Functional Haplotypes and Evolutionary Insight into the Granule-Bound Starch Synthase II (GBSSII) Gene in Korean Rice Accessions (KRICE_CORE)

Granule-bound starch synthase 2 (GBSSII), a paralogous isoform of GBSSI, carries out amylose biosynthesis in rice. Unlike GBSSI, it mainly functions in transient organs, such as leaves. Despite many reports on the starch gene family, little is known about the genetics and genomics of GBSSII. Haplotype analysis was conducted to unveil genetic variations (SNPs and InDels) of GBSSII (OS07G0412100) and it was also performed to gain evolutionary insight through genetic diversity, population genetic structure, and phylogenetic analyses using the KRICE_CORE set (475 rice accessions). Thirty nonsynonymous SNPs (nsSNPs) were detected across the diverse GBSSII coding regions, representing 38 haplotypes, including 13 cultivated, 21 wild, and 4 mixed (a combination of cultivated and wild) varieties. The cultivated haplotypes (C_1–C_13) contained more nsSNPs across the GBSSII genomic region than the wild varieties. Nucleotide diversity analysis highlighted the higher diversity values of the cultivated varieties (weedy = 0.0102, landrace = 0.0093, and bred = 0.0066) than the wild group (0.0045). The cultivated varieties exhibited no reduction in diversity during domestication. Diversity reduction in the japonica and the wild groups was evidenced by the negative Tajima’s D values under purifying selection, suggesting the domestication signatures of GBSSII; however, balancing selection was indicated by positive Tajima’s D values in indica. Principal component analysis and population genetics analyses estimated the ambiguous evolutionary relationships among the cultivated and wild rice groups, indicating highly diverse structural features of the rice accessions within the GBSSII genomic region. FST analysis differentiated most of the classified populations in a range of greater FST values. Our findings provide evolutionary insights into GBSSII and, consequently, a molecular breeding program can be implemented for select desired traits using these diverse nonsynonymous (functional) alleles.

The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 – 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

Haplotype Variations and Evolutionary Analysis of the Granule-Bound Starch Synthase I Gene in the Korean World Rice Collection

Granule-bound starch synthase I (GBSSI) is responsible for Waxy gene encoding the, which is involved in the amylose synthesis step of starch biosynthesis. We investigated the genotypic and haplotypic variations of GBSSI (Os06g0133000) gene, including its evolutionary relatedness in the nucleotide sequence level using single-nucleotide polymorphisms (SNPs), indels, and structural variations (SVs) from 475 Korean World Rice Collection (KRICE_CORE), which comprised 54 wild rice and 421 cultivated represented by 6 ecotypes (temperate japonica, indica, tropical japonica, aus, aromatic, and admixture) or in another way by 3 varietal types (landrace, weedy, and bred). The results revealed that 27 of 59 haplotypes indicated a total of 12 functional SNPs (fSNPs), identifying 9 novel fSNPs. According to the identified novel fSNPs, we classified the entire rice collection into three groups: cultivated, wild, and mixed (cultivated and wild) rice. Five novel fSNPs were localized in wild rice: four G/A fSNPs in exons 2, 9, and 12 and one T/C fSNP in exon 13. We also identified the three previously reported fSNPs, namely, a G/A fSNP (exon 4), an A/C fSNP (exon 6), and a C/T fSNP (exon 10), which were observed only in cultivated rice, whereas an A/G fSNP (exon 4) was observed exclusively in wild rice. All-against-all comparison of four varietal types or six ecotypes of cultivated rice with wild rice showed that the GBSSI diversity was higher only in wild rice (π = 0.0056). The diversity reduction in cultivated rice can be useful to encompass the origin of this gene GBSSI during its evolution. Significant deviations of positive (wild and indica under balancing selection) and negative (temperate and tropical japonica under purifying selection) Tajima's D values from a neutral model can be informative about the selective sweeps of GBSSI genome insights. Despite the estimation of the differences in population structure and principal component analysis (PCA) between wild and subdivided cultivated subgroups, an inbreeding effect was quantified by FST statistic, signifying the genetic relatedness of GBSSI. Our findings of a novel wild fSNPS can be applicable for future breeding of waxy rice varieties. Furthermore, the signatures of selective sweep can also be of informative into further deeper insights during domestication.

