Modern methods of genome analysis (RGA- and MADS-labeling): use for biodiversity assessment and identification of genotypes of genebanks and botanic gardens collections

Author: E.Z. Kochieva, N.N. Ryzhova (Center "Bioengineering" RAS, Moscow, Russia).

At present, the value of collections of plant genetic resources represented in national genebanks and botanical gardens has increased many times, since they are the basis for many scientific studies, including the problems of assessing the biodiversity of natural communities, as well as taxonomy and phylogeny. In addition, due to the danger of depletion of genetic resources and possible genetic erosion in places of growth of species, genebanks and botanical gardens may become the sole custodian of unique genetic material. Therefore, the requirements for the organization of collections of genetic resources tightened. Rational organization of genetic collections has now become a prerequisite for the reliable conservation and efficient use of genetic resources. The key role in this is played by the assessment of biodiversity presented in the collection, as well as the nature and level of plant genetic diversity representing each sample of the collection.

Characteristics of samples of genetic collections of genebanks and botanical gardens are usually based on morphophysiological signs, less often biochemical. Over the past few decades, ex situ collections have extremely increased in number and volume as a result of attempts to conserve plant genetic resources. A set of recommendations for the effective management of national genebanks has been developed (FAO, 1998).

The solution of problems at the present stage is seen in the use of molecular methods of DNA analysis of collections, which is proposed by FAO as a basic characteristic of the genetic diversity presented in the collections.

Molecular analysis of genetic diversity within the sample and between samples of the collection allows you to simultaneously solve a number of tasks that are relevant for proper organization and characterization of genetic collections, such as identifying duplicated samples, verifying the taxonomic status of samples, creating a genebank of DNA samples, molecular certification of samples .

It should be noted that the level of morphological polymorphism does not always correlate with genetic variability and, thus, the molecular assessment of the collection can help realistically assess the level of biodiversity represented in the collections, as well as identify new potential sources of genetic diversity.

Currently, the molecular characteristics of collection samples were based on the method of complex genomic analysis, involving the use of different molecular marking methods (AFLP, RAPD, ISSR, analysis of gene families, analysis of nucleotide polymorphism of individual genes and non-coding sequences of both the nuclear genome and plastid DNA) . This approach allows us to fully characterize the genetic diversity of unique, functionally significant regions of the genome, as well as repetitive or evolutionarily neutral loci.

The purpose of this work was the use of the recently developed domain-directed labeling method [2, 3] for the analysis of biodiversity of two families of adaptive-significant genes: the resistance gene family and the MADS-homeosis genes of wild-growing and cultivated species of the Solanum genus (section Petota) and the genus Capsicum presented in the collections of the GNU GNC VIR (Russia) and CGN (Netherlands).

Modifications have been developed for labeling gene families characterized by the presence of specific conservative domains, such as the NBS-LLR domains of the resistance genes and the MADS-box domains of homeotic genes. These modifications were designated as RGA marking and MADS marking.

RGA labeling of sample collections. The analysis included 136 samples of 42 species of the genus Solanum (sect Petota) and 68 samples of 11 species of the genus Capsicum. Specific primers developed on the basis of the conserved NBS domain sequences made it possible to obtain 304 RGA fragments of the NBS-LRR family of potato resistance genes, 293 (89) of which turned out to be polymorphic. As a result, each sample was characterized by a specific set of RGA fragments, and species-specific RGA spectra were obtained for samples of each type (Fig. 1).

Subsequent sequencing of polymorphic fragments confirmed their belonging to the RGA family of genes.

The level of polymorphism of RGA fragments detected in wild species of the genus Solanum varied. The calculated coefficients of interspecific genetic distances (GD) reached 0.56–0.78. The lowest variability of RGAs was shown for S. tuberosum (GD 0.10-0.14). Analysis of 33 S. tuberosum varieties revealed a rather low level of polymorphism of the RGA-family of genes compared with wild-growing potato species. About 75 fragments of the RGA spectra were monomorphic for both all S. tuberosum varieties and the wild-growing sample of S. tuberosum ssp. tuberosum.

As a result of the labeling of the Capsicum resistance genes, 163 polymorphic genome fragments were obtained. In addition, each type of pepper was characterized by a specific set of RGA markers, apparently reflecting the specificity of resistance to phytopathogens. A number of markers were present in the spectra of only individual pepper samples. An unexpectedly high level of polymorphism (GD 0.32–0.37) of RGA sequences was shown for wild-growing C. annuum representatives from Nicaragua and Guatemala, while the level of polymorphism of C. annuum varieties was extremely low (GD 0.03–0.08).

MADS - labeling collection samples. For the first time, the method of labeling the gene family of floraal organogenesis — the homeotic MADS-box genes — was used to assess the biodiversity of the genetic resources of Solanum and Capsicum, represented in the CGN and VIR collections.
The use of this method in the analysis of genomic diversity of 11 species of Capsicum allowed us to identify both species and genotype-specific fragments of the genome of pepper. On the basis of the obtained spectra, interspecific genetic distances were determined and the levels of diversity of individual types of collection were determined. Virtually every sample of the collection, with some exceptions, was characterized by a unique spectrum of MADS fragments.

According to the results of the analysis, representatives of wild-growing C. annuum differed in high values ​​of the variability of MADS-containing sequences (GD 0.16-0.25), comparable to interspecific genetic differences in Capsicum. At the same time, the variability of MADS-containing sequences in the studied C. annuum varieties was extremely low (GD 0.01–0.03).

In general, the polymorphism of Capsicum detected MADS fragments was lower than that of RGA fragments. For example, for C. chacoense, the percentage of polymorphic MADS fragments was 26.4, while the percentage of polymorphic RGA fragments in the spectra of this species was 43. Similar values ​​are obtained for specimens of C. chinense and C. baccatum species. An exception was the form of C. frutescens: MADS spectra of samples of this type gave 56.8 polymorphic bands against RGA polymorphism, when the number of variable fragments in samples of this type was only 37.7.

The data on the low variability of MADS – box sequences is consistent with the ideas about the nature of this family of genes, which, in contrast to the rapidly evolving RGA family of genes, are more conservative [1].

Thus, using the methods of RGA- and MADS-marking, the potential of variability of families of resistance genes and genes of plant organogenesis of representatives of Solanum and Capsicum species of the genebank collection of the GNU GNR VIR and CGN was studied for the first time. This method can be recommended for assessing the biodiversity of various species and varieties, characterizing the overall variability and plasticity of genomes, as well as assessing the genomic erosion of plant genetic resources presented in the collections of genebanks and botanical gardens.

This work is supported by grants RFBR 07-04-01123 and 08-04-00300.

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