Next GEM Next Generation Mutagenesis. Guri Johal. Department of Botany and Plant Pathology. Purdue University

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1 Next GEM Next Generation Mutagenesis Guri Johal Department of Botany and Plant Pathology Purdue University June 24, 2015

2 The focus of this approach is how to enhance genetic variation, which drives almost everything we do in genetics, genomics and breeding A key source of genetic variation is: Mutations Spontaneous or Intentional or induced

3 Intentional induction of mutations Hermann Mueller Lewis Stadler Mueller and Stadler, working independently on Drosophila and barley, respectively, were the first to show that mutations can be induced in living organisms. They used X-rays in their research. Mueller got the Noble prize for this work but Stadler was overlooked

4 Induced mutagenesis Mutagenesis can be described as the process of inducing any heritable change in the genetic material which is subsequently transmitted to daughter cells where it gives rise to a mutant cell or individual (Rieger et al, 1976) Mutations can be induced by a number of means or agents; e.g., Physical agents Chemical agents Genetic mutagens

5 Among the chemical mutagens, Ethyl methanesulfonate (EMS) is the most popular Reason: it s one of the most efficient mutagen causing point mutations. It causes ethylation of guanine (G), converting it to o- 6-ethylguanine. During DNA replication, o-6-ethylguanine pairs with Thymine (T), instead of cytosine (C). As a consequence, EMS results in G/C to A/T transitions.

6 Chemical mutagenesis in plants With the exception of maize, mutagenesis is done by treating the seed with the mutagen. To do so, seed is soaked in a solution of EMS for some specific duration of time period, thoroughly washed, and then planted as the M1 material. The seed collected from these M1 plants is called the M2 seed.

7 Overview of chemical mutagenesis in Arabidopsis Dose: 0.3% or mm EMS 8-12 h duration M1 plant M2 seed Page and Grossniklaus, 2002

8 What happens when seed is treated with EMS? Genetically effective cell number (GECN)

9 Overview of chemical mutagenesis in Arabidopsis Genetically effective cell number (GECN) Page and Grossniklaus, 2002

10 M1 plants produced by chemical mutagenesis are chimeric with regard to mutations Henikoff and Comai, 2003

11 An M1 population size of roughly 125,000 plants, after EMS mutagenesis, is needed to effectively saturate the genome for all possible EMS-inducible base changes (Haughn and Somerville, 1987)

12 Production of a High-Efficiency TILLING Population through Polyploidization While the same treatment sterilized diploid Columbia, the tetraploid M1 plants produced good seed. To determine the mutation density, we searched 528 individuals for induced mutations in 15 genes for which few or no knockout alleles were previously available. We constructed tridimensional pools from the genomic DNA of M2 plants, amplified target DNA, and subjected them to Illumina sequencing. This small population provided a rich resource with approximately 25 mutations per queried 1.5-kb fragment, including on average four severe missense and 1.3 truncation mutations. The overall mutation density of 19.4 mutations Mb 1 is 4 times that achieved in the corresponding diploid accession, indicating that genomic redundancy engenders tolerance to high mutation density. Polyploidization of diploids will allow the production of small populations, such as less than 2,000, that provide allelic series from knockout to mild loss of function for virtually all genes. Comai group. Plant Physiology, 2013, Vol. 161, pp

13 Seed mutagenesis is not the method of choice in maize T T E E

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15 Thus pollen mutagenesis was invented in maize Gerry Neuffer, the inventor of pollen mutagenesis in maize he was a student of Stadler

16 A typical pollen mutagenesis protocol to generate M2 populations in maize Also called a random mutagenesis approach

17 Targeted mutagenesis in maize The example shown here is of the br2 dwarfing mutation. This is how the cross is done to generate new alleles of br2 by pollen mutagenesis with EMS. The mutant tester is the female.

18 We can t do seed mutagenesis in maize and Pollen mutagenesis efficiency is depressingly low Candela and Hake (2008)

19 The Next GEM A recurrent mutagenesis approach

20 (M0) B73 X B73 (M0) + EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

21 (M0) B73 X B73 (M0) + EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

22 (M0) B73 X B73 (M0) + EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

23 (M0) B73 X B73 (M0) + EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

24 The BC2M3 seed It is a library of maize mutations in the which mutation density is expected to be many times higher compared to the traditional pollen mutagenesis approach. This seed is almost an immortal resource of mutations and it can be dipped into at any time for any kind of mutational need. Every mutation in this resource is held in a heterozygous condition.

