DNA barcoding identifies the plants a person has eaten
Reliable technique should improve clinical trials, nutrition studies and historical research
Peer-Reviewed PublicationIMAGE: DNA BARCODING CAN NOW BE USED TO IDENTIFY WHAT PLANTS A PERSON HAS EATEN, AND IN WHAT RELATIVE AMOUNTS. THE TECHNIQUE SHOULD IMPROVE CLINICAL TRIALS AND NUTRITION STUDIES. (IMAGE, LAWRENCE A. DAVID) view more
CREDIT: LAWRENCE A. DAVID, DUKE UNIVERSITY
DURHAM, N.C. – What people say they’ve eaten and what they’ve actually eaten are often two very different lists of foods. But a new technique using DNA barcoding to identify the plant matter in human feces may get at the truth, improving clinical trials, nutrition studies and more.
Building on earlier studies that attempted to compare DNA found in feces with reported diets, researchers in the lab of Lawrence David, an associate professor of molecular genetics and microbiology in the Duke School of Medicine, have developed a genetic marker for plant-based foods that can be retrieved from poop.
“We can go back after the fact and detect what foods were eaten,” said Brianna Petrone, PhD, an MD/PhD student who led the project.
The marker is a region of DNA plants use to power chloroplasts, the organelle that converts sunlight into sugars. Every plant has this genomic region, called trnL-P6, but it varies slightly from species to species. In a series of experiments, they tested the marker on more than 1,000 fecal samples from 324 study participants across five different studies, about twenty of whom had high-quality records of their diet.
In findings appearing June 27 in the Proceedings of the National Academy of Sciences, the researchers show that these DNA markers can indicate not only what was consumed, but the relative amounts of certain food species, and that the diversity of plant DNA found in feces varies according to a person’s diet, age, and household income.
David’s lab relied on a reference database of dietary plants that contains markers for 468 species typically eaten by Americans to connect versions of trnL-P6 detected in poop to specific plant sources. After some tweaking, their barcode was able to distinguish 83 percent of all major crop families.
Petrone said the subset of crop families that could not currently be detected tended to be consumed in other parts of the world. The lab is now working to add crops such as pearl millet and pili nuts to their database.
They also haven’t tracked meat intake yet, though the technology is capable of that as well, David said. “That relative ratio of plant to animal intake is probably one of the most important nutritional factors we might look at.”
The scientists first tried the marker out on fecal samples from four individuals in a weight loss intervention where they knew exactly what study participants had been fed a day or two before. Knowing the patients had been given a dish called mushroom wild rice pilaf for example, they looked for the markers of its components: wild rice, white rice, portobello mushrooms, onion, pecans, thyme, parsley and sage.
In this and a second intervention group, they found that barcoding could not only identify the plants, it also could identify relative amounts consumed for some kinds of plants. “When big portions of grains or berries were recorded in the meal, we also saw more trnL from those plants in stool,” Petrone said.
Then they looked at samples from 60 adults who had taken part in two studies of fiber supplementation and kept track of what they were eating with surveys. The number of plants detected by trnL was in good agreement with dietary diversity and quality estimated from participants’ survey responses.
Next, they applied the barcoding to a study 246 adolescents with and without obesity with diverse racial, ethnic, and socioeconomic backgrounds. There was only a minimal record of diet in this cohort.
“Dietary data collection was challenging because some traditional surveys are 140 pages long and take up to an hour to fill out, families are busy, and a child might not be able to fill it out alone,” David said. “But because they had banked stool, we were able reanalyze those samples and then gather information about diet that could be used to better understand health and lifestyle patterns between kids. What really struck me was that we could recapitulate things that were known as well get new insights that might not have been as obvious.”
They found 111 different markers from 46 plant families and 72 species in the adolescents’ diet. Four kinds of plants were eaten by more than two thirds of subjects: wheat, found in 96 percent of participants, chocolate (88%), corn (87%) and the potato family (71%), a group of closely related plants that includes potato and tomatillo.
David said the barcode isn’t able to distinguish individual members of the cabbage family – the brassica – such as broccoli, Brussels sprouts, kale, and cauliflower, which are closely related.
Still, the large adolescent cohort showed that dietary variety was greater for the higher-income study participants. The older the adolescents were however, the lower their intake of fruits, vegetables and whole grain foods, potentially because of a known pattern of older children eating with their families less often.
