1) Name three gene transfer methods that can be used to introduce DNA into plants.
2) Briefly describe three genetically modified plants that are currently in the field/on the market (outside Europe). Which traits do they contain?
3) What is Bt-toxin? Which organism is the original source of Bt-genes? Briefly explain its mechanism of toxicity for insects.
4) What does „codon-modification/optimization“ of a gene sequence mean? Why is it done?
5) Briefly describe three general strategies to achieve herbicide resistance in plants.
6) Can herbicide resistance also be achieved by conventional mutation breeding? How? Does GMO-regulation apply in such case?
7) Describe the mechanism of CRISPR/Cas technology. What is it used for?
Recombinant endonucleases (such as Zn-finger nucleases) can be used to modify plant genes (genome editing). Please explain briefly how this works.
9) Explain how a PCR works.
10) Briefly describe the use of particle bombardment for gene transfer into plants.
11) How does oligonucleotide-directed mutagenesis work? Describe a commercial product generated with this technology.
12) Explain the terms Cisgene and Transgene.
13) Your aim is to abolish a specific enzymatic activity in a given plant. You have got the sequence of the corresponding gene encoding the enzyme. Develop and describe a strategy to achieve this goal.
14) What is the role of the guide RNA in CRISPR/Cas-based gene editing?
15) Explain briefly how RNA-induced silencing works. How can this be used to generate plants with increased virus resistance?
16) Describe briefly the use of antisense (RNAi) technology for generating amylose-free potatoes.
17) Describe briefly the use of CRISPR/Cas-based technology for generating amylose-free potatoes.
18) Select two strategies for generating amylose-free potatoes. Describe them briefly, compare them, and point out advantages and disadvantages
19) The procedure for CRISPR/Cas-based gene editing can be done by transferring a DNA-construct encoding the Cas enzyme and the gRNA into the plant cell. Is the integration of the DNA strictly necessary? If not, describe the two options (with/without integration) and their pros and cons. Can CRISPR/Cas-based gene editing also work without DNA-transfer? How is this done?
20) Plant transformation constructs often contain a selectable marker. Why? Give an example for a selectable marker.
21) What is the aim of "reverse breeding"? Which function needs to be suppressed to achieve this goal?
22) Explain RNAi-based strategies to protect plants from insect pests.
23) How can CRISPR/Cas-based gene editing be used to introduce precise point mutations?
24) Describe the properties of golden rice.
Chapter 2: biotechnology in plant breeding, molecular markers, genetic and physical mapping and breeding application of markers (Buerstmayr)
25) Give your personally preferred definition of Plant Breeding and explain why you like it.
26) In which typical phases can the activity of Plant Breeding be structured? For which of these can Biotechnology help breeders: give three examples that you appreciate most.
27)For which of these can Biotechnology help breeders: give three examples that you appreciate most.
28) When, Where and By Whom did domestication of the first crop plants begin in human history?
29) Was domestication a single event in human history?
30) Mention and put in a time-line minimum ten (10) important milestones that mark major steps/achievement in the history of plant breeding, starting from domestication until today.
31) What is tissue culture in a plant breeding context? Name at least four typical applications of tissue culture in a plant breeding context.
32) What are Doubled Haploids, how can these be produced? Which is the typical genetic constitution of a doubled haploid plant? Advantages and disadvantages of this method in breeding.
33) From which cells can haploid plants be derived, and how? Will a haploid plant typically be fertile and form seeds?
33) What is the genetic status of a doubled haploid plant in terms of homozygosity or heterozygosity? Is there an alternative procedure to achieve the same zygosity-status as with doubled haploids? If you know one name and describe it.
34) Explain and describe the terms GENE and ALLELE. Mention a few examples.
35) What are morphological markers, name several examples. What is their advantage? What their disadvantage?
36) What are biochemical markers, name a few examples.
37) What do DNA markers typically measure? What are advantages of DNA markers, compared to e.g. morphological or biochemical markers?
38) Which are the principle types of DNA polymorphisms that we can utilize as DNA markers?
39) Mention at least four important types of DNA markers based on the methodology used for detection/analysis.
