BackgroundDetailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.ResultsIn this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.ConclusionThe MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.

Abstract. Sormin SYM, Purwantoro A, Setiawan AB, Teo CH. 2021. Application of inter-SINE amplified polymorphism (ISAP) markers for genotyping of Cucumis melo accessions and its transferability in Coleus spp. Biodiversitas 22: 2918-2929. An unambiguous characterization of melon (Cucumis melo L.) accessions based on their morphological traits is often laborious and affected by environment when compared with molecular marker genotyping. Short interspersed nuclear elements (SINEs) are highly abundant non-autonomous and non-coding retrotransposons that are widely scattered over all chromosomes of eukaryotes. They can serve as a good molecular marker for routine genotyping in plant breeding and marker-assisted selection. This study aimed to apply inter-SINE amplified polymorphism (ISAP) markers for genotyping of Cucumis melo accessions and its transferability in Coleus spp. Twenty-one C. melo accessions, one C. metuliferus E. Mey. ex Naudin, and 15 accessions of Coleus spp. were used for ISAP marker development. A copy of cucumber-specific long interspersed nuclear element (LINE) and multiple copies of melon-specific SINE were identified and isolated. ISAP primers were designed from the highly conserved region of the SINEs and LINE. The melon and cucumber-specific ISAP markers showed a higher degree of polymorphism (87.5%-100%) than potato ISAP markers (60%-100%) in all the tested melon accessions. The unweighted pair group method with arithmetic average (UPGMA) dendrogram generated from polymorphic ISAP bands clearly distinguish the Cucumis melo accessions from their distantly related wild species C. metuliferus. The transferable nature of Cucumis ISAP marker system was demonstrated in Coleus species, where the marker differentiates the tested accessions.

Abstract. Fatmawati Y, Setiawan AB, Purwantoro A, Respatie DW, Teo CH. 2021. Analysis of genetic variability in F2 interspecific hybrids of mung bean (Vigna radiata) using inter-retrotransposon amplified polymorphism marker system. Biodiversitas 22: 4880-4889. Mung bean (Vigna radiata L. Wilczek) categorized as one of the pivotal annual crops of Vigna genera is commonly cultivated in rotation with the cereal crops during the drought season. Conversely, to ameliorate its stunted productivity, the interspecific hybridization technique has been introduced between the mung bean and the common bean to promote genetic improvement with the breeding projects in Indonesia. However, since mung bean is a self-pollinated crop and has a narrow genetic base, the selection and improvement of a specific trait using marker-assisted selection is more challenging. Hence, a precautionary investigation is imperative to evaluate the progenies resulting from interspecific hybridization using an ideal marker. This study aimed to investigate the genetic variability of the F2 population of the interspecific mung bean hybrids using retrotransposon-based markers, particularly Inter-Retrotransposon Amplified Polymorphism (IRAP) markers. In this study, we identified retrotransposon from the mung bean genome and determined the Long Terminal Repeat (LTR) sequence using the LTR Finder. The IRAP primers were designed from a conserved region of the LTR sequence. One hundred of the F2 interspecific hybrids generated from the crossing between mung bean and common bean were successfully discriminated by IRAP markers. The IRAP marker showed high heterozygosity and moderate Polymorphic Information Content (PIC) values. The IRAP markers were able to detect genetic variability in the F2 progenies resulting from the interspecific hybridization. Cluster analysis showed that 100 of the F2 progenies were grouped into three clusters. This study demonstrated that retrotransposon-based markers can offer an effective approach for evaluating the segregation in the F2 population of intercross hybrids in the mung bean.

Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that <i>Menolird18</i>, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in <i>Cucumis melo</i>, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, <i>Menolird18</i> was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of <i>Menolird18</i> were found in centromeric and rDNA regions of mitotic chromosomes suggests that <i>Menolird18</i> is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of <i>Menolird18</i> in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (<i>C. melo</i>) and cucumber (<i>C. sativus</i>) genomes.