GENETIC IMPROVEMENT IN POPCORN

– Popcorn consumption in Brazil has grown significantly over the years, and genetic improvement is essential to obtain sustainable gains in multiple traits to supply this increasing demand. Thus, the objective of this review was to contribute information concerning the process of popcorn breeding in tropical regions, germplasm availability, popcorn breeding plans, the main characteristics related to popcorn quality and yield, and advances and perspectives in the process of popcorn improvement. The main focus of breeding programs is to obtain hybrids from inbred lines with high popping expansion (>40 mL g -1 ) and yield (>4.000 kg ha -1 ). The genetic improvement performed in Brazil has presented significant advances, mainly due to work developed in public institutions with the development of new hybrids that present more significant popping expansion and yield. However, the number of cultivars is still low, and most of them are controlled by private companies. Therefore, intrapopulation methods are recommended to develop open-pollinated varieties with high popping expansion, and this trait can be used as an early predictor of promising inbred lines to obtain superior hybrids for grain quality. Furthermore, popping expansion can be quickly recovered in backcrosses involving the cross of common maize with an inbred popcorn line.


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The main characteristic of popcorn is its grain hardness and small length, which can burst and turn into popcorn after heating (Sawazaki, 1995). This phenomenon occurs because, due to vaporization of moisture and gelatinization of starch grain subjected to temperatures >150ºC. In addition, the expansion of water molecules in the form of steam creates internal pressure in the grain to the point where the pericarp ruptures, exposing the endosperm, thus forming the white and soft mass known as popcorn (Zinsly & Machado, 1987).
The importance of popcorn comes from its profitability in the market, with growing consumption and widespread acceptance of the final product. In addition, its consumption is usually associated with leisure time, such as fairs and movie theaters (Amaral Júnior et al., 2013). In the USA, approximately 15 billion quarts (almost 17 billion liters) of popcorn are consumed annually (Matzke, 2021). In 2016, the popcorn market was around $9.06 billion, and it is projected to reach $15 billion by the end of 2023 (Dawande, 2018).
Between 2014 and 2019, an increase of 223% in areas sown with popcorn was observed in Brazil (Kist et al., 2019), which has become the secondlargest producer of popcorn in the world, producing approximately 320,000 tons. Approximately 70% of this production is intended for domestic consumption and the remainder for export (Blecher, 2019).
However, there is still a vast market for popcorn cultivation in Brazil.
Genotypes with superior characteristics are required to promote the popcorn production chain from both the agronomic and commercial approaches of the final product. All improvement breeding methods applied to standard corn can also be used in popcorn, emphasizing popcorn quality evaluation.
According to Sawazaki (1995), knowledge of all the factors that affect popcorn quality and consumer preferences is fundamental for the success of the improvement breeding program.
However, over the years, the supply of seeds of improved cultivars of popcorn in Brazil has not followed the improvement shown by common maize.
The This review aims to contribute to information concerning the process of genetic improvement of popcorn in tropical regions, emphasizing germplasm availability, popcorn breeding programs, main characteristics related to popcorn quality and yield, methodologies for popcorn quality evaluation, and advances and perspectives in the process of popcorn breeding.

Germplasm
Some authors agree that the popcorn was one of the first improved races during the maize domestication process (Wellhausen et al., 1951;Contreras et al., 2006) because popcorn presents some characteristics considered as wild, such as smaller grain size, higher prolificacy, thicker pericarp (Ziegler, 2001), and, in some races, narrow grains (Silva et al., 2020).
Since the germplasm choice is crucial in conducting a breeding program, information found 3 in the literature regarding some of the most relevant popcorn germplasm at national and global levels will be presented below.

South American Races
The term race refers to a group of related individuals with enough characteristics in common to permit their recognition as a group (Anderson & Cutler, 1942).
The center of origin of popcorn is Latin America, where a great diversity of indigenous races exist, but only three races are well documented. The first is the Pororo race from Bolivia (Ramírez et al., 1960), which has white, small, rounded kernel, low to medium height plants, with tillers, and high prolificacy (Vencovsky, 1987). The second is the Pisankalla race, originating in Bolivia, Paraguay, and Argentina, producing white and narrow grains (Ramírez et al., 1960).

