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SALAD analysis selected the followingsequences for tree construction. Sequences are represented by the following abbreviations (accession numbersunigene names and chromosome location are in parentheses): barley HvPPO (AB, chr. HL), HvPPO (AB, chr. HL); wheat TaSSPPOA (AB chr. AL), TaSSPPOB (AB chr. BL), TaSSPPOD (AB chr. DL), TaPpoAa (EF chr. AL), TaPpoAb (EF chr. AL), TaPpoDa (EF chr. DL), TaPpoDb (EF chr. DL); rice OsPPO (LOC_Osg, chr.), OsPPO (LOC_Osg, chr.); maize ZmPPO (EU chr.), ZmPPO (EU chr.); sorghum SbPPO (Sbg, chr.), SbPPO (Sbg, chr.), SbPPO (Sbg, chr.), SbPPO (Sbg, chr.), SbPPO (Sbg, chr.), and B. distachyon BdPPO (Bradig, chr.). (A) Phylogenetic tree. Amino acids on the central tyrosinase domain (Pfam) have been compared and also the tree was constructed using the Neighbor oining system. Numbers indicate bootstrap values. PPO proteins were classified into two big groups labelled with group A and group B. (B) Motif evaluation by SALAD database.reactionnegative accessions were Odel complexity will alsoDOI: .acs.est.b Environ. Sci. Technol. , Environmental genotyped. Figureportrays the geographical distribution of mutant PPO genes that have been found in domesticated barley accessions using a adverse phenolreaction of awns. Of ,accessions had been classified into one of the five nonfunctional types discovered by sequencing. Nonetheless, the remainingaccessions have been indistinguishable from phenol reactionpositive accessions by marker analyses and are classified as other people. These unclassified accessions, which have been collected in India, China, and Nepal, demand further sequencing inside the promoter region. Kind IV was most prevalent and wasdistributed in wide places ranging from Turkey, Iran, and Afghanistan, to Xinjiang. This distribution pattern indicates that sort IV was disseminated by human activities along the Silk Road. Sorts I and III were locally distributed in Pakistan; type I accessions are all naked caryopsis (nud), and variety III accessions all have brief rachilla hair (srh). Kind II was only identified in Spain and this variety carried quick rachilla hair (srh). Variety V was discovered only in Korea. These final results show that lossoffunction mutations on the PPO gene occurred independently as barley spread from the two centres of origin, namely the Close to East and southern areasBarley polyphenol oxidases and the phenol reaction of Central Asia (Morrell and Clegg, ; Saisho and Purugganan,). The phenol reaction of the representative phenol reactionnegative barley accessions and NILs were tested. The outcomes showed that PPO mutants are not stained in awns (Fig. A), grains (Fig. B), hulls or rachises, although these tissues are strongly stained within the phenol reactionpositive manage. It truly is especially exciting that a closer examinat.Bootstrap values. Barley PPOs were clustered within the same significant clade (labelled with group A), which contains the rice chromosomePPO (OsPPO). Yet another rice chromosomePPO (OsPPO) formed a different significant clade (group B) like no barley or wheat clone. These phylogenetic information validate that the two barley PPO homologues isolated in this study represent accurate PPO genes using a conserved domain of tyrosinase. The two linked HvPPO and HvPPO in barley are likely to possess been derived by duplication. Two sorghum PPOs (SbPPO and SbPPO) which are linked on chromosomeare sister for the barley and wheat clade. It remains unknown whether PPO gene duplication in barley (chromosome H) and sorghum (chromosome) on syntenic chromosomes possess a common origin or not.Taketa et al.Fig. . Phylogenetic analysis of PPO proteins in six grass species.