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Engineered minichromosomes in plants
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ScienceDirect Engineered minichromosomes in plants James A Birchler Platforms for the development of synthetic chromosomes in plants have been produced in several species using telomere mediated chromosomal truncation with the simultaneous inclusion of sites that facilitate further additions to the newly generated minichromosome. By utilizing truncated supernumerary or B chromosomes, the output of the genes on the minichromosome can be amplified. Proof of concept experiments have been successful illustrating that minichromosome platforms can be modified in vivo. Engineered minichromosomes can likely be combined with haploid breeding if they are incorporated into inducer lines given that the observations that basically inert chromosomes from haploid inducer lines can be recovered at workable frequencies in otherwise haploid plants. Future needs of synthetic chromosome development are discussed. Addresses Division of Biological Sciences, University of Missouri, Columbia, MO 65211, United States Corresponding author: Birchler, James A ([email protected])
Current Opinion in Plant Biology 2014, 19:76–80 This review comes from a themed issue on Physiology and metabolism Edited by Sarah E O’Connor and Thomas P Brutnell
http://dx.doi.org/10.1016/j.pbi.2014.05.009 1369-5266/Published by Elsevier Ltd.
Introduction Artificial chromosomes were first assembled in yeast by combining centromere sequences, a selectable marker, and an origin of replication and were capped by telomeres at the ends [1]. The centromeres of yeast, however, are unusual in that they are point centromeres rather than the repetitive arrays that are typical of multicellular eukaryotes including plants. Artificial chromosomes have been generated in mammalian cells by transformation of centromere arrays and selectable markers but usually the result is a megabase conglomerate of the input [2]. Alternatively, artificial chromosome constructs have been produced by the use of telomere mediated chromosomal truncation that cleaves off the chromosome arms and leaves the endogenous centromere that is subsequently modified [3,4]. Current Opinion in Plant Biology 2014, 19:76–80
The use of centromere repeats to produce artificial chromosomes in plants has been attempted but there is no convincing demonstration of their function after reintroduction. Centromere sequences do not necessarily cause the formation of a kinetochore and many examples of inactive centromeres have now been described [5–8]. In a careful test of centromere sequence function in maize and rice, the sequences were successfully re-introduced into the genome but were not active [9]. A paper published some years ago claimed to have produced artificial chromosomes from centromere sequences transformed into maize cells [10]. A full critique of this work has been published [11] and will not be re-iterated here other than to note that it is likely that the genetic marker followed was inserted into a normal chromosome and if the FISH images were authentic they must represent conglomerates of input DNA on the order of megabases. Thus, while theoretically possible, it is not presently clear if the build-up approach to producing artificial chromosomes will work in plants. Here, we will focus on the telomere-mediated truncation method in which endogenous centromeres are used to attach selectable markers and site-specific recombination cassettes [12] that permit the modification of the minichromosome and provide the potential to grow the chromosome. The method of telomere-mediated truncation, as noted above, uses telomere repeats inserted into one side of the transformation construct (Figure 1). The presence of this array causes chromosome breakage at the site of potential integration, leaving the other end of the construct attached and with the presence of a telomere at the broken end. Using truncation, both B [13,14] and A [15] chromosomes have been cut down to the point that basically an endogenous centromere is present with added transgenes. In this case, telomere sequences in the transformed constructs condition the formation of telomere structures and the distal ends of chromosomes are lost. Because the telomere sequence is identical in the vast majority of plants, this technique can be and has been applied widely across taxa and published reports have noted success with telomere-mediated truncation in Arabidopsis [16,17], barley [18], and rice [19]. Indeed, vectors developed for one species can be used either directly or with slight modification in others. The precise mechanism of telomere-mediated truncation is not known. It is known that there is an apparent requirement for only one side of the introduced fragment of DNA to contain telomere repeats [16,17,20]. These constructs can be stably integrated in some transformations and these insertions do not appear to cause www.sciencedirect.com
Minichromosomes Birchler 77
Figure 1
Microprojectile Bombardment B-chromosomes
(a)
Microprojectile Transformation
NUCLEUS
Transgene Telomere Repeat Array (b)
T-DNA
Agrobacteriummediated Transformation Current Opinion in Plant Biology
Generalized scheme for the production of engineered minichromosomes in maize. (a) At the left is depicted a stylized maize cell containing two supernumerary B chromosomes (labeled). Embryos of such a genotype are subjected to microprojectile bombardment with input DNA carrying telomere repeats (dark blue) affixed to transgenes (black) to be added to the truncated chromosome. On the right is depicted a severely truncated B chromosome but that has an endogenous centromere with the added transgenes placed at the site of truncation and then these transgenes are capped with the added telomere array. (b) The same type of progression is used during Agrobacterium-mediated transformation with the same type of input DNA, which is present in a T-DNA with the telomere repeats inside the right border. Truncation removes the distal portion of the chromosome and places the desired transgenes at the terminal position. Figure modified from Ref. [11] by Nathan Swyers.
