Morphological and phenological variability of Guaraní maize germplasm from Northeastern Argentina (NEA)

Abstract Morphological and phenological variability of Guaraní maize germplasm from Northeastern Argentina (NEA) are potential sources of genetic resources, constituting valuable repositories of alleles associated with tolerance and resistance to biotic and abiotic stresses. Here, we studied the morphological and phenological variability in maize populations of 14 Guaraní landraces from NEA and analysed the relationship among them. These landraces were characterised according to the endosperm consistency of grain as Popcorn (Pc), Floury (F) and Floury with corneal periphery (F-Pc). There were inter-population differences in phenological characters as time from germination to tassel emergence, from germination to spike emergence and from germination to anthesis (length of vegetative cycle -LVC). The morphological and phenological data here reported were used to carry out a cluster analysis, in which the F and Pc populations appeared as two separate groups and the F-Pc populations were included in the F group. Thus, the well-defined F (including F-Pc) and Pc genetic pools should be prioritised within the framework of breeding plans as well as ex situ and in situ conservation programs. For these purposes it is essential the morphological, phenological and genetic characterisation the maize Guaraní landraces.


Introduction
Maize is a major cereal crop worldwide, adapted to different climatic conditions and farming practices.It is cultivated from Alberta in Canada to the province of Chubut in Argentina, and from sea level to 4,000 m.a.s.l (Samayoa et al. 2018).The first extensive study of maize morphological diversity in Latin America was led by Sergey Bukasov from the Soviet Institute of Applied Botany, as part of Nikolai Vavilov's efforts to investigate the diversity and utility of crops worldwide.The analysis of over 8,000 maize populations sampled globally revealed that at extreme latitudes (e.g., Argentinian Patagonia region) plants are short, have few leaves and a short vegetative cycle, whereas in tropical regions (e.g., as Yucatan and southern Colombia) plants are tall, have many leaves and show a long vegetative cycle (Kuleshov 1930(Kuleshov , 1933)).
Maize landraces are classified by the characteristics of the ear, grain, tassel, whole plant and their geographic distribution (Goodman & Brown 1988).Cámara Hernández et al. (2011) described morphologically and phenologically about 50 Argentine native maize landraces cultivated by small farmers for their own consumption and cultural practices and provided a guide to their identification.Two centres of maize diversity have been recognised in Argentina based on the geographical distribution of genetic factors: the Northwestern Argentina Region (NWA), which represents an expansion of the Peruvian Andes, and the Northeastern Argentina Region (NEA), also called Chaco-Mesopotamian plain (Horovitz 1935).So far, 28 landraces have been reported from NWA and 23 from NEA, 15 of which are cultivated by the Guaraní indigenous communities from the subtropical forests in Misiones Province (Cámara Hernández et al. 2011).
The Guaraní's landraces grow in an area of little topographic variation and relatively stable environmental conditions.Their agronomic traits (e.g., marked differences in length of the vegetative cycle, resistance to fungi and high soil humidity) make them a potentially important source of germplasm for genetic improvement (Melchiorre et al. 2006(Melchiorre et al. , 2020;;Realini 2017;Realini et al. 2016).The Guaraní communities cultivated native landraces maintaining the use of long-standing agricultural practices in small parcels (Keller 2007).In the last decades, however, the use of commercial high-yielding varieties and the expansion of the agricultural frontier led to loss of native maize biodiversity in NEA (Bracco et al. 2012;Cámara Hernández et al. 2011).Genetic and karyotype studies of Guaraní maize revealed the existence of two gene pools, the Popcorn and the Floury landraces, which differ in endosperm consistency.The F-Pc landraces, endosperm floury with corneal periphery, has been included in the Floury gene pool (Bracco et al. 2016;Realini et al. 2016Realini et al. , 2018)).
The study and conservation of Guaraní landraces is important because they are well adapted to the environmental conditions in NEA and meet the needs and traditions of local communities (Melchiorre et al. 2020).
The determination of clearly defined maize genetic groups is useful for implementing ex situ and in situ conservation and breeding programs.The objective of this study was to analyse the phenotypic diversity and the relationship among Guaraní maize landraces from NEA based on morphological and phenological traits.

