elocation-id: e3805
The genus Chenopodium contains two species of importance in the diet of Mesoamerica and South America, namely Chenopodium quinoa Willd. (Quinoa) and Chenopodium berlandieri subsp. nuttalliae, the genetic resources of which have not been characterized despite their great nutritional potential and adaptability. In order to molecularly characterize germplasm of red chia, huauzontle (Chenopodium berlandieri subsp. nuttalliae) and quinoa (Chenopodium quinoa Willd.), we molecularly studied 48 genotypes from the Germplasm Banks of the National Institute of Nuclear Research and the Plant Genetic Resources Laboratory of Brigham Young University. To determine the genetic variability, 14 microsatellite markers (SSRs), specific for Chenopodium, were used. Genetic affinity was assessed using the Jaccard similarity coefficient and the analysis of results was performed using the UPGMA method. The results indicate that, within the studied genotypes of both species, 175 alleles were produced, ranging from 8 (KGA16, QCA88) to 16 (QCA37, QAAT74, QCA57), these being the ones that obtained the most alleles per locus. The dendrogram showed that, at a coefficient of 0.9, four main groups were formed, where groups 1 and 2 join advanced lines of quinoa and red chia, mutants of red chia and huauzontle, groups 3 and 4 joins chia and huauzontle, and group five includes all the germplasm of the Plant Genetic Resources Laboratory of BYU, mostly made up of subspecies of Chenopodium zsachei, boscianum and zinatum. It was concluded that there is a great genetic affinity between quinoa, huauzontle and red chia, which opens the possibility of inter- and intraspecific crosses for the genetic improvement of both species.
Chenopodium berlandieri subsp. nuttalliae, Chenopodium quinua, molecular markers, SSR.
In recent years, there has been a growing interest in recovering and valuing crops with high protein content and nutritional value, which have a promising potential for exploitation and that contribute to reducing malnutrition, an example of which is red chia (Chenopodium berlandieri subsp. nuttalliae), huauzontle (Chenopodium berlandieri subsp. nuttalliae) and quinoa (Chenopodium quinoa Willd), edible grain pseudocereals (García, 2017).
Pseudocereals are of great relevance since they are a biological resource of high nutritional value and great hardiness since they tolerate cold climates, drought, salinity, and poor soils (Eisa et al., 2012; Jacobsen et al., 2012). Due to these characteristics, they constitute a cultivation alternative for the marginal regions of the country (De la Cruz et al., 2010).
Within this group are quinoa, red chia, huauzontle, and species of the genus Amaranthus (Xingú-López, 2010). These crops had great food, economic and religious importance among pre-Hispanic civilizations since they constituted the basis of the diet, like corn (Zea mays L.) and beans (Phaseolus vulgaris L.); however, upon the arrival of the Spaniards, their cultivation and consumption were left behind and even prohibited, surviving in very remote areas (Ramírez et al., 2011). Red chia, huauzontle, and quinoa have exceptional nutritional qualities (12.5 to 16.7% protein, 5% lipids, and 58 to 76.2% carbohydrates) (Yasui et al., 2016).
The study of genetic diversity is very important for the conservation, evaluation and use of genetic resources for plant breeding and to determine the authenticity of cultivars or varieties, facilitating sustainable agriculture practices, which can lead to food sovereignty (Xingú, 2010).
There are currently several molecular techniques that allow us to know the genetic variability in natural populations. Thus, there are several types of molecular markers that are used in genetic improvement to obtain estimates of genetic distances between populations, varieties, lines or hybrids, as well as to establish kinship relationships between lines or varieties by detecting polymorphisms in single or multiple loci of dominant or co-dominant type (Xingú, 2010).
Molecular markers are also used for the genetic characterization of Chenopodium germplasm since they have been employed to differentiate genotypes under environmental conditions that confused their phenotypes (Nolasco et al., 2013). Simple sequence repeats (SSRs) are one of the frequently used molecular markers for genotyping crops (Jarvis et al., 2008).
Simple sequence repeats (SSR) microsatellites, also known as short or simple sequences, are repeats of mono-, di-, tri- and tetranucleotides made up of 2 to 10 base pairs as a basic unit, and are found throughout the genome of eukaryotic organisms in both coding and non-coding regions. Their technique requires little DNA, without having a high quality of purity, and provides highly polymorphic results, with its interpretation being relatively simple (Allende, 2014).
In order to determine the genetic variability in the germplasm of red chia, huauzontle (Chenopodium berlandieri subsp. nuttalliae), and quinoa (Chenopodium quinoa Willd.) collected in producing areas of the State of Mexico and wild materials from the United States, 48 materials were molecularly characterized, which include the collection of the National Institute of Nuclear Research of Mexico and the Plant Genetic Resources Laboratory of Brigham Young University, USA, using 14 primers for microsatellites developed specifically for Chenopodium by Maughan et al. (2013).
