The resurrection of sweet corn inbred SC11-2 using marker aided breeding for β-carotene

Sweet corn has dominated the urban market due to its sweetness, tenderness, and ease of digestibility. It's import and export values have dramatically increased during the past 10 years as a fresh, processed, and preserved commodity. However, the commercially available sweet corns are deficient in β-carotene. In our study, we introgressed the favorable allele of crtRB1 (responsible for high β-carotene) into the recurrent sweet corn inbred SC11-2 from maize donor parent UMI1230β1⁺ to develop the β-carotene-rich sweet corn genotype by marker aided breeding. The crtRB1 3′TE InDel marker was utilized for foreground selection of favorable genotype. A total of 103 polymorphic SSR markers were employed for background selection, resulting in a 96% recovery of recurrent parent genome (RPG). We recorded high β-carotene content (9.878–10.645 μg/g) in the introgressed lines compared to the recurrent parent, SC11-2 (0.989 μg/g). The sugar content ranged from 18 to 19.10% and was on par with the recurrent parent (20.40%). These biofortified inbreds can be used as a donor in maize breeding programs to develop sweet corn genotypes with high β-carotene content.

Introduction

Hidden hunger, caused by micronutrient malnutrition, is one of the prime focuses in this era. Around 30 vitamins and minerals cannot be synthesized in our body in sufficient quantity, but it has to be supplied through diet and are termed “essential micronutrient.” Micronutrient malnutrition has appeared to be a serious health issue worldwide (Bouis et al., 2019). Malnutrition affects all age groups irrespective of geographical location and economic status. Nearly two billion people suffer from micronutrient deficiency (Global Nutrition Report, 2018). Vitamin A deficiency (VAD) is a major cause of malnutrition, and it leads to several diseases in humans viz., measles, respiratory diseases, night blindness, xerophthalmia, growth retardation, generative disorders, and low immunity (Lozano-Alejo et al., 2007; Bouis and Saltzman, 2017).

Economic value of sweet corn increased due to its demand for fresh and processed vegetables. Sweet corn is preferred due to its sweet taste, tender nature, easy digestibility, and nutritional value (Mehta et al., 2017). Sweet corn demand is higher in urban cities where it is consumed as snacks, soups, and for preparing main courses. It is an integral part of the diet in the countries of South East Asia (Feng et al., 2015; Yang et al., 2018). Frozen sweet corn is estimated to have a global import of $433.60 million and an export of $426.25 million. Preserved sweet corn is estimated to have an import of $957.09 million and $908.31 million in export (FAOSTAT, 2022). Traditionally cultivated sweet corn varieties possess a significantly low level of provitamin A (Feng et al., 2015). Thus, traditional sweet corn does not make a significant contribution to the daily Provitamin A requirement in human beings.

Biofortification, medicinal supplementation, dietary diversity, and food fortification are widely used to combat malnutrition worldwide. Biofortification is an approach for increasing nutrient density in food crops by breeding. Biofortification is the preferred method to develop nutrition-rich staple crops that provide a sustainable solution to control malnutrition (Pfeiffer and McClafferty, 2007). The favorable allele related to β-carotene hydroxylase1 (crtRB1) increases the accumulation of proA in maize kernel endosperms by limiting the conversion of β-carotene into further downstream products. β-carotene acts as the precursor for vitamin A development (Yan et al., 2010). The proA biofortified sweet corn could resolve the problem of VAD, especially in urban areas. Hence, we need superior sweet corn donor plants with crtRB1 favorable allele to develop biofortified sweet corn hybrids.

Sweet corn is developed naturally through recessive mutation in genes related to the starch biosynthesis pathway. Mutation in the Shrunken2 (Sh2) allele, present in chromosome 3L of sweet corn, leads to the formation of recessive shrunken2 (sh2) (Baveja et al., 2021). Due to this sh2 loci, sweet corn accumulates 2–4-folds more sucrose and reducing sugar than maize (Nelson, 1980). This also determines the phenotypic appearance of the sweet corn kernel by shrinking it (Lertrat and Pulam, 2007). The development of commercial sweet corn lines predominantly uses the sh2 gene (Wong et al., 1994). Marker-assisted selection (MAS) has been successfully used for developing agronomically superior cultivars (Singh et al., 2011). MAS of the target allele shortens the breeding cycle and makes the breeding program more efficient and precise (Ribaut and Hoisington, 1998). Marker-assisted backcross breeding (MABB) is the most effective approach for transferring the gene of interest and improving the nutritional traits in maize (Singh et al., 2012; Babu et al., 2013; Chandran et al., 2019; Pukalenthy et al., 2019, 2020; Sagare et al., 2019; Sarankumar et al., 2019; Natesan et al., 2020; Das et al., 2021; Qutub et al., 2021; Talukder et al., 2022).