truncation in subsequent generations. It is thus likely that the truncation process occurs as an interference of the normal mechanism of incorporation of introduced DNA into plant chromosomes but truncation does not occur in every case.
New developments A recent development for producing engineered minichromosomes plays on the fact that cobombardments of mixed DNA fragments will often produce co-integrations. Thus, by mixing free telomere arrays with a construct carrying genes of interest and a selectable marker, two groups have demonstrated that truncated chromosomes could be recovered [15,19]. The co-integration must occur in such an orientation that the selectable marker is retained on the chromosome and the position of the telomere array is terminal. A second advance involves the use of telotrisomics as starting material for transformation with the intent of telomere-mediated truncation. Telotrisomics are extra chromosomes that consist of a chromosome arm that www.sciencedirect.com
results from misdivision of the centromere. A complete collection, for example, is available for rice. The concept is that with truncation of this extra chromosome, there will be no normal chromosome with a terminal deletion and thus the truncation event can be recovered and passed to the next generation. Such an approach is useful in species for which there are no supernumerary B chromosomes or for which there are no tetraploid varieties available — the two previous means of recovering truncated chromosomes. This approach was successful in producing engineered minichromosomes in rice [19]. It should be noted that truncated chromosomes could also be recovered by using diploid material as the target of transformation. Such a case was found in maize by using the cobombardment of the transforming construct and free telomeres [15]. The minichromosome recovered had an endogenous A centromere together with the added genes and was present in cells with the normal number of chromosomes. It is apparently the case that the free telomeres fractured the chromosomes to such an extent Current Opinion in Plant Biology 2014, 19:76–80
78 Physiology and metabolism
that both arms were removed. At the time of formation, one would anticipate that a monosomic condition would be generated for the fragmented chromosome. However, the recovered minichromosome was found in an otherwise diploid plant suggesting that any such monosomic state was selected against in competition with cells that regain two copies of the chromosome via nondisjunction, for example. Clearly, the exact mechanism cannot be reconstructed after the fact but the stated scenario seems reasonable.
background that contains one or more normal B chromosomes. Using this fact, engineered truncated mini B chromosomes were subjected to an accumulation regime [27]. Multiple copies could be recovered ranging up to 19. Thus, in addition to engineering high expression of genes on the minichromosome, it is possible in the case of those derived from B chromosomes that the output can be amplified further if gene silencing mechanisms can be avoided.
Behavior of small chromosomes With the production of truncated chromosomes or engineered minichromosomes now found in several plant species, future developments will focus on how to modify these chromosomes in vivo and add to them. Previous methods for modifying transgenes have relied on site-specific recombination systems. Such systems involve a recombinase that recognizes two target sites and catalyzes an exchange. The product of this reaction can either be an inversion of the DNA between the target sites or excision of the intervening DNA as a circle depending on the relative orientation. This technique has been used to remove selectable markers from transgene cassettes present in normal chromosomes [21–23]. Gaeta and colleagues used the Cre-lox system to demonstrate that the selectable marker on an engineered minichromosome could be removed in vivo. This demonstration served as a proof of concept that sequences could be removed in vivo from engineered minichromosomes.
