…In barley, the black grain trait has long been known to be controlled by a single dominant locus Blp on chromosome 1H (Costa et al, 2001). Several previous studies have attempted to identify the Blp gene (Bungartz et al, 2016;Glagoleva et al, 2017Glagoleva et al, , 2022Jia et al, 2017;Long et al, 2019;Shoeva et al, 2016) and have narrowed it to an extremely small genetic region, containing only 21 annotated genes (Long et al, 2019). However, the corresponding candidate gene is still hiding in mystery.…”