40) What are restriction-endonucleases? What is their typical feature ? For which molecular markers do we need restriction-endonucleases?
41) What is the principle behind gel-electrophoresis of DNA? How does it work? Which equipment do you need for gel-electrophoresis?
42) What is an RFLP marker? Describe the principle and the essential steps one needs for an RFLP analysis.
43) What is a dominant marker and what is a -dominant marker? Name a few examples in each category.
44) What is meant by PCR? Which ingredients are required for a PCR reaction?
45) Describe the principle and the steps of the PCR reaction.
46) Think about PCR reaction: at which rate are DNA molecules amplified with resect to their molecule size?
47) Do SSR markers usually detect SNPs or INDELs? What is the diffeence between SNP and INDEL polymorphism?
48) What is an SSR marker?
49) Which separation and detection methods can be used for analyzing SSR markers?
50) Are SSR markers usually dominant or co-dominant markers?
51) Are SSR markers usually bi-allelic or multi-allelic?
52) Why are SSR markers still quite popular in plant breeding application.
53) What is meant by the term SNP marker?
54) Name a few methods for SNP marker detection with and/or without electrophoresis. Describe the principle of one method.
55) What is the principle of the KASPar system for SNP detection?
56) What is meant with the term Genotyping by Sequencing (GBS)? Describe the principle of this method.
57) Name at least two methods that can be used for high throughput SNP marker genotyping. Describe advantages / disadvantages of at least two such methods.
58) Linkage mapping depends on which biological phenomenon that takes place during meiosis?
59) Your task is to develop a linkage map. How would you perform this task? Design and describe step-by-step what you need to do and achieve in order to generate a new linkage map from the beginning to completion.
60) You were given the task to identify markers and map quantitative trait loci (QTL) for starch content in sorghum. How would you perform this task? Design, describe, and possibly illustrate step-by-step what you need to do and achieve in order to map relevant QTL for these traits in this crop species from the beginning to completion.
61) Name and describe at least three possible population types useful for linkage mapping. Describe advantages/disadvantages of these population types.
62) Your task is to generate a linkage map in an F2 population of 250 F2 plants. Will it make a difference if you use a marker system with allows DOMINANT marker scoring or which allows CO-DOMINANT marker scoring? Which is the difference between a so called dominant marker versus a co-dominant marker? Which marker system would you prefer for making a genetic map in an F2 population (dominant or co-dominant)? and justify your an answer.
63) In a mapping project you have genotyped your mapping population of 250 DH lines with 40 SSR markers and 500 SNP markers (using a SNP chip method) and for two morphological markers with a monogenic segregation pattern: flower color, seed color. Your task is to calculate a genetic map in this population. --> Reply true or false: A) I can integrate only the DNA markers (which are SSR and SNP markers) and calculate a map combining these two molecular marker types BUT I cannot map the morphological markers at the same time. TRUE or FALSE? ----------------------------- B) I can integrate the all markers (=morphological and SSR and SNP markers) in one combined data set and use this to calculate a genetic map combining all three marker types in one go. TRUE or FALSE? ------------------------------
C) I cannot combine these marker data. The only solution is that I have to calculate three separate genetic maps: one for the SSR markers, one with the SNP markers, and another one with the morphological markers. TRUE or FALSE? ----------------------------- >> Explain and justify your answer....
64) What is an immortal mapping population, name a few examples, describe advantages / disadvantages of an immortal population.
65) What is a temporary mapping population, name a few examples, advantages / disadvantages of this population type for mapping?
66) You want to create a mapping population with 100 progeny from a cross of two homozygous parents. Let's say you could generate either 100 recombinant inbred lines (RIL) using single seed descent (SSD) or 100 doubled haploid lines (DH) descending from F1 plants. Does the population type (RILs vs. DH) have a impact on the expected resolution of the genetic map ? (A) YES (B) NO >> Explain your answer....
67) What if I make a cross between two homozygous parents, then perform 2 generations of random mating before I start with selfing lines to generate a recombinant inbred line (RIL) population of 100 RILs? Will the resolution of a linkage map from such a population increase or decrease or remain largely the same, relative to a 'normal' RIL population without two generations of random mating? (mark correct answer(s)) (A) increase (B) decarese (C) remain largely the same >> Explain and jusitfy your answer....