The current study describes the revival of sweetcorn-inbred SC11-2. We used a MABB strategy, introgressed the crtRB1 gene into sweetcorn inbred SC11-2, and developed agronomically superior lines rich in β-carotene. The newly developed lines will be valuable as a donor for the sweet corn biofortification program.

Materials and methods

Plant genetic materials and experimental site

A sh2 based sweet corn inbred SC11-2 developed at the Department of Millets, Tamil Nadu Agricultural University, Coimbatore, India was used as a recurrent parent. UMI1230β1⁺ was used as a donor parent from the Center for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India. The donor parent was used to introduce the favorable target allele of crtRB1 into the recurrent parent SC11-2. All the field experiments were done at Eastern Block Farm, Tamil Nadu Agricultural University, Coimbatore, India, during 2019–2020.

Generation of backcross and self-progenies

The F₁s were generated by a single cross between the recurrent parent, SC11-2, and the donor parent, UMI1230β1⁺, during Rabi, 2019 (November–March). The true F₁s were selected using crtRB1 gene-specific markers. In Kharif 2020 (June–September), the BC₁F₁ population was developed by backcrossing the F₁s with the recurrent parent. Only the shrunken seeds from the BC₁F₁ cobs were selected for forwarding into the next generation. These shrunken seeds were grown, and the second backcross was performed on the crtRB1 heterozygous plants in Rabi, 2020 (November–March) to develop the BC₂F₁. We obtained all the seeds as shrunken in this generation. The selected crtRB1 heterozygous plants in BC₂F₁ were selfed to produce the BC₂F₂ generation. The crtRB1 positive homozygous plants were selected from the segregating population of BC₂F₂ using the crtRB1 gene-specific marker and were selfed again to develop the stabilized BC₂F₃ population (Figure 1).

FIGURE 1

Figure 1. The selected flint kernel in the F₁ generation is backcrossed with the recurrent parent (SC11-2). The shrunken kernel is selected (highlighted with green color box) in BC₁F₁ and these shrunken kernels are selected and selfed in the subsequent generations.

Foreground selection with gene-specific marker

Genomic DNA was extracted from 3 weeks old young seedlings of parents, backcrossed populations, and selfed BC₂F₂ population following the standard CTAB method (Murray and Thompson, 1980). crtRB1 gene-specific 3′TE InDel marker (Supplementary Table 1) was used for foreground selection (Yan et al., 2010). The crtRB1 gene exhibits three allelic forms due to crtRB1-3′TE polymorphism. Allele 1 (543 bp, without TE insertion), which is attributed to increase β-carotene by decreasing transcript expression of the crtRB1 gene, is the favorable allele. Allele 2 (296 + 875 bp; with 325 bp TE insertion) and allele 3 (296 + 1,221 + 1,880 bp; 1,250 bp TE insertion) are the unfavorable alleles. crtRB1 gene was amplified using polymerase chain reaction (PCR) (Eppendorf, Hamburg) following the procedure as described by Natesan et al. (2020). We screened for favorable allele at 543 bp and unfavorable allele at 296 bp. Under heterozygous conditions, both alleles were amplified. The PCR amplicons were resolved in 3% agarose gel at 120 V for 2 h. The resolved products in the agarose gel were visualized using a gel documentation system (BioRad, California, USA). This marker was used to select heterozygous plants in the BC₁F₁ and BC₂F₁ generations, and homozygotes in the BC₂F₂ generation.

