Genetic Variation and Unintended Risk in the Context of Old and New Breeding Techniques

Figures & data

Figure 1. Timeline of major events in crop breeding. BCE, Before the Common Era; CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein; DNA, deoxyribonucleic acid; US, United States. References: ¹Robbins, 1922; ²Laibach, 1925; ³Stadler, 1928 b; ⁴Blakeslee and Avery, 1937; ⁵Watson and Crick, 1953; ⁶Carlson et al., 1972; ⁷Caplan et al., 1983; ⁸Burr et al., 1983. Image sources: corn, John Doebley; hand pollination, tissue culture, wheat field, DNA fingerprinting, DNA sequence, and gene editing obtained from iStock; Gregor Mendel, wikipedia; wheat embryos, Andriy Bilichak, Agriculture and Agri-Food Canada; radiation, colchicine and double helix obtained from pngegg.com; protoplasts, Mnolf; petri dish, Udaya Subedi, Agriculture and Agri-Food Canada – Dr. Stacy Singer; tomato, pngfuel.com.

Figure 2. Schematic representation of common plant breeding techniques. A) Introgression breeding involves the crossing of two plant genotypes, the selection of offspring with a desirable trait, and multiple rounds of backcrossing. B) Mutation breeding involves the treatment of plant tissue with physical or chemical mutagens, selection of a genotype with a desirable trait from a large mutagenized population, and multiple rounds of backcrossing. C) Transgenic, cisgenic and RNAi approaches involve the insertion of an exogenous sequence of DNA into the plant genome, which results in the production of either a protein or double-stranded RNA molecule, and consequently the improved trait. D) Simple NHEJ-based CRISPR/Cas gene editing involves the introduction of Cas and sgRNA into plant cells, resulting in a double stranded DNA break at the target site and the subsequent generation of an indel at a precise genomic location, which leads to trait improvement. CRISPR/Cas, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein; DNA, deoxyribonucleic acid; RNAi, ribonucleic acid interference. Image sources: plums, apricots, grapefruit, daffodils, bacteria, rice plant, potato plant, and apple tree obtained from Pixabay; radiation, scissors, proteins, double helix and Cas obtained from pngegg.com; pluots, dreamstime; chromosomes, iStock; callus on media, Udaya Subedi, Agriculture and Agri-Food Canada – Dr. Stacy Singer; citrus seeds, pixnio.com; greenhouse, Government of Newfoundland and Labrador, Department of Fisheries, Forestry and Agriculture; Golden Rice, Golden Rice Project (www.goldenrice.org.); potato blight, Ronald Hutten, Laboratory of Plant Breeding, Wageningen University – Dr. Henk Schouten; nonbrowning apple, webcomicms.net; wheat immature embryos, derived from Hayta et al., 2019; protoplasts, Mnolf; gel image, Agriculture and Agri-Food Canada – Dr. Stacy Singer; edited wheat, National Agriculture and Food Research Organization and Okayama University (for more information, please refer to the following article: https://doi.org/10.1016/j.celrep.2019.06.090).

Figure 3. Means by which DNA mutations are incurred spontaneously in plants. Although many naturally occurring mutations have no observable effect on plant growth and appearance, some do, and it is these that are important for plant adaptation and the breeding of new crop cultivars using traditional techniques. DNA, deoxyribonucleic acid; TE, transposable element.

Table 1. Examples of approximate ranges in genetic variability derived spontaneously and from various breeding methods as determined by whole genome sequencing in rice and Arabidopsis.

Type	Arabidopsis (∼135 Mb)	Ref^h	Rice (∼430 Mb)	Ref
Between generations^a	1–5 SNVs and indels	1,2	41 SNVs and indels	16
Intra-varietal^b	1,200–4,700 SNVs 1,300–3,600 indels^f 390 larger variations^g	3–5	23,000–110,000 SNVs 9,100 indels	17, 18
Inter-varietal^c	350,000–790,000 SNVs 44,000–210,000 indels 2,300–53,000 larger variations	3, 5, 6–9	25,000–2,500,000 SNVs 1,400–470,000 indels 15,000–17,000 larger variations	17, 19–28
Tissue culture	9–65 SNVs 2–6 indels 0 larger variations	10	67–37,000 SNVs 12–17,000 indels	16, 19, 29–31
Radiation	5–75 SNVs 3–44 indels 0–21 larger variations	11, 12	21–630,000 SNVs 0–70,000 indels 0–11,000 larger variations	28, 32–39
Chemical mutagen	460 SNVs	13	1,500–26,000 SNVs	40, 41
Transgenic^d	0–3 SNVs 0–4 indels small deletions and filler at T-DNA junctions rare SVs flanking T-DNA very rare T-DNA splinters	14	80–490 SNVs 22–360 indels small amount of filler at T-DNA junctions rare translocations or deletion	21,30, 42
CRISPR/Cas^e	indel at target location very rare translocation at target site 0 off-target mutations	4, 15	indel at target location 0–10 off-target mutations (off-targets only found for very small number of sgRNAs)	16, 17

^aFrom parent to progeny.