Minichromosome copy number amplification Supernumerary or B chromosomes of maize [24] are a favored target for producing engineered minichromosomes because they are basically genetically inert causing little in the way of phenotypic consequence until their numbers approach about 15. They are not present in most inbred lines of maize and their absence has no impact on the phenotype just as their presence has little to none. Thus, their truncation can be easily recognized and the truncation events can be readily recovered. The normal B chromosome is maintained in populations because of its accumulation mechanism. This mechanism consists of nondisjunction at the second pollen mitosis [25] that produces the two maize sperm and the preferential fertilization of the egg as opposed to the polar nuclei by the sperm with two B chromosomes [26]. The centromere is the apparent target of nondisjunction but will only occur when the distal tip of the B chromosome is present in the same nucleus. Thus, the truncated B chromosomes lose the property of nondisjunction and subsequently behave as normal chromosomes — a useful circumstance for engineered minichromosomes. However, the truncated B chromosomes can be induced to undergo nondisjunction if they are present in a genetic Current Opinion in Plant Biology 2014, 19:76–80
Coping with the meiotic transmission of small chromosomes is an area for future development in the field. Small chromosomes often lack sister chromatid cohesion in meiosis I and thus the chromatids separate at this division whereas normal sized chromosomes do not [28,29]. This fact precludes a 100% transmission if a pair of minichromosomes is present because there will be microspores at the end of meiosis that will be missing a minichromosome becaue of sister separation in meiosis I and random distribution in meiosis II. Also small chromosomes often cannot find their pairing partners in meiosis, which is a fact that also militates against complete transmission from a pair of minichromosomes because they independently assort rather than segregate from each other. These considerations can potentially be overcome. One way would be to use a truncated B chromosome that is sufficiently long to find its pairing partner and to maintain sister chromatid cohesion. Truncated B’s of about half size or larger appear to fulfill these criteria [28,29]. A second potential means to overcome these issues would be to place a gametophytic selection on a minichromosome. If a plant has one such minichromosome and it was used as a male parent, then there would be an effective complete transmission to the next generation of the mini. Gametophytic selection might work via placing a normal copy of a nuclear male sterile gene on the minichromosome or alternatively using a gametophytically acting nuclear restorer of fertility for a cytoplasmic male sterility system such as cms-S in maize. It should be noted, however, that for crop species that have difficult breeding programs but use vegetative propagation for field planting such as sugarcane, cassava, banana, potato, and others, these issues are irrelevant. Breeding programs could select an optimal genotype carrying the desired minichromosome and then expand it for field propagation. The current state of the art needs no further adjustments for these types of crops.
Combining minichromosomes and haploid breeding Recent findings suggest that minichromosomes can be combined with haploid breeding to facilitate transgene transfer to many new varieties or to assess how a transgene behaves in a different genetic background. Haploid www.sciencedirect.com
Minichromosomes Birchler 79
breeding is now used extensively in maize to develop new genetic materials. Haploid inducer lines have been optimized from the original stock 6 line discovered by Coe [30]. When they are used as a male parent, the paternal chromosomes are lost in a fraction of the progeny producing maternal haploids. However, cytological and genetic evidence suggested that occasionally chromosomal material from the inducer lines is maintained and incorporated into the haploids that are doubled [31–33]. Recently, it was discovered that the B chromosome of maize when present in the inducer lines could be recovered in progeny maternal haploids as an extra chromosome [34]. Our laboratory has confirmed this result [M McCaw and JA Birchler, unpublished]. Given this result, it is likely that minichromosomes introgressed into inducer lines could be transferred to haploids. Using this procedure, it would then be possible to incorporate the minichromosome into new inbred lines much more quickly than by repeated backcrossing. Also, the mere ability to place engineered minichromosomes into haploids would allow the evaluation of the interaction of the included transgenes with various established or novel genomic constitutions. Haploid oat is produced by using maize pollen onto oat and a related type of phenomenon might be at work in those oat–maize addition lines that often result in a plant in which a haploid oat is carrying an extra maize chromosome [35]. Haploid inducer lines that function via modified centromeric histone exhibit the retention of chromosomes from the inducer at a certain frequency and thus they might be used in the same fashion with engineered minichromosomes [36].