David said the barcode is readily able to identify the diversity of plants found in a sample as a proxy for dietary diversity, a known marker of nutrient adequacy and better heart health.
David said that in each of these cohorts, the genomic analyses had been carried out on samples that had been collected years in the past, so the technique opens up the possibility of reconstructing dietary data for studies that have already been finished.
The authors think the new methodology should be a boon for all sorts of studies of human nutrition. “We are limited in how we can track our diets, or participate in nutrition research or improve our own health, because of the current techniques by which diet is tracked,” David said. “Now we can use genomics to help gather data on what people eat around the world, regardless of differences in age, literacy, culture, or health status.”
The team anticipates extending the technique to studies of disease across the globe, as well as monitoring food biodiversity in settings facing climate instability or ecological distress.
Funding for this work came from the National Institute of Diabetes and Digestive and Kidney Diseases (grants 5R24DK110492-05 and 5R01DK116187-05), the Burroughs Wellcome Fund Pathogenesis of Infectious Disease Award, the Duke Microbiome Center, the Springer Nature Limited Global Grant for Gut Health, the Chan Zuckerberg Initiative, the Triangle Center for Evolutionary Medicine, the Integrative Bioinformatics for Investigating and Engineering Microbiomes Graduate Student Fellowship, and the Ruth L. Kirschstein National Research Service Award to the Duke Medical Scientist Training Program. This work used a high-performance computing facility partially supported by grants from the North Carolina Biotechnology Center (2016-IDG-1013 and 2020-IIG-2109).
CITATION: “Diversity of Plant DNA in Stool is Linked to Dietary Quality, Age and Household Income,” Brianna L. Petrone, Ammara Aqeel, Sharon Jiang, Heather K. Durand, Eric P. Dallow, Jessica R. McCann, Holly K. Dressman, Zhengzheng Hu, Christine B. Tenekjian, William S. Yancy, Jr., Pao-Hwa Lin, Julia J. Scialla, Patrick C. Seed, John F. Rawls, Sarah C. Armstrong, June Stevens, Lawrence A. David. PNAS, June 27, 2023.
DOI: 10.1073/pnas.2304441120
Online: https://www.pnas.org/cgi/doi/10.1073/pnas.2304441120
DNA Barcoding can now be used to identify what plants a person has eaten, and in what relative amounts. The technique should improve clinical trials and nutrition studies. (Image, Lawrence A. David)
CAPTION
DNA Barcoding can now be used to identify what plants a person has eaten, and in what relative amounts. The technique should improve clinical trials and nutrition studies. (Image, Lawrence A. David)
CREDIT
Lawrence A. David, Duke University
JOURNAL
Proceedings of the National Academy of Sciences
METHOD OF RESEARCH
Observational study
SUBJECT OF RESEARCH
People
ARTICLE TITLE
Diversity of Plant DNA in Stool is Linked to Dietary Quality, Age and Household Income
ARTICLE PUBLICATION DATE
27-Jun-2023
Illinois study reveals genetic secrets of America's favorite snack
IMAGE: MULTIPLE TYPES OF POPCORN ILLUSTRATE SOME OF THE GENOMIC DIVERSITY FOUND IN A UNIVERSITY OF ILLINOIS STUDY. THE RESULTS COULD FACILITATE FUTURE BREEDING EFFORTS TO CREATE YUMMIER AND EASIER-TO-GROW VERSIONS OF AMERICA'S FAVORITE SNACK. view more
CREDIT: MADSEN SULLIVAN, UNIVERSITY OF ILLINOIS
URBANA, Ill. – In its simplest form, popcorn is pretty uncomplicated. Most supermarket varieties offer the choice of two kernel colors, yellow or white, and two kernel shapes, pointed or pearl. When popped, the flake typically expands into one of two shapes: mushroom or butterfly. But there’s more to popcorn than meets the eye. New research from the University of Illinois Urbana-Champaign reveals a wealth of untapped diversity lurking in popcorn’s genetic code.
Analyzing 320 publicly available popcorn lines, crop sciences researchers found variation at more than 308,000 locations across the genome. This diversity may or may not translate into more popcorn variety for consumers, but some of the differences could be important for improving the agronomic performance of the crop.