^bBetween plants of the same variety/cultivar/accession.

^cBetween plants of different varieties/cultivars/accessions (conventionally bred) of the same species.

^dDerived from Agrobacterium-mediated transformation. While transgenic Arabidopsis plants were produced in a manner that did not involve tissue culture (via floral dip), mutations found in rice transformants include those occurring as a result of somaclonal variation.

^e Off-target mutations were assessed by screening sites bearing an appropriate PAM.

^f The length of insertions and deletions considered as indels varies among studies.

^g Larger variations refer to copy number variations, and structural rearrangements such as large deletions, duplications, inversions or translocations.

^hReferences: ¹Ossowski et al., 2010; ²Willing et al., 2016; ³Uchida et al., 2011; ⁴Feng et al., 2014; ⁵Ossowski et al., 2008; ⁶Gan et al., 2011; ⁷Lu et al., 2012; ⁸Kasulin et al., 2017; ⁹Pucker et al., 2016; ¹⁰Jiang et al., 2011; ¹¹Belfield et al., 2012; ¹²Kazama et al., 2017; ¹³Tabata et al., 2013; ¹⁴Schouten et al., 2017; ¹⁵Peterson et al., 2016; ¹⁶Tang et al., 2018; ¹⁷Zhang, Wang, et al., 2014; ¹⁸Xu et al., 2011; ¹⁹Qin et al., 2018; ²⁰Du et al., 2017; ²¹Kawakatsu et al., 2013; ²²Park et al., 2019; ²³Takagi et al., 2013; ²⁴Yamamoto et al., 2010; ²⁵Arai-Kichise et al., 2011; ²⁶Arai-Kichise et al., 2014; ²⁷Jeong et al., 2013; ²⁸Zhang, Sun, et al., 2020; ²⁹Zhang, Zhang, et al., 2014; ³⁰Wei et al., 2016; ³¹Miyao et al., 2012; ³²Li, Numa, et al., 2019; ³³Hwang et al., 2020; ³⁴Li, Shimizu, et al., 2019; ³⁵Cheng et al., 2014; ³⁶Zhenga et al., 2017; ³⁷Zheng, Li, et al., 2020; ³⁸Li, Zheng, et al., 2016; ³⁹Yang et al., 2019; ⁴⁰Sevanthi et al., 2018; ⁴¹Abe, Takagi, et al., 2018; ⁴²Kashima et al., 2015.

Figure 4. CRISPR/Cas applications in plants that do not require the extended presence of a transgene. Red base pairs denote random indel mutations at the targeted site, whereas green base pairs represent specific alterations made at targeted loci. Cas, CRISPR-associated protein; dCas, catalytically dead Cas; DNA, deoxyribonucleic acid; HDR, homology-dependent repair; nCas, Cas nickase; NHEJ, nonhomologous end-joining; PAM, protospacer adjacent motif; pegRNA, prime editing guide RNA; RNA, ribonucleic acid; sgRNA, single guide RNA; uORF, upstream open reading frame.

Table 2. Examples of CRISPR/Cas-mediated improvement of various traits in crop species through targeted gene disruption via the generation of NHEJ-derived indels.