Background selection with SSR markers

A total of 201 SSR markers covering 10 chromosomes of the maize genome were retrieved from the maize genome database (www.maizegdb.org). The SSR markers were custom synthesized from Eurofins Genomics India Pvt. Ltd., Bangalore, India. These SSR markers were used to screen the recurrent and donor parents to identify the parental polymorphism. The identified polymorphic markers were employed on foreground favorable plants of BC₁F₁, BC₂F₁, and BC₂F₂ to calculate the recovery of the recurrent parent genome (RPG). The polymerase chain reaction (PCR) and agarose gel electrophoresis were done following the method used by Chandrasekharan et al. (2022). Recovery of RPG for the selected plants was estimated using the formula:

$\begin{array}{ll}\text{RPG }(\text{in\%})=\left\{(\text{A}+0.5\text{H})/(\text{A}+\text{B}+\text{H})\right\}\times 100\%& (1)\end{array}$

“A” is the number of SSR markers homozygous to the donor plant, “B” is the number of SSR markers homozygous to the recurrent parent, and “H” is the number of SSR markers in the heterozygote state. Polymorphic information content (PIC) for each pair of SSR markers was estimated using the formula described by (Botstein et al., 1980; Supplementary Table 2).

Characterization of morphological traits

Morphological traits were recorded among the foreground positive plants with high recovery of RPG in BC₁F₁, BC₂F₁, and BC₂F₂ generation following the descriptors suggested by the International Board for Plant Genetic Resources (CTA, 1992). The traits recorded were 50% days to tasseling, 50% days to silking, plant height, ear height, tassel length, number of tassels, leaf length, leaf breadth, cob girth, number of kernel row per cob, number of kernel row, and single plant yield.

Sugar and β-carotene contents

Selfed cobs from BC₂F₃ generation were harvested 24 days after pollination (DAP) to check the total soluble solids (TSS) using the portable Brix meter (Neuffer and Sheridan, 1997). Total sugar was estimated following the Anthrone method (Hodge, 1962), and reducing sugars were estimated by the Nelson-Somogyi method (Nelson, 1944; Somogyi, 1952). Non-reducing sugar was estimated by the difference between the estimated value of total sugar and that of reducing sugar. To estimate the β-carotene, the matured BC₂F₃ cob kernels were dried in dark conditions before grinding into fine powder. The extraction solvents for the estimation were prepared according to the Harvest Plus protocol (Rodriguez-Amaya and Kimura, 2004). Quantification of β-carotene in the introgressed lines, recurrent, and donor parents were performed using Shimadzu HPLC Analytical C18G column 250 × 4.6 mm. Samples were eluted using YMC carotenoid C18 column. A photodiode array detector was used to detect the peaks. The mobile phase consisting of Acetonitrile: Methanol: Ethyl acetate at a ratio of 80:10:10 was made to pass through the column, under high pressure, at a flow rate of 1mL min⁻¹. Five standards (0.1, 1, 10, 50, and 100 μg/g) (Kurilich and Juvik, 1999) of β-carotene (M/s. Sigma Aldrich India) were used to make the standard curve. β-carotene content was calculated in the inbreds and parents at 453 nm. The chromatograph was compared with the standard curve to calculate the β-carotene level in the kernels of the inbreds and the parents.

Statistical analysis

The Chi-square test was performed to test the goodness of fit in the segregation pattern of the crtRB1 alleles in BC₁F₁, BC₂F₁, and BC₂F₂ generations. A t-test was performed to assess the significance of the increase in β-carotene level in the introgressed lines over the recurrent parent.

Results

Marker polymorphism

The recurrent (SC11-2) and donor (UMI 1230β1⁺) parents were screened with a crtRB1 gene-specific marker which revealed an unfavorable allele (296 bp) in the recurrent parent and a favorable allele (543 bp) in the donor. Out of 201 SSR markers used for screening parental polymorphism, 103 markers showed polymorphism (51.24%), and the number of polymorphic markers in each chromosome ranged from 5 to 10. The polymorphic markers were used to screen the foreground favorable plants from BC₁F₁, BC₂F₁, and BC₂F₂ generation to trace the high RPG plants.

