New wild diploids in Paspalum notatum Flügge ( Poaceae ) : potential accessions for use in breeding

Paspalum notatum is a polymorphic species with two accepted varieties, according to the ploidy level. The ploidy level is closely related to the reproductive mode of this species. Usually, diploid cytotypes have sexual reproduction, and tetraploid cytotypes have an apomictic reproduction. Apomixis limits genetic recombination, hindering the development of new cultivars. The identification of sexual plants represents many possibilities in breeding programs. This study aimed to analyze the chromosome number in P. notatum accessions under agronomic evaluation. Ploidy level was determined in 25 accessions. Two ploidy levels were detected, resulting in 21 tetraploid and four diploid accessions. The four diploid accessions originate from Argentina and Uruguay, in a region close to the origin of Pensacola, which is a diploid sexual cultivar of P. notatum. These new diploid accessions may be useful in the development of breeding programs of Paspalum species.


INTRODUCTION
Paspalum L. comprises about 350 species distributed in tropical and subtropical American natural pastures (Zuloaga and Morrone 2005).Among the species of this genus, Paspalum notatum Flügge is considered as one of the most promising forage plants in Southern Brazil due to its yield, quality, resistance, and growth (Canto-Dorow et al. 1996, Pozzobon andValls 1997).
In the genus Paspalum, polyploidy is associated with apomixis (Moraes-Fernandes et al. 1974, Delgado et al. 2014), which is defined as an asexual mode of reproduction by seeds (Martinez et al. 2007).This reproduction mode is an obstacle to the genetic breeding because it hinders genetic recombination and consequently cultivars protection (Huber et al. 2016).Since apomixis prevents crosses, the genetic breeding in Paspalum is limited to the identification and selection of superior tetraploid accessions, without any breeding process (Aguilera et al. 2011).
The existence of sexual genotypes is an imperative prerequisite for any breeding program of polyploid apomictic grasses (Sartor et al. 2009).In other Paspalum species, cytogenetic analyses have revealed diploid accessions (Pozzobon et al. 2008, Pozzobon et al. 2013).In P. notatum, sexual tetraploids plant were obtained by the chromosome duplication of cultivar Pensacola (Quarin et al. 2001, Weiler et al. 2015, Machado et al. 2017).However, in some cases, the mode of reproduction changes after duplication, and tetraploidized plants become apomictic (Quarin et al. 2001, Krycki et al. 2016).Quarin et al. (2001) state that the apomixis gene is present at the diploid level, but it is unexpressed in the diploid plant.The ploidy-dependence may occur at a locus that controls the apomixis using a secondary locus that involves a higher allele dosage to affect the expression of the main locus.The expression of apomixis in this duplicated plant is possibly a gene-dosage effect.
The search of diploids in germplasm banks associated with morphologic and molecular analyses could distinguish new diploid accessions of P. notatum Pensacola.Moreover, the analysis of their agronomic performance could indicate their use in breeding programs of this species.Based on these considerations, this study aimed to determine the ploidy level in a collection of P. notatum accessions subject to agronomic and morphologic evaluation.

MATERIAL AND METHODS
A cytogenetic study was carried out on 25 P. notatum accessions.These materials belong to the Germplasm Bank of the United States Department of Agriculture (USDA) and were collected from the years of 1950 to 1970 in Southern Brazil, Argentina, Uruguay, and Paraguay (Fig. 1).For this study, plants were obtained from an experiment for the agronomic evaluation conducted by Fachinetto et al. (2012).Ploidy level was determined from the gametic chromosome number and pollen mother cells (PMC) analysis.During the summer of 2010, young inflorescences of two individuals randomly chosen from each accession were collected and fixed in Carnoy 3:1 (ethanol: acetic acid) for 24 hours, at room temperature, and stored in ethanol 70% under refrigeration (Pereira et al. 2014, Moreira et al. 2017).Slides were prepared by isolating the anthers, squashing, and staining with 1% propionic carmine (Pagliarini et al. 2002, Simioni andVale 2009).Cells were analyzed in an optical microscope, totaling ten cells per individual.Gametic chromosome numbers were determined in diakinesis/ metaphase I and anaphase I when the chromosomes showed visible and adequate spread.The ploidy level was determined considering x = 10 chromosomes, described as the basic number for the species (Dahmer et al. 2008).
In this study, the diploid accessions were not considered as an escape of Pensacola due to their origin.Accessions 66N, 67N, and 92N were collected in Santa Fe, Argentina, while 87N was collected in Paysandú, Uruguay (Figure 1, Table 1).These four accessions were considered as wild diploids since they were collected in the origin region of Pensacola (Burton 1967).
These accessions are part of a larger P. notatum collection and were evaluated for agronomic, morphological, and molecular aspects.Dry matter yield was higher in three diploid accessions (66N, 67N and 92N); however, these accessions did not differ from 87N when compared with Pensacola.66N and 67N produced about four times more, and 92N produced about seven times more than Pensacola.Furthermore, these accessions showed superior persistence to winter conditions than Pensacola (Fachinetto et al. 2012).Moreover, morphological analyses indicated that the four diploid accessions grouped separately from each other and Pensacola (Fachinetto et al. 2017).
This separation into different morphological and molecular groups may be due to the higher variability since these accessions are probably of sexual reproduction.P. notatum var.saurae has narrower leaves, small spikelets, and more racemes per inflorescence (Quarin et al. 1984, Canto-Dorow et al. 1996), being classified together with plants from sexual reproduction.So far, all the diploid plants of this species (wild or cultivated) are typically sexual (Espinoza and Quarin 1997, Daurelio et al. 2004, Pozzobon et al. 2008).Delgado et al. (2014) stated that genetic determinants of apomixis found in diploids are not sufficient for a considerable expression of the trait, as found in P. rufum, and that the gene expression of this trait is related to other factors.
Apomixis is a type of asexual reproduction in which seeds formation occurs without fertilization.The resulting individuals are genetically identical to the plant that originated them.Asexual reproduction allows the genotypes to be fixed, and the desired traits to be maintained.Due to the absence of recombination, this phenomenon maintains the presence of gene blocks and linked genes, allowing the continuous exploration of heterosis, besides eliminating the need for plant isolation during seed production.However, plants that reproduce exclusively by apomixis hinder breeding processes for preventing genetic recombination between individuals, making them highly dependent on mutations to evidence genetic variability (Carvalho et al. 2008).These diploids, which probably present sexual reproduction, could be used for crossing between each other or even with Pensacola, without chromosome doubling.These findings would ensure sexual accessions for crossing since chromosome doubling can lead to apomixis (Quarin et al. 2001, Krycki et al. 2016).
Sexual plants with superior traits could be used in new recombinations within the breeding program, in the formation of new individuals, maximizing the benefits of hybrid vigor (Burton et al. 1973, Jank et al. 2011, Lopes et al. 2018).
After the crossing between the diploids and the obtainment of plants with favorable traits and heterosis, chromosome doubling could lead to apomictic tetraploid plants to be used to keep desired traits.The findings of this work may contribute to the Paspalum species, especially P. notatum, conferring new possibilities of crossing and insertion of genetic variability in breeding programs.