Crop	Target	Trait	Possible outcome	Off-target^a	Ref^b
Disease or pest resistance
Tomato Grapevine	MLO	↑ resistance to powdery mildew	↓ losses related to a fungal pathogen	0 ND	1,2
Rice	OsERF922	↑ resistance to rice blast	↓ losses related to a fungal pathogen	ND	3
Watermelon	Clpsk1	↑ resistance to Fusarium	↓ losses related to a fungal pathogen	0	4
Cucumber Cassava Rice Tomato	eIF4E	↑ resistance to viruses	↓ losses due to viral pathogens	0 1 0 ND	5–8
Apple	MdDIPM4	↓ fire blight susceptibility	↓ losses due to a bacterial pathogen	0	9
Tomato	CCD8	↑ resistance to broomrapes	↓ losses due to a parasitic weed	0	10
Rice	CYP71A1	↓ infestation by rice brown planthopper	↓ losses due to an insect pest	ND	11
Abiotic stress tolerance
Tomato	SlLBD40	↑ drought tolerance	↑ climate change resilience	ND	12
Tomato	SlMAPK3	↑ heat stress tolerance	↑ climate change resilience	ND	13
Rice	OsRR22	↑ salinity tolerance	↑ growth on marginal land	ND	14
Yield
Brassica napus	BnaMAX1	↑ branching and siliques	↑ yield per plant	0	15
Rice	Gn1a	↑ grain number	↑ yield per plant	3	16
Rice	Gs3	↑ grain size	↑ yield per plant	3	16
Nutritional value
Tomato	SGR1	↑ lycopene content	↓ risk of cancer and cardiovascular disease	0	17
Banana	LCYɛ	↑ β-carotene content	↓ vitamin A deficiency	0	18
Tomato	SlGAD2 and 3	↑ γ-aminobutyric acid	↓ blood pressure	ND	19
Toxicity, anti-nutritional or allergenicity
Rice	OsNramp5	↓ cadmium accumulation	↓ heavy metal toxicity	0	20
Wheat	α-gliadin-encoding genes	↓ gluten	↓ allergenicity	0	21
B. napus	BnITPK	↓ phytic acid	↑ mineral absorption	ND	22
Ease of harvesting
Wheat	TaQsd1	↓ preharvest sprouting	↓ losses due to preharvest rain	ND	23
B. napus	BnA03.IND	↓ silique shatter-resistance	↓ seed losses prior to harvest	ND	24
Quality for downstream use
Corn	Wx1	↑ amylopectin content	↑ usefulness for industrial purposes	0	25
Potato	PPO	↓ browning	↑ consumer approval	0	26
Camelina sativa Rice Soybean B. napus	FAD2	↑ oleic acid content	↑ usefulness for cooking and bio-fuel production	0 ND 0 0	27–30

^aNumber of off-target mutations based on biased assessments of between 1 and 140 of the most likely off-target sites with at least one nucleotide mismatch with the sgRNA in each case.

^bReferences: ¹Nekrasov et al., 2017; ²Wan et al., 2020; ³Wang et al., 2016; ⁴Zhang, Liu, et al., 2020; ⁵Chandrasekaran et al., 2016; ⁶Gomez et al., 2019; ⁷Macovei et al., 2018; ⁸Yoon et al., 2020; ⁹Pompili et al., 2020; ¹⁰Bari et al., 2019; ¹¹Lu et al., 2018; ¹²Liu et al., 2020; ¹³Yu et al., 2019; ¹⁴Zhang, Liu, et al., 2019; ¹⁵Zheng, Zhang, et al., 2020; ¹⁶Li, Li, et al., 2016; ¹⁷Li, Wang, et al., 2018; ¹⁸Kaur et al., 2020; ¹⁹Nonaka et al., 2017; ²⁰Tang et al., 2017; ²¹ Sánchez-León et al., 2018; ²²Sashidhar et al., 2020; ²³Abe et al., 2019; ²⁴Zhai et al., 2019; ²⁵Gao et al., 2020; ²⁶González et al., 2019; ²⁷Jiang et al., 2017; ²⁸Abe, Araki, et al., 2018; ²⁹al Amin et al., 2019; ³⁰Okuzaki et al., 2018.

MLO, MILDEW RESISTANT LOCUS O; OsERF922, Oryza sativa ETHYLENE RESPONSIVE FACTOR 922; Clpsk1, Citrullus lanatus PHYTOSULFOKINE 1; eIF4E, EUKARYOTIC TRANSLATION INITIATION FACTOR 4E; MdDIPM4, Malus x domestica DspA/E-INTERACTING PROTEIN M4; CCD8, CAROTENOID CLEAVAGE DIOXYGENASE 8; CYP71A1, CYTOCHROME P450 71A1; SlLBD40, Solanum lycopersicum LATERAL ORGAN BOUNDARIES 40; SlMAPK3, S. lycopersicum MITOGEN-ACTIVATED PROTEIN KINASE 3; OsRR22, O. sativa RESPONSE REGULATOR 22; BnMAX1, Brassica napus MORE AXILLARY GROWTH 1; Gn1a, Grain number 1a; Gs3, Grain size 3; SGR1, STAYGREEN 1; LCYɛ, LYCOPENE Ɛ-CYCLASE; SlGAD2 and 3, S. lycopersicum GLUTAMATE DECARBOXYLASE 2 and 3; OsNramp5, O. sativa natural resistance associated macrophage protein 5; BnITPK; B. napus INOSITOL TETRAKISPHOSPHATE KINASE; TaQsd1, Triticum aestivum Quantitative trait locus on seed dormancy 1; BnA03.IND, B. napus A03 INDEHISCENT; Wx1, Waxy 1; PPO, POLYPHENOL OXIDASE; FAD2, FATTY ACID DESATURASE 2.

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