68) What if I make a cross between two parents and then: A) I perform 2 generations of random mating before I start with selfing the lines with single seed descent (SSD) to the F5 B) I start selfing immediately from the F1 and self by SSD to the F5. I then choose for genetic mapping 200 random F5 plants in population A) or B). The genetic resolution of the obtained linkage maps from population A and B will be I) exactly the same □ TRUE □ FALSE II) The resolution of the map from population A will be better □ TRUE □ FALSE III) The resolution of the map from population B will be better □ TRUE □ FALSE Explain and justify your answer!
69) How do you calculate the recombination rate between two loci (e.g. two markers or genes)?
70) Are two markers possibly linked if the experimentally obtained recombination rate between them is:
0.501? □ YES □ NO
0.01 □ YES □ NO
0.0005 □ YES □ NO
0.49 □ YES □ NO
0.05 □ YES □ NO
71) Two loci are 1 cM apart when the recombination rate between these two loci is .. (complete the sentence) ………
72) Why do we use mapping functions to convert recombination rates in genetic distances in Centimorgan?
73) Describe and explain the difference between: recombination rate (r) and genetic distance in cM
74) Is the recombination frequency typically uniform along a chromosome?
75) You should map a gene of interest in a bi-parental cross. Your aim is to map this gene with a precision of about 0.1 cM. You decided to develop a doubled haploid (DH) population for mapping. (A) What is the minimum number of DH lines in your mapping population in order to achieve this goal? (B) Could you alternatively use a recombinant inbred line (RIL) population as well ? (C) How many RILs would you need to obtain an expected average map resolution of 0.1 cM?
76) What is the basic principle behind a genetic similarity coefficient based on molecular markers?
→ Genotypes that often have marker alleles in common are closer related to each other than genotypes that possess often different marker alleles.
77) Mention / describe two methods for illustrating / visualizing genetic relatedness (similarity, diversity) based on molecular markers in plant populations.
78) For which reasons / questions could it be quite interesting to know about diversity / relatedness in a plant breeding context, name and describe minimum three examples.
79) Describe briefly two multivariate statistical approaches useful to estimate and to visualize and illustrate genetic diversity in a plant population based on molecular markers.
80) How can one map a gene responsible for a monogenic trait in a plant population, using genetic mapping. Describe and illustrate the principle and steps needed.
81) What is the principle difference between a qualitative trait compared to a quantitative trait? Give a few examples.
82) What are the two principle reasons for quantitative variation of a character in a segregating population?
83) You should find out among e.g. n putative F1 plants, which are real F1s and which are accidental selfings. Both: the egg-cell parent and the pollen parent appear phenotypically very similar and it is impossible to discriminate them based in their phenotypic appearance. Is it really necessary for the breeder to know which plants are selfings and which are true F1s? (A) □ YES it is or (B) □ NO it os not. > Explain your answer to the above question. > Describe a strategy to solve this problem.
84) What is the meaning of the term QTL? What is the basic definition of a QTL?
85) Which questions can we answer when we apply QTL analysis? And mention at least two questions we cannot answer with QTL ananlysis.
86) Illustrate and explain the principle steps needed for a QTL analysis.
87) What is a temporary mapping population, what is an immortal mapping population? Give a few examples.
88) Your task is mapping QTL for grain yield and seed color in soybean. You already made a cross between two homozygous parents, one with back seeds and low yield and one with yellow seeds and high yield, in the past. You can choose between two population types for mapping these characters: ------------- A) an F2 population with 300 F2 plants and B) a recombinant inbred line population (RIL) with 300 F6-derived RIL lines. ------------- Will it make a difference which population you choose for which trait? Which one would you prefer for mapping QTL for grain yield ? Which one would you choose for mapping the trait seed color? Explain why!
89) What is the general principle behind statistical methods used for QTL analysis? Name and explain at least two typical statistical methods for QTL estimation (QTL mapping).
90) Let's say you have mapped a gene for an important disease resistance trait in a plant population using genetic mapping. Your mapping was successful and you found markers that identity the chromosome and flank this gene at a distance of 2cM left and 3 cM right of this gene. Do you already know with this information the sequence and function of the disease resistance gene under investigation? ............... If not, what could you do in order to find the gene that causes this resistance trait?