Finally, the Brachytic Pop
Corn race is found in mountainous regions of the Andes and has brachytic plants, oval ears, small and thin grains, generally dark red, with white endosperm, and colorless aleurone (Brieger et al., 1958).

Indigenous open-pollinated variety (OPV)
Indigenous Composite: Obtained from more than 40 open-pollinated varieties (OPV) of indigenous corn collected in various regions of South America, recombined for more than eight generations, followed by mild selection. As a result, it has substantial genetic variability involving several characteristics and excellent material for popcorn improvement (Zinsly & Machado, 1987).
Guarani: This OPV has significant variability in terms of grain color characteristics. It has mediumheight plants with tillers, high prolificacy, and a semiearly production cycle (Sawazaki, 1995). Therefore, the Guarani OPV is critical in providing alleles of interest for improving popcorn, being utilized as a parent in hybrids such as IAC-112.

Improved OPV
South American Mushroom (SAM): The Instituto Agronômico de Campinas (IAC) developed it in 1941, and its pedigree came from Argentina.

Composite CMS-42 and Composite CMS-43:
They originated from 25 OPV of yellow round grains and 33 white grains, respectively, and were obtained from the germplasm bank of Embrapa. These have resistance to Exserohilium turcicum and Puccinia spp. with tremendous genetic variability for plant and grain characteristics (Pacheco et al., 1998).  (Zinsly & Machado, 1987).
Zélia: A three-way cross hybrid adapted to tropical and temperate environments developed by Pioneer. Its kernel color is yellow, has a precocious cycle and good yield potential (>3000 kg ha -1 ), and high popping expansion (>35 mL g -1 ) (Scapim et al., 2002).

IAC-268:
A three-way hybrid developed by the Agronomic Institute of Campinas (IAC), has a high PE (>45 mL g -1 ) and yield (>4.500 kg ha -1 ). It has a light yellow kernel and stands out with high resistance to foliar diseases and Pratylenchus brachyurus nematode (Paterniani et al., 2020).

Exotic Germplasm
Exotic germplasm for applied breeding programs means all germplasm that is not immediately useful without selection for adaptation for a given area (Hallauer & Miranda-Filho, 1988).
Jap Hulless: It is a variety having good popcorn quality; its plants have short and thick cobs, rows of 30-40 kernels, and long, thin, whitish-colored kernels (Sawazaki, 1995).

South American: Originally from Argentina
and introduced in the United States in 1920, the popcorn formed by popping its kernels is mushroomtype and has suitable PE, with good popcorn attractiveness due to its aleurone coloration being mottled golden yellow (Sawazaki, 1995).

Super gold (Sunburst, Yellow Pearl):
Developed by the Kansas Experiment Station through mass selection for PE on the Queen Golden variety, it has small, conical spikes, small and glassy grains, with suitable PE (Sawazaki, 1995).

Current Germplasm
At the National Register of Cultivars (RNC) of the Ministry of Agriculture, Livestock and Food Supply -Brasil (2021), there are 137 registered cultivars (Table 1). However, a few cultivars have been effectively seeded in commercial crops.  (Sawazaki et al., 2000;Miranda et al., 2012). The restricted access to these seeds, According to Lee (1995), heterotic groups can be defined as a germplasm collection that, when crossed with germplasm from another group, tends to exhibit higher levels of heterosis than when crossed with members of its group. Santacruz-Varela et al. The PE is one of the most critical traits for popcorn improvement. Thus, in the next section, we will discuss its definition, cause, and the type of inheritance governing this trait.

