Ines Van den houwe

A platform for efficient genotyping in Musa using microsatellite markers

Establishment of a standardized platform for genotyping banana (Musa spp.) using a set of previously published SSR markers is described. The platform will serve a broad Musa research and breeding community and support the conservation and use of genetic diversity.

Molecular and cytological characterization of the global Musa germplasm collection provides insights into the treasure of banana diversity

Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank.

A genome-wide association study on the seedless phenotype in banana (#Musa# spp.) reveals the potential of a selected panel to detect candidate genes in a vegetatively propagated crop

Banana (Musa sp.) is a vegetatively propagated, low fertility, potentially hybrid and polyploid crop. These qualities make the breeding and targeted genetic improvement of this crop a difficult and long process. The Genome-Wide Association Study (GWAS) approach is becoming widely used in crop plants and has proven efficient to detecting candidate genes for traits of interest, especially in cereals. GWAS has not been applied yet to a vegetatively propagated crop. However, successful GWAS in banana would considerably help unravel the genomic basis of traits of interest and therefore speed up this crop improvement. We present here a dedicated panel of 105 accessions of banana, freely available upon request, and their corresponding GBS data. A set of 5,544 highly reliable markers revealed high levels of admixture in most accessions, except for a subset of 33 individuals from Papua. A GWAS on the seedless phenotype was then successfully applied to the panel. By applying the Mixed Linear Model corrected for both kinship and structure as implemented in TASSEL, we detected 13 candidate genomic regions in which we found a number of genes potentially linked with the seedless phenotype (i.e. parthenocarpy combined with female sterility). An additional GWAS performed on the unstructured Papuan subset composed of 33 accessions confirmed six of these regions as candidate. Out of both sets of analyses, one strong candidate gene for female sterility, a putative orthologous gene to Histidine Kinase CKI1, was identified. The results presented here confirmed the feasibility and potential of GWAS when applied to small sets of banana accessions, at least for traits underpinned by a few loci. As phenotyping in banana is extremely space and time-consuming, this latest finding is of particular importance in the context of banana improvement.

Molecular and Cytogenetic Characterization of Wild Musa Species.

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The worlds largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny.

Thermotherapy, Chemotherapy, and Meristem Culture in Banana

Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the international exchange and conservation of banana germplasm-a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole(®) is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate.

Molecular and cytogenetic study of East African Highland Banana

East African highland bananas (EAHBs) are staple food crop in Uganda, Tanzania, Burundi, and other countries in the African Great Lakes region. Even though several morphologically different types exist, all EAHBs are triploid and display minimal genetic variation. To provide more insights into the genetic variation within EAHBs, genotyping using simple sequence repeat (SSR) markers, molecular analysis of ITS1-5.8S-ITS2 region of ribosomal DNA locus, and the analysis of chromosomal distribution of ribosomal DNA sequences were done. A total of 38 triploid EAHB accessions available in the Musa germplasm collection (International Transit Centre, Leuven, Belgium) were characterized. Six diploid accessions of Musa acuminata ssp. zebrina, ssp. banksii, and ssp. malaccensis representing putative parents of EAHBs were included in the study. Flow cytometric estimation of 2C nuclear DNA content revealed small differences (max ~6.5%) in genome size among the EAHB clones. While no differences in the number of 45S and 5S rDNA loci were found, genotyping using 19 SSR markers resulted in grouping the EAHB accessions into four clusters. The DNA sequence analysis of the internal transcribed spacer region indicated a relation of EAHB clones with M. acuminata and, surprisingly, also with M. schizocarpa. The results suggest that EAHB cultivars originated from a single hybrid clone with M. acuminata ssp. zebrina and ssp. banksii being its most probable parents. However, M. schizocarpa seems to have contributed to the formation of this group of banana.