A null allele of granule bound starch synthase (Wx-B1) may be one of the major genes controlling chapatti softness

Chapatti (unleavened flatbread) is a staple food in northern India and neighboring countries but the genetics behind its processing quality are poorly understood. To understand the genes determining chapatti quality, differentially expressed genes were selected from microarray data of contrasting chapatti cultivars. From the gene and trait association studies, a null allele of granule bound starch synthase (GBSS; Wx-B1) was found to be associated with low amylose content and good chapatti quality. For validation, near-isogenic lines (NILs) of this allele were created by marker assisted backcross (MAB) breeding. Background screening indicated 88.2 to 96.7% background recovery in 16 selected BC3F5 NILs. Processing quality and sensory evaluation of selected NILs indicated improvement in chapatti making quality. Traits that showed improvement were mouthfeel, tearing strength and softness indicating that the Wx-B1 may be one of the major genes controlling chapatti softness.

Competition between Granule Bound Starch Synthase and Starch Branching Enzyme in Starch Biosynthesis

Background Starch branching enzymes (SBE) and granule-bound starch synthase (GBSS) are two important enzymes for starch biosynthesis. SBE mainly contributes to the formation of side branches, and GBSS mainly contributes for the synthesis of amylose molecules. However, there are still gaps in the understanding of possible interactions between SBE and GBSS. Results Nineteen natural rice varieties with amylose contents up to 28% were used. The molecular structure, in the form of the chain-length distribution (CLDs, the distribution of the number of monomer units in each branch) was measured after enzymatic debranching, using fluorophore-assisted carbohydrate electrophoresis for amylopectin and size- exclusion chromatography for amylose. The resulting distributions were fitted to two mathematical models based on the underlying biosynthetic processes, which express the CLDs in terms of parameters reflecting relevant enzyme activities. Conclusions Finding statistically valid correlations between the values of these parameters showed that GBSSI and SBEI compete for substrates during rice starch biosynthesis, and synthesis of amylose short chains involves several enzymes including GBSSI, SBE and SSS (soluble starch synthase). Since the amylose CLD is important for a number of functional properties such as digestion rate, this knowledge is potentially useful for developing varieties with improved functional properties.

Proteomic analyses of Citrus petiole responses to early Huanglongbing Disease

Backgroud Huanglongbing (HLB) is currently one of the most destructive citrus disease worldwide. It is caused by Candidatus Liberibacter asiaticus (CLas), a nonculturable alpha-proteobacterium, which it resides exclusively in the phloem tissues. Therefore, understanding the early CLas-responsive proteins in citrus petiole where pathogenic bacteria colonized will help to investigate plant resistance to the pathogen.Results In this study, a comparative proteomic approach was applied to identify the petiole proteins associated with the response to CLas infection. A total of 777 proteins were differentially expressed in response to CLas. Among them, 499 proteins were up-regulated and 278 were down-regulated. Among the most highly up-regulated differentially expressed proteins (DEPs) were salicylate carboxymethyltransferase, ubiquitin carboxyl-terminal hydrolase 13, trans-resveratrol di-O-methyltransferase, linoleate 13S-lipoxygenase 2-1, granule-bound starch synthase 1 and thaumatin-like proteins. While the most highly down-regulated DEPs were oxygen-evolving enhancer proteins, ribulose bisphosphate carboxylase/oxygenase activase, peroxidases and photosystem reaction center subunits. The results of qPCR analysis of a number of indicated DEPs and western blotting further validated four representative DEPs, including salicylate carboxymethyltransferase, linoleate 13S-lipoxygenase 2-1, granule-bound starch synthase 1 and photosystem I reaction center subunit showed that most of detected DEPs were positively correlated with their mRNA and protein levels.Conclusions Our comparative proteomic analysis first profiling reveals early and primary proteome alterations in CLas-infected citrus petiole, where pathogens reside in. The DEPs results demonstrate that CLas infection could promote the carbohydrate metabolism, depress the photosystem and activate/inhibit defense responses.