Accelerated development of SC11-2 with crtRB1 gene

F₁ generation

The crtRB1 gene-specific marker was used to confirm heterozygosity in the F₁s of SC11-2 × UMI 1230β1⁺. A total of 46 plants were screened in this generation, of which 42 were found to be heterozygous for crtRB1. The heterozygote F₁ plants were backcrossed with the recurrent parent (SC11-2), and well-set BC₁F₁ cob designated as DBT 17-1 was selected for advancing it to the BC₁F₁ generation.

BC₁F₁ generation

A total of 96 plants from DBT 17-1 cob were screened in this generation using the crtRB1 gene-specific foreground marker. A total of 35 plants with unfavorable allele (296 bp) and 61 plants in heterozygous condition (296 + 543 bp) were confirmed (Figure 2). The segregation pattern for crtRB1 alleles deviated significantly from the expected Mendelian ratio (1:1) (Table 1). Recovery of RPG ranged from 72.30 to 76.60% in the crtRB1 heterozygous plants (Table 2). Three plants designated as DBT 17-1-1, DBT 17-1-6, and DBT 17-1-47, with higher RPG recovery, were selected and advanced to the next generation by backcrossing with the recurrent parent, SC11-2.

FIGURE 2

Figure 2. Foreground selection in backcross [(A) BC₁F₁, (B) BC₂F₂] and selfed generation [(C) BC₂F₃] of SC11-2 × UMI 1230β1⁺ using crtRB1 gene specific marker crtRB13′TE. (M) Ladder (100 bp), (P₁) SC11-2, (P₂) UMI 1230β1⁺, (1–10) progenies from BC₁F₁, BC₂F₂, and BC₂F₃ generation.

TABLE 1

Table 1. Segregating pattern of crtRB1 alleles in the backcross and selfed populations derived from SC11-2 × UMI 1230β1⁺.

TABLE 2

Table 2. Recovery percentage of recurrent parent genome in backcross and selfed populations derived from SC11-2 × UMI 1230β1⁺.

BC₂F₁ generation

In this generation, 110 plants were screened for the crtRB1 foreground marker, where 53 and 57 plants were detected as unfavorable and heterozygous, respectively (Figure 2). The segregation ratio of the alleles in this generation was as per the Mendelian ratio of 1:1 (Table 1). Recovery of RPG ranged from 84.30 to 89.36% (Table 2) in this generation. Three heterozygous plants, DBT 17-1-1-1, DBT 17-1-1-29, and DBT 17-1-1-71, with high recovery of RPG, were further selected and selfed to advance to BC₂F₂ generation.

BC₂F₂ generation

In BC₂F₂ generation, 152 segregating plants from DBT 17-1-1-1 cob were screened for crtRB1 alleles (Figure 2). The segregation of the crtRB1 alleles into 42 favorable positives (543 bp), 78 heterozygous (543+296 bp), and 32 unfavorable (296 bp) alleles were as per the Mendelian ratio of 1:2:1 (Table 1). Recovery of RPG in the foreground positive plants ranged from 91.20 to 94.68% (Table 2). Three positive plants with high recovery of RPG and phenotypic similarity with the recurrent parent were selected and selfed to forward to BC₂F₃ generation. The plants were designated as DBT 17-1-1-1-13, DBT 17-1-1-1-35, and DBT 17-1-1-1-101.

BC₂F₃ generation

In this population, a crtRB1 gene-specific marker was used to ascertain the presence of a positive allele (543bp) (Figure 2). Recovery of RPG was 94–96% in this generation. Biochemical analysis for sugar estimation and β-carotene was carried out in kernels of four positive plants with high recovery of RPG. The plants were designated as DBT 17-1-1-1-35-1, DBT 17-1-1-1-35-2, DBT 17-1-1-1-35-3 and DBT 17-1-1-1-35-4 (Table 1, Figure 3, Supplementary Figure 1).

FIGURE 3

Figure 3. Background selection using SSR markers for selected improved lines from BC₂F₃ generation of SC11-2 × UMI 1230β1⁺. (M) Ladder (100 bp), (P₁) UMI 1230β1⁺, (P₂) SC11-2, (1) DBT 17-1-1-1-35-1, (2) DBT 17-1-1-1-35-2, (3) DBT 17-1-1-1-35-3, (4) DBT 17-1-1-1-35-4.