In the future, new properties could be conferred to plants by the introduction of whole biochemical pathways. Such properties might include means to modify the use of chemical fertilizers, to develop insect, microbial, or viral resistances, to adapt crops to new field conditions, to improve nutritional qualities, and to increase yield among the many possibilities. This technology might also allow the use of plants as factories to generate foreign proteins or metabolites in mass quantities. In basic studies, chromosomes could be produced to specification to understand the parameters needed for normal chromosome structure, behavior, and the mechanism of recombination. Introduction of biochemical pathways onto engineered minichromosomes could facilitate the study of many aspects of metabolism. Adding regulatory factors could be used to study gene expression. In a broader sense, the development of new genetic engineering techniques will facilitate genomics studies in general.
1.
Murray AW, Szostak JW: Construction of artificial chromosomes in yeast. Nature 1983, 305:189-193.
Next step
2.
Basu J, Willard HF: Artificial and engineered chromosomes: non-integrating vectors for gene therapy. Trends Mol Med 2005, 11:251-258.
3.
Farr CJ, Stevanovic M, Thomson EJ, Goodfellow PN, Cooke HJ: Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis. Nat Genet 1992, 2:275-282.
4.
Heller R, Brown KE, Burgtof C, Brown WR: Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage. Proc Natl Acad Sci U S A 1996, 93:71257130.
5.
Han F, Lamb J, Birchler JA: High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize. Proc Natl Acad Sci U S A 2006, 103:3238-3243.
6.
Han F, Gao Z, Birchler JA: Centromere inactivation and reactivation reveals both epigenetic and genetic components for centromere specification. Plant Cell 2009, 21:1929-1939.
7.
Birchler JA, Han F: Maize centromeres: structure, function, epigenetics. Annu Rev Genet 2009, 43:287-303.
8.
Gao Z, Fu S, Dong Q, Han F, Birchler JA: Inactivation of a centromere during the formation of a translocation in maize. Chromos Res 2011, 19:755-761.
9.
Phan BH, Jin W, Topp CN, Zhong CX, Jiang J, Dawe RK, Parrott WA: Transformation of rice with long DNA-segments consisting of random genomic DNA or centromere-specific DNA. Transgenic Res 2007, 16:341-351.
