Insight into the Chromosome Structure of the Cultivated Tetraploid Alfalfa (Medicago sativa subsp. sativa L.) by a Combined Use of GISH and FISH Techniques.

Egizia Falistocco

doi:10.3390/plants9040542

Abstract

Cytogenetic research in Medicago sativa subsp. sativa L., the cultivated tetraploid alfalfa (2n = 4x = 32), has lagged behind other crops mostly due to the small size and the uniform morphology of its chromosomes. However, in the last decades, the development of molecular cytogenetic techniques based on in situ hybridization has largely contributed to overcoming these limitations. The purpose of this study was to extend our knowledge about the chromosome structure of alfalfa by using a combination of genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) techniques. The results of self-GISH (sGISH) suggested that a substantial part of the repetitive fraction of the genome of subsp. sativa is constituted by tandem repeats typical of satellite DNA. The coincidence of sGISH and C-banding patterns supported this assumption. The FISH mapping of the Arabidopsis-type TTTAGGG telomeric repeats demonstrated, for the first time, that the alfalfa telomeres consist of this type of sequence and revealed a massive presence of interstitial telomeric repeats (ITRs). In the light of this finding M. sativa appears to be a suitable material for studying the origin and function of such extra telomeric repeats. To further exploit this result, investigation will be extended to the diploid subspp. coerulea and falcata in order to explore possible connections between the distribution of ITRs, the ploidy level, and the evolutionary pathway of the taxa.

Highlights

Medicago sativa subsp. sativa L. (2n = 4x = 32), the cultivated alfalfa, is one of the most important forage crops in the world and the most intensively studied species of its genus
The chromosome structure in subsp. sativa was investigated based on the distribution patterns of repetitive and heterochromatin by sativa using a combination of Self-genomic in situ hybridization (sGISH) and Giemsa C-banding
The chromosome structure in subsp. sativa was investigated based on the distribution patterns and C-banding, and the metaphase chromosomes of the alfalfaand ecotype areC-banding displayed in of repetitive DNA

Summary

Introduction

Cytogenetic research on it has lagged behind other crop species mostly due to the characteristics of its chromosomes, which are small and very uniform in shape and size, making it difficult to perform detailed analyses. Progress achieved in molecular cytogenetic techniques based on in situ hybridization, such as fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), has opened new opportunities for the study of plant species with this type of chromosome [1]. The FISH procedure using simultaneously 45S and 5S rDNA probes provided the first cytogenetic markers for chromosome identification in the tetraploid subsp. The GISH procedure, a very effective way for examining the organization of repetitive DNA in plant genomes, has never been applied. It is known that a major component of the Plants 2020, 9, 542; doi:10.3390/plants9040542 www.mdpi.com/journal/plants

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