“This could be helpful if popcorn companies wanted to bring in material to diversify their germplasm, which is super important for things like disease resistance and herbicide tolerance. More work has to be done to identify traits of interest, but this dataset opens up those possibilities,” says study co-author Tony Studer, associate professor and popcorn breeder in the Department of Crop Sciences, part of the College of Agricultural, Consumer and Environmental Sciences at Illinois.
Studer’s team documented the genetic differences in a process known as genotyping by sequencing, which narrows the focus of genetic sequencing efforts to the most information-packed parts of the genome. Differences, or polymorphisms, among corn lines occurred at the level of single nucleotides, the building blocks of DNA.
Popcorn populations
Having identified hundreds of thousands of differences, the team could then group corn lines by patterns of single nucleotide polymorphisms (SNPs), allowing the researchers to make inferences about relatedness. According to the analysis, North American popcorns fall into two groups: One primarily composed of yellow pearl types, with white pointed and Latin American types falling into a second group.
“Grouping popcorn based on genetic similarities allows us to look at the diversity present in each group and better predict the performance of crosses between lines. In addition, if a gene is found to improve performance, knowing its group membership will help breeders incorporate it into their program, something I hope popcorn companies will take advantage of to improve their products both on the consumer side and the grower side,” says Madsen Sullivan, doctoral student in crop sciences and the study’s first author.
The results showed a high level of inbreeding among yellow pearl popcorns. That means lower genetic diversity and greater relatedness among that group. Although this has resulted in better popping traits, material from the other group will likely contain versions of genes that could be useful but are not present in the yellow pearl popcorns.
The analysis also provides a starting point from which to uncover popcorn’s long history of movement across North America and the world. Studer says the first people to consume corn likely ate it popped, not buttered on the cob. He’s working to trace popcorn’s early origins in a follow-up study.
Demystifying popcorn’s herbicide tolerance
With popcorn’s genetic code spelled out, the researchers were eager to tackle a longstanding mystery related to herbicide application labels. Nicosulfuron has been killing weeds in cornfields since the early 1990s, but it’s only labeled for yellow-kerneled hybrids; farmers are specifically warned against using it on white-kerneled popcorn.
“That was a red flag to me, because kernel color should have nothing to do with herbicide sensitivity,” says co-author Marty Williams, USDA-ARS ecologist and affiliate professor of crop sciences at Illinois. “Kernel color is controlled by genes in a completely different part of the genome.”
Williams worked with Studer’s team to test 294 popcorn genotypes from both populations, the yellow pearl group and the white pointed and Latin American group; incidentally, neither group is exclusively yellow or white, despite their names. The researchers applied nicosulfuron to the test group as well as sensitive and tolerant popcorn and sweet corn hybrids as controls.
While nicosulfuron did injure more white-kerneled popcorns, the effect had nothing to do with kernel color itself. Instead, nicosulfuron sensitivity correlated with genetic heritage and population structure. The pointed and Latin American types were more sensitive than yellow pearls. In dent corn, nicosulfuron is detoxified by a gene known as nsf1. The researchers immediately looked for the same gene in popcorn, assuming it would be active in the tolerant genotypes.
“We expected nsf1 to come up in popcorn, but instead we found a whole different set of genes that seemed to relate to nicosulfuron tolerance,” Studer says. “That opens up the possibility of an alternate mechanism for herbicide tolerance in popcorn, and we’re planning to follow up on that.”
Next steps
Williams recommends popcorn breeders use this research to improve tolerance to nicosulfuron, and perhaps other herbicides, in their existing and new cultivars. Then herbicide labels ultimately could be updated to reflect tolerance throughout the crop, regardless of kernel color.
Could the results of the genome study improve popcorn’s agronomic traits? Studer says it will take more work to screen the dataset for desirable traits, but eventually elite popcorn lines could be developed and marketed by popcorn companies.
The study, “Genetic diversity of North American popcorn germplasm and the effect of population structure on nicosulfuron response,” is published in Crop Science [DOI: 10.1002/csc2.21039]. Madsen Sullivan, Marty Williams, and Tony Studer are authors. The research received funding from the University of Illinois, and Sullivan was supported by a fellowship from the Illinois Corn Growers Association.
JOURNAL
Crop Science
ARTICLE TITLE
Genetic diversity of North American popcorn germplasm and the effect of population structure on nicosulfuron response
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