Popping expansion (PE)
PE is the ratio between the volume of popped popcorn and the volume or mass of kernels used for popping. Its units can be expressed as volume/volume (mL mL -1 ) or volume/grams (mL g -1 ), according to the units of measurement of popcorn kernel sample (Miranda et al., 2003). Different systems for assessing popcorn expansion should be used in individual and family selections. The hot air popcorn pumper is an efficient system in which 10 g of the kernel can be used to evaluate plants, and up to 90 g of the kernel can be used to evaluate families in the trials. The microwave oven is equivalent to the hot air popcorn pumper. Kraft paper bags can be used in the microwave oven. For plant evaluation, 10 g of a kernel with 140 seconds is recommended. For progenies evaluation, 30 g to 90 g of a kernel with 220 seconds can be used (Matta & Viana, 2001 Seed moisture is one of the factors that most influence PE. The moisture associated with maximum PE depends on the cultivars and the method adopted for its determination (Luz et al., 2005). The ideal average moisture of grains for marketing should be around 13.5% to 14%, which provides good PE (Sawazaki et al., 2000).
Concerning the genetic inheritance of PE, there is a predominance of additive variance ( Table 2). The selection of superior individuals is a good strategy that can be adopted in a breeding program using simple methods and that the selection gains for the trait will be effective

Factors affecting kernel and popcorn quality
The improvement process of popcorn is slower than that of common maize, mainly because of the additional need to work with characteristics related to popcorn quality. Information about the legislation involved in classifying grains and the main characteristics related to popcorn quality are presented below.

Characteristics related to grain quality
In Brazil, popcorn classification follows Normative Instruction Nº61/2011 (Brasil, 2011) that defines its classification according to quality and identity requirements. The quality requirements for popcorn are defined according to the color of the grains (yellow, white, colored, or mixed) and the maximum tolerance limits and are set out in Table 3.
Straw stalking is highly correlated with the percentage of fungal grain infections. Poorly compacted varieties have a higher percentage of infected ears than those with better compactness.
Another factor that favors reductions in the incidence of fungal attacks is stalk development. This factor causes the cob to yield after the ripening period, with its tip facing downwards, preventing water entry (Sawazaki, 1995) and preventing fungus development.
Factors related to harvest, processing, and grain storage influence the popcorn quality. Pacheco et al. (1996)

Characteristics related to popcorn quality
Other factors that should be considered during the improvement process of popcorn are those related to its consumption, such as the presence of unpopped kernels, color, texture, and flavor (Leonello et al., 2009;Sawazaki et al., 1984;Sweley et al., 2013).

Pericarp thickness:
The pericarp presents a significant association with PE is responsible for maintaining the internal pressure in the kernel that is generated with heating; therefore, its thickness and integrity are responsible for the explosion and size of the popcorn (Zinsly & Machado, 1987;Mohamed et al., 1993;Sawazaki, 2001

Plant characteristics correlated with yield
In a breeding program, the indirect Selection  Table 4.
Through path analysis, Saito et al. (2021) concluded that pericarp thickness has a direct effect on PE and that the thermal diffusivity of the pericarp has an indirect effect on pericarp thickness, on PE.
The authors also performed a study of canonical correlations to identify better the associations that allow for an increase in the PE of popcorn and found the canonical pair significant and high (r = 0.9541).
This result verified that PE was directly proportional to pericarp thickness and thermal diffusivity but inversely proportional to grain length and yield, indicating that increased PE is associated with greater pericarp thickness and thermal diffusivity, shorter grain length, and lower yield.

Intrapopulation improvement in popcorn populations
In this topic, the main improvement methods applied to popcorn and some of its particularities will be presented.

Mass Selection
It is recommended that mass selection be carried out in two stages. The first represents a less intense selection for agronomic characters of interest,  and the second constitutes a more rigorous selection for PE (Zinsly & Machado, 1987;Sawazaki, 1995).
According to Sawazaki (1995), the success of this method depends on existing genotypic correlations involving the selected traits in the first phase and the PE.

Hybrids from inbred lines
One of the objectives of breeding programs involving popcorn, in the long-term, is to obtain hybrids from inbred lines, thus enabling the exploitation of hybrid vigor. Howeveer, the method generally used in obtaining homozygous inbred lines is artificial self-mating, generating depression by inbreeding depression (Lima et al., 1984;Simon et al., 2004;Scapim et al., 2006). Thus, the value of a population as a source of inbred lines is dependent on the inbreeding depression when compared to the various characteristics, especially yield, limiting the generation of superior inbred line for the subsequent establishment of vigorous hybrids (Lima et al., 1984;Arnhold et al., 2007).
For PE, the loss by inbreeding is lower than that of yield due to the lower genetic complexity of the characters and the predominance of additive effects resulting in a more outstanding contribution of loci being in homozygosity than loci in heterozygosity for this trait (Dofing et al., 1991;Pacheco et al., 1998;Larish & Brewbaker, 1999;Miranda et al., 2008;Arnhold et al., 2010). For example, Simon et al.

