Development of Transgenic Sugarcane Resistant to Stem Borer by Transforming cry1Ab-cry1Ac Fusion Gene through Agrobacterium tumefaciens Transformation Method

<p>Bulu Lawang (BL) is a sugarcane variety preferred by farmers in Indonesia due to its high yield, but this cultivar is susceptible to shoot and stem borer insect pests. Genetic engineering using cry1Ab and cry1Ac fusion gene is an effort to generate BL varieties resistant to the insect pests. This study aimed to 1) transform T-DNA containing cry1Ab-cry1Ac fusion gene into sugarcane genome by using Agrobacterium tumefaciens method, 2) obtain selection media composition of callus transformants, and 3) obtain transformation efficiency comparison of A. tumefaciens strains EHA105 and GV3101. The research was conducted at the Laboratory of Cell and Tissue Biology, the Laboratory of Molecular Biology, and the greenhouse of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor from March to August 2019. Research activities consisted of four parts namely 1) callus induction and Agrobacterium culture preparation containing pCambia5300-cry1Ab-cry1Ac ///// pCambia-5300_OaRbcS-prom-cTP-cry1Ab-cry1Ac plasmid, 2) callus incubation in cocultivation and resting media, 3) selection and differentiation of shoots on regeneration media, and 4) molecular analysis using PCR method. Results showed that the composition of media, both for selection and regeneration processes of putative plant transformants, was the key to the success of this experiment. A. tumefaciens strain EHA105 resulted in higher transformation efficiency (11.1%) compared to that of strain GV3101 (9.0%). Molecular analysis showed that cryIAb-cryIAc fusion gene was successfully inserted into the sugarcane genome suggesting that the transgenic plant containing cry1Ab-cry1Ac fusion gene was obtained. The putative transgenic plants need further assay through bioassay tests to verify its resistance phenotype to the insect pests.</p>

Base Excision Repair in Sugarcane – A New Outlook

The base excision repair (BER) pathway has been associated with genome integrity maintenance. Owing to its central role, BER is present in all three domains of life. The studies in plants, considering BER, have been conducted using Arabidopsis and rice models. Therefore, future studies regarding BER are required in other organisms, particularly in crops such as sugarcane, to understand its mechanism, which may reflect the uniqueness of DNA repair in monocots. Our previous results have revealed that sugarcane is an interesting plant for studying this pathway considering the polyploidy genome and genome evolution. This chapter aimed to characterize the BER pathway in sugarcane by using different bioinformatics tools, for example, screening for BER homologs in the sugarcane genome to identify its members. Each sequence obtained was subjected to structural analysis, and certain differences were identified when Arabidopsis was compared to other monocots, including sugarcane. Moreover, ROS1, DEM, and DML3 were not identified as a complete sequence in the sugarcane EST database. Furthermore, FEN1 is present as two sequences, namely FEN1A and FEN1B, both featuring different amino acid sequence and motif presence. Furthermore, FEN1 sequence was selected for further characterization considering its evolutionary history, as sequence duplication was observed only in the Poaceae family. Considering the importance of this protein for BER pathway, this sequence was evaluated using protein models (3D), and a possible conservation was observed during protein–protein interaction. Thus, these results help us understand the roles of certain BER components in sugarcane, and may reveal the aspects and functions of this pathway beyond those already established in the literature.

Regional genomic heritability mapping for agronomic traits in sugarcane

BackgroundThe identification of genomic regions involved in agronomic traits is the primary concern for sugarcane breeders. Genome-wide association studies (GWAS) leverage the sequence variations to bridge phenotypes and genotypes. However, their effectiveness is limited in species with high ploidy and large genomes, such as sugarcane. As an alternative, a regional heritability mapping (RHM) method can be used to capture genetic signals that may be missed by GWAS by combining genetic variance from neighboring regions. We used RHM to screen the sugarcane genome aiming to identify regions with higher heritability associated with agronomic traits. We considered percentage of fiber in sugarcane bagasse (FB), apparent percentage of sugarcane sucrose (PC), tonnes of pol per hectare (TPH), and tonnes of stalks per hectare (TSH).MethodsSequence-capture data of 508 sugarcane (Saccharum spp.) clones from a breeding population under selection were processed for variant calling analysis using the sugarcane genome cultivar R570 as a reference. A set of 375,195 single nucleotide polymorphisms were selected after quality control. RHM was conducted by splitting the sugarcane genome into windows of 2 Mb length.ResultsWe selected the windows explaining > 20% of the total genomic heritability for TPH (64 windows - 5,654 genes) and TSH (72 windows - 6,050 genes), and > 15% for PC (16 windows - 1,517 genes) and FB (17 windows - 1,615 genes). The top five windows that explained the highest genomic heritability ranged from 20.8 to 24.6% for FB (629 genes), 18.0 to 22.0% for PC (452 genes), 53.8 to 66.0% for TPH (705 genes), and 59.5 to 67.4% for TSH (413 genes). The functional annotation of genes included in those top five windows revealed a set of genes that encode enzymes that integrate carbon metabolism, starch and sucrose metabolism, and phenylpropanoid biosynthesis pathways.ConclusionsThe selection of windows that explained the large proportions of genomic heritability allowed us to identify genomic regions containing a set of genes that are related to the agronomic traits in sugarcane. These windows spanned a region of 58.38Mb, which corresponds to 14.28% of the reference assembly in the sugarcane genome. We contend that RHM can be used as an alternative method for sugarcane breeders to reduce the complexity of the sugarcane genome.

A miniature inverted-repeat transposable element, AddIn-MITE, located inside a WD40 gene is conserved in Andropogoneae grasses

Miniature inverted-repeat transposable elements (MITEs) have been associated with genic regions in plant genomes and may play important roles in the regulation of nearby genes via recruitment of small RNAs (sRNA) to the MITEs loci. We identified eight families of MITEs in the sugarcane genome assembly with MITE-Hunter pipeline. These sequences were found to be upstream, downstream or inserted into 67 genic regions in the genome. The position of the most abundant MITE (Stowaway-like) in genic regions, which we call AddIn-MITE, was confirmed in a WD40 gene. The analysis of four monocot species showed conservation of the AddIn-MITE sequence, with a large number of copies in their genomes. We also investigated the conservation of the AddIn-MITE’ position in the WD40 genes from sorghum, maize and, in sugarcane cultivars and wild Saccharum species. In all analyzed plants, AddIn-MITE has located in WD40 intronic region. Furthermore, the role of AddIn-MITE-related sRNA in WD40 genic region was investigated. We found sRNAs preferentially mapped to the AddIn-MITE than to other regions in the WD40 gene in sugarcane. In addition, the analysis of the small RNA distribution patterns in the WD40 gene and the structure of AddIn-MITE, suggests that the MITE region is a proto-miRNA locus in sugarcane. Together, these data provide insights into the AddIn-MITE role in Andropogoneae grasses.