This characterization made it possible to determine the degree of variability within species, as well as the affinity within quinoa, huauzontle and chia, to design genetic improvement strategies through hybridization that allow combining desirable traits. This study also corroborated evolutionary work since it is considered that Chenopodium quinoa Willd. and Chenopodium berlandieri were independently domesticated, the latter in Mesoamerica and North America, whereas the former in South America (Maughan et al., 2024).
A total of 48 genotypes were evaluated, including varieties (a group of plants resulting from breeding work) and collections (samples taken in the field from cultivated and wild specimens) from the Germplasm Bank of the National Institute of Nuclear Research (ININ, for its acronym in Spanish) and the Germplasm Bank of the Plant Genetic Resources Laboratory of Brigham Young University.
Seed of the genus Chenopodium was used: three collections of Chenopodium berlandieri subsp. nuttalliae var. huauzontle (H3, H16, and H18 from the Toluca Valley), five collections of Chenopodium berlandieri subsp. nuttalliae var. red chia (J. Silva, D. Oros, R. Rguez, P. Bravo, and Zumbaro from the shores of Lake Pátzcuaro, Michoacán), two advanced lines of Chenopodium quinoa donated by the National Germplasm Bank of the College of Postgraduates (640304 and 11L240), four lines of Chenopodium quinoa obtained by radiation mutagenesis (ININ136, ININ240, ININ311, and ININ333), one F1 line from the cross (42AdeM x Red Chia), and 33 collections of Chenopodium berlandieri ssp. donated from the Germplasm Bank of the BYU Plant Genetic Resources Laboratory (Table 1 and 2).
For DNA extraction, the tissue used was healthy leaf tissue from 10 individual plants 30 days after sowing (das) established under greenhouse conditions. The leaf tissue sample (four leaves) was introduced into Eppendorf microtubes to be placed in a LABIST FDL1R-1a freeze-drying chamber with a freezing dryer at 0.7 atm pressure for 24 h.
The freeze-dried leaf tissue was ground in a Retsch-Mill 200. DNA extraction was performed according to the procedures described by Maughan et al. (2013). The extracted DNA was quantified with a GBC Nanodrop spectrophotometer and diluted to 30 ng μl-1 in TE buffer solution (Tris 10 mM, EDTA 1 mM, pH 7.5).
All plants were grown in the greenhouse of the Plant Genetic Resources Laboratory of Brigham Young University in Provo, Utah, USA, in 15 cm pots at 25 °C under halogen lamps with a photoperiod of 12 h.
Fourteen microsatellite primers (Table 3) developed by Mason et al. (2005), specific for Chenopodium, were used for the study of genetic diversity of the 48 genotypes, namely: QCA37, KGA20, QAAT74, QAAT50, QAAT70, QGA02, QCA14, KGA16, QCA57, QCA88, QAAT76, QAAT78, QCA38, and QAAT24.
PCR amplifications were performed in 12 μl reactions consisting of 3 μl (30 ng μl-1) of DNA, 0.5 μl of every 10 μm of forward and reverse primers, 6 μl of MyTaq HS Red Master Mix (Bioline, Taunton, Massachusetts, USA) and 2 μl of H2O. PCR reactions were performed using a C1000 or T100 thermal cycler (Bio-Rad, Applied Biosystems, Foster City, California, USA) with the following parameters: 95 °C for 60 s, 35 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 10 s, and a final extension cycle of 72 °C for 60 s.
Electrophoresis of the amplified products was performed with 1.5% agarose gel (250 ml of TBE, 7.5 g of agarose, and 12.5 μl of midori green). DNA samples were run in an electrophoresis chamber with Bio-Rad power supply (Power-PAC 300, Berkely, Ca.) for 30 min. At the end of this time, the gel was washed with distilled water and placed in a Bio-Rad Universal Hood II ultraviolet (UV) light transilluminator and the gels were recorded and stored in a database using the Quantity one program.
For the statistical analysis, a binary matrix of absence (0) and presence (1) was generated. Diffuse bands were not considered, genetic similarity between individuals was assessed using the Jaccard similarity coefficient. The cluster analysis was performed using the UPGMA method. The corresponding dendrogram was generated using the statistical package of the numerical taxonomy system for personal computers (NTSYS), PC 2.02 version, to determine the similarities between the genotypes in question.
The results indicate that, within the population of 48 genotypes from the Germplasm Banks of the National Institute of Nuclear Research and the Plant Genetic Resources Laboratory of BYU, they produced 175 alleles at 14 SSR loci. These alleles vary from 8 (KGA16, QCA88) to 16 (QCA37, QAAT74, QCA57), these being the loci with the highest number of observed alleles.
The primer QAAT74, according to Ormeño’s (2015) work, is one of those with the highest number of observed alleles, whereas QCA88 presented the fewest alleles and was used in Donaire’s (2018) work, where a high number of alleles was also recorded. This indicates that, in each job where they are used, they act differently.
The analysis was performed using data obtained from 14 loci recorded for 48 genotypes. From the results obtained, it is possible to have an approximate idea about the genetic diversity of the samples analyzed contained in the information from the microsatellites. The objective of cluster analysis is to form groups where the individuals in each group are more similar to each other than to the individuals of another group (Allende, 2017). To visualize the relationships between populations according to their distance, a hierarchical dendrogram was constructed.