The next step in the development of effective synthetic chromosomes would be to have means to grow the chromosome to one’s own specifications. Techniques have been developed for the addition of new DNA into sites already present in the genome so it should be straightforward to adapt those methods to minichromosome vectors. With the ability to amend minichromosomes, a path could be envisioned by which there could be continuous additions to a chromosome. If it becomes possible to produce minichromosomes with large cargos or to recombine large cargos onto them [37], synthetic chromosomes could be grown in large increments. If gametophytic selection for enhanced transmission from one generation to the next could be incorporated into the minichromosome, a method to place the minichromosome into virtually all progeny would be established. Finally, the transfer of minichromosomes from a haploid inducer into numerous target lines will eliminate linkage drag and facilitate the placement of minichromosomes into many different varieties. The consolidation of these features into the same vectors should produce a minichromosome with high potential for routine and effective use. www.sciencedirect.com
Acknowledgements Research on this topic supported by NSF Plant Genome grant DBI 0701297 and IOS 1339198. The author thanks Nathan Swyers for producing Figure 1.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
10. Carlson SR, Rudgers GW, Zieler H, Mach JM, Luo S, Grunden E, Krol C, Copenhaver GP, Preuss D: Meiotic transmission of an in vitro-assembled autonomous maize minichromosome. PLoS Genet 2007, 3:e179. Current Opinion in Plant Biology 2014, 19:76–80
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11. Gaeta RT, Masonbrink RE, Krishnaswamy L, Zhao C, Birchler JA: Synthetic chromosome platforms in plants. Annu Rev Plant Biol 2012, 63:307-330. A review of the development and potential of synthetic chromosomes in plants. 12. Dale EC, Ow DW: Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 1990, 91:79-85. 13. Yu W, Lamb JC, Han F, Birchler JA: Telomere-mediated chromosomal truncation in maize. Proc Natl Acad Sci U S A 2006, 103:17331-17336. 14. Yu W, Han F, Gao Z, Vega JM, Birchler JA: Construction and behavior of engineered minichromosomes in maize. Proc Natl Acad Sci U S A 2007, 104:8924-8929. 15. Gaeta RT, Masonbrink RE, Zhao C, Sanyal A, Krishnaswamy L, Birchler JA: In vivo modification of a maize engineered minichromosome. Chromosoma 2013, 122:221-232. First demonstration of in vivo modification of an engineered minichromosome. Used site-specific recombination to remove the selectable marker. 16. Teo CH, Ma L, Kapusi E, Hensel G, Kumlehn J, Schubert I, Houben A, Mette MF: Induction of telomere-mediated chromosomal truncation and stability of truncated chromosomes in Arabidopsis thaliana. Plant J 2011, 68:28-39. Demonstration of telomere mediated chromosomal truncation in Arabidopsis using tetraploids as the target. 17. Nelson A, Lamb J, Kobrossly P, Shippen D: Parameters affecting telomere-mediated chromosomal truncation in arabidopsis. Plant Cell 2011, 23:2263. Demonstration of telomere mediated truncation in Arabidopsis and characterization of truncation events. 18. Kapusi E, Ma L, Teo CH, Hensel G, Himmelbach A, Schubert I, Mette MF, Kumlehn J, Houben A: Telomere-mediated truncation of barley chromosomes. Chromosoma 2012, 121:181-190. Demonstration of telomere mediated chromosomal truncation in barley. 19. Xu C, Cheng Z, Yu W: Construction of rice mini-chromosomes by telomere-mediated chromosomal truncation. Plant J 2012, 70:1070-1079. First demonstration of engineered minichromosomes in rice. Used telotrisomics and cobombardment of free telomeres and genes to be added to truncate the extra chromosome. 20. Chiurazzi M, Signer ER: Termini and telomeres in T-DNA transformation. Plant Mol Biol 1994, 26:923-934. 21. Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L: Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 2003, 107:1157-1168. 22. Srivastava V, Ow DW: Marker-free site-specific gene integration in plants. Trends Biotechnol 2004, 22:627-629. 23. Akbudak MA, Srivastava V: Improved FLP recombinase, FLPe, efficiently removes marker gene transgene locus developed by Cre-lox mediated site-specific gene integration in rice. Mol Biotechnol 2011, 49:82-89.