Morphological traits

A high degree of resemblance was found between the introgressed inbred and recurrent parent line concerning morphological characters and yield attributing traits. Four improved lines showed phenotypic resemblance from 87.63% (Cob girth) to 100% (Tassel length and number of kernel rows per cob). Among the four lines, DBT 17-1-1-1-35-3 showed maximum resemblance with the recurrent parent. For instance, days to tasselling, days to silking, plant height, ear height, number of tassel branches, lead length, cob length, and single plant yield showed more than 95% resemblance with SC11-2 (Figure 4, Table 3).

FIGURE 4

Figure 4. The morphological traits [(A) tassel, (B) silk, and (C) cob] in parents and improved line (DBT 17-1-1-1-35-1) from BC₂F₃ generation of SC11-2 × UMI 1230β1⁺.

TABLE 3

Table 3. Details of morphological traits and recovery percentage in the BC₂F₃ improved lines of SC11-2 × UMI 1230β1⁺ with respect to the recurrent parent SC11-2.

Sugar and β-carotene contents

TSS was estimated at 24DAP using the Brix meter and was found to be 19.00% in the recurrent parent kernels and 10.00% in the donor plant kernels. The introgressed inbred had an average TSS ranging from 16 to 18.5%. Total sugar (TS) in the recurrent parent was 20.40%, and in the donor parent, it was 5.25, whereas TS in the introgressed lines ranged from 18.35 to 19.65%. Reducing sugar (RS) in the recurrent parent and donor parent was estimated to be 3.5 and 2.1%, respectively, whereas RS in the introgressed lines was between 2.45 to 3.35%. Non-reducing sugar (NRS) was estimated to be 16.90% in the recurrent parent and 3.15% in the donor parent. NRS in the introgressed lines ranged from 15.30 to 17.20% (Figure 5, Table 4). The β-carotene level in the matured kernels of the recurrent parent was 0.989 and 11.608 ppm in the donor parent kernel. The β carotene level in the kernels of the introgressed lines ranged from 9.878 to 10.645 ppm. This result suggests that there was an average of 10.37-fold higher β-carotene levels in the introgressed lines compared to the recurrent line (Figure 5, Table 4).

FIGURE 5

Figure 5. Total sugar (%) and β-carotene (μg/g) content in parents and improved lines from BC₂F₃ generation of SC11-2 × UMI 1230β1⁺. Error bars indicate means ± SE. Asterisks (**) indicate significant different from the recurrent parent β-carotene (μg/g) content using t-test (p < 0.01).

TABLE 4

Table 4. Details of sugar and β-carotene contents in parents and BC₂F₃ improved lines of SC11-2 × UMI 1230β1⁺.

Discussion

Sweet corn demand has increased over the last decade and is preferred for its tenderness and sweet taste all over the world (Mehta et al., 2017). Sweet corn is eaten fresh in the milky stage which helps to retain the nutritional quality (Cabrera-Soto et al., 2018). Many commercially grown sweet corn is deficient in proA to meet the minimal daily requirement (Feng et al., 2015). Development of vitamin A biofortified sweet corn will provide essential vitamin A rich food (Hossain et al., 2019). In this study we developed vitamin A biofortified sweet corn inbred by marker assisted introgression of crtRB1 favorable allele in a promising sweet corn inbred, SC11-2.