The dendrogram in Figure 1 shows that, at a similarity coefficient of 0.9, five groups were formed; in contrast, in Ormeño (2015), where only 16 genotypes were evaluated, six groups were formed and in Xingú (2018), where 38 genotypes were evaluated, 10 fewer than in this study, six groups were formed.
Group 1 consisted of four genotypes, two advanced lines (11L240 and 640304) of quinoa, an F1 single cross between quinoa and red chia (42AdeMXCR), and a red chia (D. Oros).
Group 2 consisted of seven genotypes, one red chia (Zumbaro), three mutant quinoas (ININ311, ININ136, and ININ240), two huauzontle genotypes (H-3 and H-18), and a collection of BYU from Chenopodium berlandieri (HBYUMEX); in contrast to Allende (2014), there is a separation between the huauzontles and the chias.
Groups 3, 4 and 5 only had two genotypes each. Group 3 had a huauzontle (H-16) and a red chia (J. Silva). Group 4 had two genotypes of red chia (R. Rguez and P. Bravo). Group 5 was made up of a collection of BYU from C. berlandieri (BYU14108) and a mutant quinoa (ININ333).
The following conclusions were derived from the present research: Great genetic affinity was detected between the species C. quinoa Willd. and C. berlandieri since the primers designed for quinoa adequately amplified for huauzontle. A high genetic affinity was detected between the cultivated genotypes of C. quinoa and C. berlandieri subsp. nuttalliae local breeds red chia and huauzontle, which were only in groups 1, 2, 3 and 4.
The genetic affinity between cultivated accessions allows us to predict favorable results in genetic improvement work by hybridization between C. quinoa Willd and C. berlandieri subsp. nuttalliae. The dendrogram shows two very interesting groups, such as groups 1 and 2, where the advanced quinoa lines join with red chia and mutant quinoas with red chia and huauzontle and groups 3 and 4 had all the germplasm from the Plant Genetic Resources Laboratory of BYU.
De la Cruz, T. E.; Rubluo, I. A.; Palomino, G. H.; García, A. J. M. and Laguna, C. A. 2007. Characterization of Chenopodium germplasm selection of putative mutants and its cytogenetic study. In: Ochat, S.; Mohan, J. S. Ed. Breeding of neglected and underutilized crops species and herbs. Science Publishers. Enfield, NH, USA. 123-36 pp.
Jacobsen, S. E.; Jensen, C. R. and Liu, F. 2012. Improving crop production in the arid Mediterranean climate. Field Crop Res. 128:34-47. https://doi.org/10.1016/j.fcr.2011.12.001.
Jarvis, D. E.; Kopp, O.; Jellen, E. N.; Marllory, M. C.; Pattee, J.; Bonifacio, F. A.; Coleman, C. E.; Stevens, M. R.; Fairbanks, D. J. and Maughan, P. J. 2008. Simple sequence repeats marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.). J. Genet. 87(1):39-51. https://doi.org/10.1007/s12041-008-0006-6.
Mason, S. L.; Stevens, M. R.; Jellen, E. N.; Bonifacio, F. A.; Fairbanks, D. J.; Coleman C. E.; McCarty, R. R.; Rasmussen, A. G. and Maughan, P. J. 2005. Development and use of microsatellite markers for germplasm characterization in quinoa (Chenopodium quinoa Willd.). Crop Sci. 45(4):1618-1630. https://doi.org/10.2135/cropsci2004.0295.
Maughan, P. J.; Jarvis, D. E.; Cruz-Torres, E.; Jaggi, K. E.; Warner, H. C.; Marcheschi, A. K.; Gomez-Pando, L.; Fuentes, F. ; Mayta-Anko, M. E.; Curti, R.; Rey, E.; Tester, M. and Jellen, E. N. 2024. North American pitseed goosefoot (Chenopodium berlandieri) is a genetic resource to improve Andean quinoa (C. quinoa). Scientific reports. 14:1-13. https://doi.org/10.1038/s41598-024-63106-8.
Ramírez, V. M. L.; Espitia, R. E.; Carballo, C. A.; Zepeda, B. R.; Vaquera, H. H. and Córdova T. L. 2011. Fertilization and plant density in varieties of amaranth (Amaranthus hypochondriacus L.). Revista Mexicana de Ciencias Agrícolas. 2(6):855-866. http://www.redalyc.org/articulo.oa?id=263121473005.
Yasui Y.; Hirakawa, H.; Oikawa, T.; Toyoshima, M.; Matsuzaki, C.; Ueno, M.; Mizuno, N.; Nagatoshi, Y.; Imamura, T.; Miyago, M.; Tanaka, K.; Mise, K.; Tanaka, T.; Mizukoshi, H.; Mori, M. and Fujita, Y. 2016. Draft genome sequence of an inbred line of Chenopodium quinoa, an allotetraploid crop with great environmental adaptability and outstanding nutritional properties. DNA Res. 23(6):535-546. https://doi.org/10.1093/dnares/dsw037.