Current Opinion in Plant Biology 2014, 19:76–80
24. Carlson WR: The B chromosome of maize. CRC Crit Rev Plant Sci 1986, 3:201-226. 25. Roman H: Mitotic nondisjunction in the case of interchanges involving the B-type chromosome in maize. Genetics 1947, 32:391-409. 26. Roman H: Directed fertilization in maize. Proc Natl Acad Sci U S A 1948, 34:36-42. 27. Masonbrink RE, Birchler JA: Accumulation of multiple copies of maize minichromosomes. Cytogenet Genome Res 2012, 137:50-59. Demonstration that engineered minichromosomes derived from the B chromosome of maize can be amplified in copy number using the B chromosome accumulation mechanism. 28. Han F, Gao Z, Yu W, Birchler JA: Minichromosome analysis of chromosome pairing, disjunction and sister chromatid cohesion in maize. Plant Cell 2007, 19:3853-3863. 29. Masonbrink RE, Birchler JA: Multiple maize minichromosomes in meiosis. Chromos Res 2012, 20:395-402. Characterization of pairing and sister chromatid cohesion properties of engineered minichromosomes of varying lengths. 30. Coe EH: A line of maize with high haploid frequency. Am Naturalist 1959, 93:381-382. 31. Deimling S, Rober FK, Geiger HH: Methodik und genetic der invivo-haploid eninducktionbeimais. Vortr PX Anzensuchtg 1997, 38:203-224. 32. Zhang Z, Qiu F, Liu Y, Ma K, Li Z, Xu S: Chromosome elimination and in vivo haploid production induced by stock 6-derived inducer line in maize (Zea mays L.). Plant Cell Rep 2008, 27:1851-1860. 33. Li L, Xu X, Jin W, Chen S: Morphological and molecular evidence for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize. Planta 2009, 230:367-376. 34. Zhao X, Xu X, Xie H, Chen S, Jin W: Fertilization and uniparental chromosome elimination during crosses with maize haploid inducers. Plant Physiol 2013, 163:721-731. Discovery that B chromosomes incorporated into haploid inducer lines of maize could be recovered in haploids. Proof of concept that minichromosomes might be transferred in a like manner. 35. Ananiev EV, Riera-Lizarazu O, Rines HW, Phillips RL: Oat–maize chromosome addition lines: a new system for mapping the maize genome. Proc Natl Acad Sci U S A 1997, 94:3524-3529. 36. Ravi M, Chan SW: Haploid plants produced by centromeremediated genome elimination. Nature 2010, 464:615-618. 37. Ow DW: Recombinase-mediated gene stacking as a transformation operating system. J. Integr Plant Biol 2011, 53:512-519. Outlines stacking methods for multiple additions to a pre-existing transgene cassette, which could be applied to minichromosomes.
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ScienceDirect Engineered minichromosomes in plants James A Birchler Platforms for the development of synthetic chromosomes in plants have been produced in several species using telomere mediated chromosomal truncation with the simultaneous inclusion of sites that facilitate further additions to the newly generated minichromosome. By utilizing truncated supernumerary or B chromosomes, the output of the genes on the minichromosome can be amplified. Proof of concept experiments have been successful illustrating that minichromosome platforms can be modified in vivo. Engineered minichromosomes can likely be combined with haploid breeding if they are incorporated into inducer lines given that the observations that basically inert chromosomes from haploid inducer lines can be recovered at workable frequencies in otherwise haploid plants. Future needs of synthetic chromosome development are discussed. Addresses Division of Biological Sciences, University of Missouri, Columbia, MO 65211, United States Corresponding author: Birchler, James A ([email protected])
Current Opinion in Plant Biology 2014, 19:76–80 This review comes from a themed issue on Physiology and metabolism Edited by Sarah E O’Connor and Thomas P Brutnell
http://dx.doi.org/10.1016/j.pbi.2014.05.009 1369-5266/Published by Elsevier Ltd.
