Peer Review #3 of "Genetic diversity, functional properties and expression analysis of NnSBE genes involved in starch synthesis of lotus (Nelumbo nucifera Gaertn.) (v0.2)"

Abstract

Background Starch branching enzyme (SBE) is one of the key enzymes in starch biosynthetic metabolism, determining amylopectin structure. Methods Full length coding sequences (CDS) of SBE genes were cloned using reverse transcription PCR (RT-PCR) technology, and neighbor-joining (NJ) tree was used for phylogenetic analysis. Single nucleotide polymorphisms (SNPs) were determined to assess the genetic polymorphisms and variation indexes between individuals and clusters. Quantitative real time PCR (qRT-PCR) was performed to analyze the spatial and temporal expression of NnSBE genes. The effect of NnSBE genes on amylopectin's fine structures was explored using affinity and the enzyme activity analysis of two isoforms in amylopectin and amylose. Results In this study, two SBE family genes, NnSBEI and NnSBEIII, were identified in lotus (Nelumbo nucifera Gaertn.). Phylogenetic analysis sorted NnSBEI into SBE family B and NnSBEIII into SBE family A. UPGMA phylogenetic tree divided 45 individuals of lotus into three classes. The homozygous haplotype (A G G A G) of NnSBEIII was observed in seed lotus. During the seed embryo development stage, NnSBEIII reached the peak in the middle of the development stage, while NnSBEI increased in the mid-late developmental stage. The different affinity activity of the two isozymes binding amylopectin and amylose assay indicated NnSBEI has higher activity and wider affinity. Discussion Genetic diversity showed that NnSBE genes received artificial selection during the process of cultivation and domestication in lotus seeds. Furthermore, the expression pattern and affinity activity analysis indicated that NnSBE genes were related to the chain length of amylopectin.