Introgression of crtRB1 gene into sweet corn inbred SC11-2

The crtRB1 3′TE favorable allele is responsible for effecting a 2–10-fold increase in β-carotene content in maize (Babu et al., 2013). In this study, we developed sweet corn inbred with crtRB1 favorable allele using maize UMI1230β1⁺ as the donor parent and sweet corn inbred SC11-2 as a recurrent parent. crtRB1-3′TE-InDel markers located within the target genes facilitated selection of individual plants with favorable alleles of crtRB1 in the segregating populations (Gupta et al., 2013; Zunjare et al., 2018). Marker assisted selection of favorable progenies in the seedling stage before pollination resulted in saving time and resources (Sarika et al., 2018). The segregation pattern of the crtRB1 alleles in BC₁F₁ showed a significant segregation distortion (SD) due to the diverse backgrounds of the parents (Babu et al., 2013; Muthusamy et al., 2014). This could be due to the presence of naturally occurring gene mutants like dek (defective kernel) (Neuffer and Sheridan, 1997), gametophytic factors (ga) (Mangelsdorf and Jones, 1926), and embryo-specific mutation (emb) (Neuffer and Sheridan, 1997). In our study, MABB led to the high recovery of RPG in the introgressed progenies. The SSR markers used for background selection were distributed across the genome. This attributed to the selection of positive progenies with high recovery of RPG (Singh et al., 2012; Gupta et al., 2013). The recovery of RPG is responsible for the similarities in the agronomical traits of the introgressed lines and their recurrent parent (Muthusamy et al., 2014). Due to the fixation of various recurring parent allele proportions in the foreground positive plants, there is diversity in RPG recovery among the introgressed progenies (Muthusamy et al., 2014). Previous studies suggest a high recovery in RPG within two generations in the MABB program (Muthusamy et al., 2014; Liu et al., 2015; Hossain et al., 2018; Sarika et al., 2018). Background selection assisted in the indirect selection of loci corresponding to different phenotypic characteristics (Hossain et al., 2018).

Kernel sweetness and nutritional quality

The selection of shrunken kernels in BC₁F₁ helped to indirectly select the sh2 allele in the progenies (Khanduri et al., 2011; Mehta et al., 2017). As a result, the introgressed lines retained their sweetness and were on par with the recurrent parent. The Sh2 gene is located in chromosome 3 and is responsible for coding a large subunit of ADP-glucose pyrophosphorylase (AGPase), which synthesizes ADP-glucose from glucose-1-phosphate and adenosine triphosphate (Baveja et al., 2022). This conversion is blocked by the recessive sh2 allele, which restricts the starch biosynthesis of amylose and amylopectin. The sweetness was estimated and found to be maximum at 24 DAP (Chhabra et al., 2019). The variation in the sweetness across the introgressed lines may be due to the presence of loci influencing the sh2 gene (Qi et al., 2009; Mehta et al., 2020).

A significant increase of β-carotene in the introgressed inbred suggests that introgression of favorable allele (543 bp) alone has a major effect on the accumulation of β-carotene in the sweetcorn kernels. The crtRB1 gene is responsible for hydroxylation of β-carotene and β-cryptoxanthin into zeaxanthin. This reduces the proA concentration in the kernels. A natural variant of crtRB1 with no TE in the 3′ UTR reduces this conversion. This leads to more accumulation of proA carotenoids in the kernels (Yan et al., 2010). In China, four sh2 based sweet corn lines were developed by introgression of lcyE (Yang et al., 2018). But they reported a low increase in the proA (2.07–3.86 ppm) over traditional inbreds. In this study, we have reported a 10.7-fold increase in β-carotene content in the developed lines compared to the original inbred. This was achieved through the MAS of plants containing the favorable allele 1 of crtRB1.

The traditional sweet corn lines are low in proA. In our study, we introgressed crtRB1 gene encoding proA into traditional sweet corn inbred using marker-aided breeding. The crtRB1 gene-specific marker facilitated the precise identification of favorable genotypes from the segregating populations. SSR-based background selection expatiated the selection process, resulting in higher recovery of recurrent parent genome within two backcrosses. The marker-assisted breeding thus helped to develop biofortified sweet corn inbred with a 10.7-fold increase in β-carotene levels. This inbred can be used as a donor parent for crtRB1 favorable allele in developing vitamin A biofortified sweet corn hybrids. ProA biofortified sweet corn can help to mitigate the problem of hidden hunger in a sustainable manner in the urban areas of developing countries.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

Author contributions

SN conceived and designed this study. IS and KR performed field and lab experiments. IS, KR, BM, and KA analyzed the data. VS, RR, and RM monitored the research and provided suggestions. IS, KR, KA, and SN wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding

Financial support from the Department of Biotechnology (DBT), Government of India (GOI), through the project entitled Incorporation of crtRB1 allele into sweet corn inbreds and north eastern landraces for development of biofortified sweet corn (BT/PR42333/NER/95/1866/2021 dt. 18.2.2022) was acknowledged. The funders had no role in the work design, data collection, and analysis, or decision and preparation of the manuscript.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsufs.2022.1004450/full#supplementary-material

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