Plant material
Twenty-six Guaraní maize populations belonging to 14 landraces were collected from Guaraní farmers in the Misiones Province, NEA (Tab.S1, available on supplementary material <https://doi.org/10.6084/m9.figshare.22151345.v1>).Each population was assigned to a maize landrace according to the classification of Cámara Hernández et al. (2011).The term landrace is used to define a taxonomic entity delimited by a set of morphological traits, and the term population refer to a group of individuals that are managed by a single local farmer and share similar characteristics (i.e., can be assigned to the same landrace).The specimens were deposited at the seed bank of the Laboratory of Genetic Resources N. I. Vavilov at the Facultad de Agronomía (FA), Universidad de Buenos Aires (UBA), and a voucher number was assigned for the Vavilov seed bank (VAV).

Methods
In up to 5 ear per population, the following traits were measured: number of grains rows and diameters of ear, cob, rachis and pith.Ten grains in three adjacent rows were removed from the middle of each ear to measure the length, thickness and weight of each grain, and then data were averaged.To classify the grain type as Floury (F), Floury with corneal periphery (F-Pc) or Popcorn (Pc), the endosperm consistency was determined after making a longitudinal cut along the grain with a razor blade, according to Cámara Hernádez et al. (2011).These grains were germinated at 28 °C for 2 to 3 d in Petri dishes containing wet filter paper.Seedlings of about 20 cm high were manually transplanted to the experimental field of the Botanical Garden Lucien Hauman (FA, UBA), (34º35'S, 58º29'W and 25 m.a.s.l.).Ten to 15 grains per ear from 1 to 5 ears belonging to each population were planted in 15 rows of 20 plants each, with a spacing of 30 cm between plants and 75 cm between rows.The crosspollinations were carried out between plants from the same population.For this purpose, the female inflorescences (spikes) of all plants and the male inflorescences (tassels) of selected plants were covered with waxed bags.The pollen grains from mature tassels were collected and dusted over the stigmas.Pollinated spikes were rebagged to prevent free pollination.
The following phenological traits were recorded for each plant: time (d) from germination to tassel emergence, to spike emergence and to anthesis, naming LVC-length of vegetative cycle as the time from germination to anthesis (Melchiorre et al. 2006).After harvest, ears were photographed, assigned voucher numbers and deposited at the N. I. Vavilov seed bank for ex situ conservation.

Statistical analysis
A UPGMA cluster analysis was performed using the mean values of morphological and phenological traits (time to spike emergence and LVC).Data were standardised and an Euclidean distance-based similarity matrix was built.Statistical analysis was done with the program Infostat, FCA, Universidad Nacional de Córdoba (Di Rienzo et al. 2012).Box-plot graph was obtained by GraphPad Prism 8.0.1 software (2018).

Results
The mean values of the morphological and phenological traits of the studied Guaraní maize populations are shown in Tables S2  and S3 (available on supplementary material <https://doi.org/10.6084/m9.figshare.22151345.v1>), respectively.Entire ears from Pipoca Colorado landrace were not available, for this reason, is not shown in Table S2 (available on supplementary material <https://doi.org/10.6084/m9.figshare.22151345.v1>).The analysis of endosperm consistency allowed to classify each population into the Floury (Fig. 1a-c), F-Pc (Fig. 1d-f) and Popcorn (Fig. 1g-i) types.The mean ear diameter ranged from 3.27 to 4.96 cm in Floury, 3.95 to 3.98 cm in F-Pc and 3.17 to 4.06 cm in Popcorn populations.The mean number of grain rows per ear ranged from 10 to 14 in Floury, 12.67 to 14 in Popcorn and 13.33 2).
The UPGMA cluster analysis based on Gower distances (cophenetic correlation = 0.8) revealed the presence of two groups (Fig. 3).One group included the Pc populations (VAV6827, VAV6826.VAV2011/09 and VAV6843) and the other comprised all the Floury and the two F-Pc populations (VAV6837 and VAV6851) (Fig. 3).
From each population, cross-pollinated ears were obtained from sister plants for the ex situ conservation purpose and some of them are show in Figure 4.

