The Banana Germplasm Genome Type Identification Of Molecular Markers System And Application

Bananas(Musa spp. sect. Eumusa) originated from intra-and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. The traditional method for determining the genome composition of Musa cultivars is based on the Simmonds-Shepherd scoring system which relies primarily on morphological characters. Several studies have investigated to differentiate the four genomes (A, B, S, T) involved in banana cultivars using genomic in situ hybridization (GISH), which also could be used to determine genome structure of banana cultivars containing A and/or B genomes. However, the method of GISThe banana can be divided into two groups, the cultivated species and the wild species. Nowadays,the cultivated banana species are formed by the intraspecific and/or interspecific hybridization of two wild diploid, Musaacuminata Colla (genome A) and Musabalbisiana Colla(genome B). Thus, they are different from each other in ploidy and genotype. In the past,the study on the genotypes of banana lied in their morphology. However, due to the impact of the environment and the stage of development stage, the results can be affected. Firstly, part of the cultivated species and the wild species were analyzed on Ploidy identification by flow cytometry. Those material were collected in thH is time-consuming and requires a high level of experimental skill. Previous investigators have found that molecular makers including PCR-RFLP of the rDNA ITS region, RAPD and IRAP, could be suitable for identifying genome composition, and have several advantages over morphological scoring system and molecular cytogenetics methods like GISH. However, those methods of differentiating triploid AAB and ABB types mostly relied on the band intensity of PCR-amplification products, regardless of the molecular maker tools they used. Many factors could strongly affect the band intensity during DNA extraction, PCR-amplification, gel electrophoresis and stain process, which inconsequently influences the accuracy of genome identification in bananas. In this study, we presented a more reliable identification strategy, which is based on a combination of molecular markers to differentiate A and B genomes, together with ploidy selection by flow cytometry. Flow cytometric analysis of nuclear DNA content was firstly used to estimate ploidy levels of bananas. Secondly, the digested DNA fragments of rDNA ITS region amplified by ITS L and ITS 4 primers and digested by Rsal, were separated and in which of 530-bp fragment was diagnostic for the presence of’A’genome while the 350-and 180-bp fragments were diagnostic for the presence of the’B’genome. If the ploidy level of the samples was diploid, their genome compositions are AA, BB, or AB, depending on the presence of different fragments tagging’A’and/or’B’genomes. If the ploidy level of the samples was triploid or tetraploid, their genome compositions are AAA, AAAA, or BBB, BBBB, depending on the presence of the only fragments tagging’A’or’B’genomes, respectively. However, if the digested ITS fragments were showed the presence of both’A’and’B’genomes, the next step of molecular identification using IRAP was required to identify the number of’B’genome complements. Thus, multiple polymorphic bands were firstly amplified with the gypsy-IRAP primer from genomic DNA of those triploid samples, in which of-350-bp fragment was tagged for the’B’genome. After that, a second PCR with Musa copia-IRAP primer pair (BFor plus BRev) from’B’specific bands was designed with Alul. And, the restriction pattern showing one or two fragments around 200 bp was linked to one or two’B’within the genome composition of those triploid samples, viz. AAB or ABB. For tetraploid bananas, one 200-bp fragment could be indentified as AAAB type, but if two 200-bp fragments were observed, it remains hardy to distinguish the AABB and ABBB types in them. The choosing PCR-RFLP of ITS as the first step to identify the genome composition, is the less fragments amplified, giving clear and distinguishable fragments linked with’A’and’B’genomes. The presented identification strategy provides a simple, fast and more accurate method to large-scale throughput demands in determining the genome constitution of mostly Musa accessions and hybrid populations generated in breeding programs.