91) Let's say you have mapped a gene for an important disease resistance trait in a plant population using genetic mapping. Your mapping was successful and you found markers that identity the chromosome and flank this gene at a distance of 1 cM left and 2 cM right of this gene. Which statement is ture? ----------------------------- (A) This is nice but only theoretical information but it is unfortunately useless for breeding, because I still do not know the exact sequence of the resistance gene. □ TRUE □ FALSE ----------------------------- (B) This is really good, I can use this information in breeding, .... ! □ TRUE □ FALSE ----------------------------- Explain and justif your answer of your answer
92) What is the principle of advanced-back cross QTL mapping? When is this approach applied?
93) What is meant with the term ‘introgression library’?
94) What is the principle of ‘association mapping’? What is the principle advantage of association mapping compared to mapping in bi-parental crossing populations?
95) What is meant with the term ‘Linkage Disequilibrium’ (LD)? What is LD mapping?
96) What is meant by the term MAS?
97) What is a MAGIC population in the context of QTL mapping? Why is this population type attractive for mapping?
98) Give a few examples when Marker Assisted Selection would be of advantage compared to traditional phenotypic selection?
99) For molecular marker assisted breeding we must know the sequence of all genes of the plant species we want to breed. Is this statement (A) □ True or (B) □ False? Explain and justify your answer!
100) What is meant by the term Genomic Selection (GS)? Which advantages are expected when using GS in plant breeding?
101) (A) For which trait architecture would you recommend marker assisted selection? (B) For which trait architecture would you recommend genomic selection? (C) Which are the typcial feature for these two approaches?
102) Illustrate and describe the components needed for using Genomic Seletion.
103) Let us say you have found markers flanking a QTL which you want to select in MAS. (A) Will the reliabiltiy of your MAS be the same or different when you use both markers simultaneously in MAS compared to using only one marker in MAS? (B) Explain and justify your answer.
104) Describe an situation when F2-enrichment could be a useful strategy in MAS. Explain and justify your answer.
105) What is the 'large p small n problem' thst is associated with Genomic Selection? With which data anaylsis strategy has thsi problem been overcome in Genomic Selection?
106) You have done a SSR marker analysis among 'putative' F1 plants from the cross female x male. Lane one on the gel is the allele from the female lane 2 from the male and all the others are the putative F1s. >> Which are true F1s and which are not true F1s? >> Plants that are not true F1s , what are these?
Chapter 3: Plant Genome Sequencing (Himmelbauer)
107) What is de novo genome sequencing and how does it differ from genome resequencing?
108) What is Pacific Biosciences (PacBio) sequencing? Explain the advantages of this technology for plant genome sequencing.
109) If an assembly is reported with an N50 size of 1 Megabase, what does it mean?
110) What is a consensus error?
111) You have downloaded a genome assembly from a public website. As a first step, you run a script on the assembly to count the number of bases. 5% of the bases are reported as unspecified bases "N". Explain the reason behind this finding.
112) How can you evaluate the completeness of a genome assembly?
113) Assemblies typically consist of contigs and scaffolds. Explain the difference.
114) You are planning to study the genome of a not yet sequenced crop plant. Your first task is to select an accession for sequencing. Explain what kind of plant material or genotype will be best suited for the work.
115) How can you identify contamination within an assembly? Name a few reasons why contaminations can occur.
116) What is genome annotation?
117) How can you discover genome variants and how can you confirm them?
118) Explain if, despite high base quality, a base call may still be wrong.
119) Explain the differences between a hierarchical shotgun strategy and a whole-genome shotgun strategy
120) You receive a file containing the scaffolds of an assembled plant genome. The assembly statistics indicates an N50-length of 1000 basepairs for the scaffolds. Explain what these assembly metrics imply. Do you think this is a high-quality genome assembly?
121) Explain the principles and features of nanopore sequencing
122) Explain what are the challenges of reconstructing the sequence of a plant genome
123) What is the Hi-C technology and how can it help to reconstruct genome sequences?
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