(2004) obtained inbreeding depression values of 27%
for PE and 68% for yield.

Backcrossing
Backcrossing is a very efficient method for transferring characters of simple inheritance aiming to correct defects in an inbred line with desirable agronomic characteristics, and used as a recurrent parent (Miranda Filho & Viegas 1987).
It is common to introduce dent corn germplasm to increase several agronomic characteristics in popcorn. However, crossings between high and low PE plants tend to produce offspring with intermediate PE characteristics. The PE can be recovered by backcrossing with parents with high PE. Studies show that PE is recovered with few backcrossings due to its mode of inheritance.

Marker-assisted selection
The recurrent selection method has been used in breeding programs for many decades (Hallauer et al., 2010;Ribeiro et al., 2016). With improvements to techniques and the acquisition of new molecular markers, it became possible to improve this breeding technique further. The availability of genetic data for each generation in the selection process and its rapid development cycle make recurrent selection assisted by markers an excellent tool to identify the maximum possible genetic variation in most regions of the genome (Pereira et al., 2006;Berilli et al., 2011;Guimarães et al., 2018). Coan et al. (2018) sought to identify genomic regions and possible candidate genes associated with resistance to ear rot caused by Fusarium spp.
in 183 tropical and popcorn maize inbred lines, and a set of 267.525 SNP-type markers were obtained using genotyping by sequencing (GBS). In this work, fourteen significant SNPs associated with the disease explained 15% to 25% of the phenotypic variation observed.

Quantitative characteristics loci (QTLS) and popping expansion
Mapping QTLs can be a valuable tool to reduce the disadvantages of phenotype-based PE determination tests. In addition, the discovery of different QTLs for PE in different germplasm sources offers ample room for efficient approaches to the use of gene pyramiding and the employment of marker-assisted selection (Lu et al., 2003;Li et al., 2007b;Senhorinho et al., 2019).
To be considered good candidates for marker-  (Stuber et al., 1992), generation (Austin & Lee, 1996Li et al., 2009), and environment (Li et al., 2003). Table 5 presents several studies that sought to determine QTLs related to PE.
Additive variance seems essential in the expression of PE, being influenced by a small number of genes (Dofing et al., 1991;Larish & Brewbaker, 1999;Lima et al., 2019). In addition, QTLs related to high PE are located on different chromosomes , and several studies indicate the presence of at least one QTL related to PE located on chromosome 1 (Lu et al., 2003;Babu et al., 2006;Li et al., 2007b). Once these molecular markers are validated, they will be helpful in markerassisted selection and better understanding of popping expansion.

The use of new statistical techniques in popcorn improvement
Since 2000, researchers have been using more refined statistical techniques to genetically improve popcorn. These include the use of multivariate analysis AMMI (Miranda et al., 2003;Gonçalves et al., 2014), the use of BLUP (Viana et al., 2011(Viana et al., , 2014Freitas et al., 2013;Vittorazzi et al., 2017), the use of Bayesian statistics for analysis of genetic parameters of PE and yield in popcorn (Rodovalho et al., 2014;Schwantes et al., 2019), Bayesian clustering via Markov chains aiming at genetic diversity studies to identify different groups of popcorn for improvement (Saavedra et al., 2013;Silva et al., 2015), and Bayesian analysis of genetic parameters for quantitative traits in families of full-sib families of popcorn .

Conclusions
When analyzing the current situation regarding popcorn cultivation in Brazil, it can be seen that the focus of breeding programs is to obtain hybrids from inbred lines with high PE and yield. However, the number of cultivars is still low, and most of them are controlled by private companies.
Intrapopulation methods are recommended to Popping expansion can be an early predictor of promising inbred lines to obtain superior hybrids for popping expansion.
Popping expansion is quickly recovered in backcrosses involving the cross of common maize with an inbred popcorn line.