Discussion
The analysis of 26 maize populations belonging to 14 Guaraní landraces from NEA revealed an important phenotypic diversity in terms of ears and grains.In this work, landraces were characterised according to the endosperm consistency of grains.Most of the endosperm is composed of starch granules at different degrees of compression.In the Floury landrace, starch granules are slightly compressed, giving grains a white colour and a floury consistency, while in the Popcorn landrace, with very compressed granules, grains have a glassy appearance and a hard consistency (Cámara Hernández & Arancibia de Cabeza 2007).The consistency of the grains and other morphological traits of the populations studied here allowed corroborating the racial classification provided by these authors.
The plants transplanted to the experimental field were used to estimate the phenological traits of the studied Guaraní maize populations.These showed differences in time from germination to tassel emergence, to spike emergence and to anthesis.In particular, a wide inter-population variability was observed for LVC, the time from germination to anthesis.The Pc populations had the longest LVC (108 -124 d), F populations were precocious or had intermediate LVC (90 -114 d), whereas F-Pc populations showed intermediate    Morphological and phenological traits were used as variables for the cluster analysis, which showed two distinct groups corresponding to the Floury and Popcorn landraces, with the F-Pc (VAV6837 and VAV6851) being included in the former group.This result is consistent with the clusters obtained by Melchiorre et al. (2006), who also included morphological and phenological traits in the analysis.It is interesting to note that these two groups of NEA maize landraces have also been identified using microsatellite markers (Bracco et al. 2012).Studies on cytogenetic characterisation showed that Popcorn populations had a higher percentage of heterochromatin and number and frequency of heterochromatic knobs, compared with Floury populations (Realini et al. 2018).Recently, Realini et al. (2021) found that heterochromatin percentage is positively correlated with the length of the vegetative period in maize populations from NEA.All these results demonstrate that the Guaraní maize from NEA presents two well-defined genetic pools, namely, the Floury (including F-Pc) and the Popcorn landraces.It is probably that selection made by Guaraní farmers for certain morphological and phenological characters, in relation to food and ritual practices, have selected indirectly the cytological characteristics related with the LVC.
Studies carried out so far indicate that future conservation programs should prioritise the protection of the Popcorn and the Floury genetic pools.The landraces belonging to these genetic pools, which are cultivated by smallholder farmers, are severely threatened by the expansion of the agricultural frontier and the introduction of commercial hybrid varieties.Therefore, part of this work was aimed at multiplying the material collected from the field and reintroducing it into the Guaraní communities in Misiones province, as a strategy for its ex situ conservation.Indeed, studies undertaken within the framework of in situ conservation programs need to be directed at improving the quality of life of local farmers through their economic growth.
The NEA maize landraces are potential sources of genetic resources, constituting useful repositories of alleles for tolerance and resistance to biotic and abiotic stresses.Thus, a comprehensive morphological, phenological and genetic characterisation is crucial for their inclusion in future breeding plans and agrobiodiversity conservation.

Figure 1
Figure 1 -a-i.Longitudinal-radial cuts of grains of Guaraní maize landraces -a-c.grains of Floury populations; d-f.grains of F-Pc populations (f. also shows longitudinal-tangential cuts); g-i.grains of Popcorn populations.Ref.: numbers correspond to voucher ears of the studied population.Bar = 1 cm.

Figure 2 -
Figure 2 -Days from germination to emergence of tassel, to anthesis (LCV) and to spike emergence for the Floury, Popcorn and F-Pc populations.Ref.: Whiskers represent the range.