25 (M0) B73 X B73 (M0) + EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

26 (M1) (Seed mutagenesis) B73 X B73 + (M0) EMS ( - pollen mutagenesis) F1M X X X X BC1M2 1/2. X. 3/4 5/6. X. 7/ /100 BC2M3 Lot 1 Lot Lot 25 5,000 seed 5,000 seed 5,000 seed

27 B73 (treat seed with EMS SM) M1 + EMS () F1M plots X X X SM SM SM SM SM SM BC1M2 Lot 1 Lot 2 Lot Lot 50 5,000 seed 5,000 seed 5,000 seed

28 B73 (treat seed with EMS SM) M1 + EMS () F1M plots X X X SM SM SM SM SM SM BC1M2 Lot 1 Lot 2 Lot Lot 50 5,000 seed 5,000 seed 5,000 seed

29 Next Gen EMS Mutagenesis (Next GEM) Additional chemical mutagens We can also use other chemical mutagens instead of EMS. ENU (N-ethyl-N-nitrosourea) Sodium Azide, both of which cause single base pair changes. Another chemical that can be used is Ultraviolet trimethylpsoralen (UV/TMP). This chemical causes small deletions only few bps in size. EMS has a strong G/C to A/T transition bias ENU mainly produces A to T and T to A transversions but also all possible transitions UV/TMP exhibits no bias, causes both single-base changes and deletions

30 Next GEM utilities or advantages Some of these are as below and I ll go through these one-by-one: To generate allelic series of mutations especially if the interest is in rather weak or subtle alleles of the mutation of interest To infuse new genetic variation into elite lines and also fix their potential drawbacks To generate and maintain dominant mutations To explore heterosis and to identify potential overdominant loci contributing to heterosis To generate new traits, such as herbicide resistance

31 Next Gen EMS Mutagenesis (Next GEM) Utilities: Facilitates the identification and recovery of allelic series of partial loss-of-function, conditional or gain-of-function mutants, and having such an allelic series is ideal for structure-function analyses. Crossing with the mutant is very easy too; by collecting pollen from the mutant and making multiple pollinations with that. Very efficient compared to 1 on 1 crossing BC2M3 plants X br2-ref/br2-ref br2*/br2-ref Since the mutant phenotype will segregate 1:1 in some testcross families, the recovery of subtle or weak alleles of br2 will be facilitated by this technique.

32 Next Gen EMS Mutagenesis (Next GEM) Utilities: Can be used to explore genes involved in heterosis For instance, by crossing the BC2M3 B73 seed with Mo17. This can also be done in a reciprocal fashion by crossing the mutagenized Mo17 seed with B73 pollen. Pollinations can be done quickly by pooling pollen and dispensing a little bit on tens of plants quickly using glassine bags.

33 Next Gen EMS Mutagenesis (Next GEM) Utilities: Can be used to identify overdominant loci contributing to heterosis. Some loci/mutations in heterozygous condition outperform either parent. A good example of an overdominant mutant locus is that of the tomato SFT locus (Single flower truss) (Krieger, Lippman and Zamir, 2010), which increased tomato yield by 60% in heterozygous condition.

34 Next Gen EMS Mutagenesis (Next GEM) Utilities: Will be ideal for identifying and maintaining dominant or partially dominant mutants. Can be used to look for herbicide tolerant/resistant genes or gene traits

35 Next GEM Utilities: BC2M3 seed are all heterozygous for mutations Take 25 or so per lot; most mutations will be represented there. Twenty five such plants from each lot can be self pollinated to generate F2 (equivalent to traditional M2) families, which can be evaluated phenotypically to look at the breadth and spectrum of visible mutations. This F2/M2 resource can also be sequenced by nextgen to generate sequence indexed reverse genetics resource. Thus, we can do both forward and reverse genetics

36 Next Gen EMS Mutagenesis (Next GEM) Various modifications We can also use other chemical mutagens instead of EMS. A few that can be used are: ENU (N-ethyl-N-nitrosourea) and Sodium Azide, both of which cause single base pair changes. Another chemical that can be used is Ultraviolet trimethylpsoralen (UV/TMP). This chemical causes small deletions only few bps in size. Pollen treatment with these chemicals can be tried/optimized. A graduate student project! EMS has a strong G/C to A/T transition bias ENU mainly produces A to T and T to A transversions but also all possible transitions UV/TMP exhibits no bias, causes both single-base changes and deletions

37 Acknowledgements Brian Dilkes, Associate Professor, Horticulture Department Rajdeep Khangura, Ph.D. student

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39 Production of a High-Efficiency TILLING Population through Polyploidization Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized it with 50 mm ethylmethane sulfonate.

40 End!