Introduction Artificial chromosomes were first assembled in yeast by combining centromere sequences, a selectable marker, and an origin of replication and were capped by telomeres at the ends [1]. The centromeres of yeast, however, are unusual in that they are point centromeres rather than the repetitive arrays that are typical of multicellular eukaryotes including plants. Artificial chromosomes have been generated in mammalian cells by transformation of centromere arrays and selectable markers but usually the result is a megabase conglomerate of the input [2]. Alternatively, artificial chromosome constructs have been produced by the use of telomere mediated chromosomal truncation that cleaves off the chromosome arms and leaves the endogenous centromere that is subsequently modified [3,4]. Current Opinion in Plant Biology 2014, 19:76–80
The use of centromere repeats to produce artificial chromosomes in plants has been attempted but there is no convincing demonstration of their function after reintroduction. Centromere sequences do not necessarily cause the formation of a kinetochore and many examples of inactive centromeres have now been described [5–8]. In a careful test of centromere sequence function in maize and rice, the sequences were successfully re-introduced into the genome but were not active [9]. A paper published some years ago claimed to have produced artificial chromosomes from centromere sequences transformed into maize cells [10]. A full critique of this work has been published [11] and will not be re-iterated here other than to note that it is likely that the genetic marker followed was inserted into a normal chromosome and if the FISH images were authentic they must represent conglomerates of input DNA on the order of megabases. Thus, while theoretically possible, it is not presently clear if the build-up approach to producing artificial chromosomes will work in plants. Here, we will focus on the telomere-mediated truncation method in which endogenous centromeres are used to attach selectable markers and site-specific recombination cassettes [12] that permit the modification of the minichromosome and provide the potential to grow the chromosome. The method of telomere-mediated truncation, as noted above, uses telomere repeats inserted into one side of the transformation construct (Figure 1). The presence of this array causes chromosome breakage at the site of potential integration, leaving the other end of the construct attached and with the presence of a telomere at the broken end. Using truncation, both B [13,14] and A [15] chromosomes have been cut down to the point that basically an endogenous centromere is present with added transgenes. In this case, telomere sequences in the transformed constructs condition the formation of telomere structures and the distal ends of chromosomes are lost. Because the telomere sequence is identical in the vast majority of plants, this technique can be and has been applied widely across taxa and published reports have noted success with telomere-mediated truncation in Arabidopsis [16,17], barley [18], and rice [19]. Indeed, vectors developed for one species can be used either directly or with slight modification in others. The precise mechanism of telomere-mediated truncation is not known. It is known that there is an apparent requirement for only one side of the introduced fragment of DNA to contain telomere repeats [16,17,20]. These constructs can be stably integrated in some transformations and these insertions do not appear to cause www.sciencedirect.com
Minichromosomes Birchler 77
Figure 1
Microprojectile Bombardment B-chromosomes
(a)
Microprojectile Transformation
NUCLEUS
Transgene Telomere Repeat Array (b)
T-DNA
Agrobacteriummediated Transformation Current Opinion in Plant Biology
Generalized scheme for the production of engineered minichromosomes in maize. (a) At the left is depicted a stylized maize cell containing two supernumerary B chromosomes (labeled). Embryos of such a genotype are subjected to microprojectile bombardment with input DNA carrying telomere repeats (dark blue) affixed to transgenes (black) to be added to the truncated chromosome. On the right is depicted a severely truncated B chromosome but that has an endogenous centromere with the added transgenes placed at the site of truncation and then these transgenes are capped with the added telomere array. (b) The same type of progression is used during Agrobacterium-mediated transformation with the same type of input DNA, which is present in a T-DNA with the telomere repeats inside the right border. Truncation removes the distal portion of the chromosome and places the desired transgenes at the terminal position. Figure modified from Ref. [11] by Nathan Swyers.
truncation in subsequent generations. It is thus likely that the truncation process occurs as an interference of the normal mechanism of incorporation of introduced DNA into plant chromosomes but truncation does not occur in every case.
New developments A recent development for producing engineered minichromosomes plays on the fact that cobombardments of mixed DNA fragments will often produce co-integrations. Thus, by mixing free telomere arrays with a construct carrying genes of interest and a selectable marker, two groups have demonstrated that truncated chromosomes could be recovered [15,19]. The co-integration must occur in such an orientation that the selectable marker is retained on the chromosome and the position of the telomere array is terminal. A second advance involves the use of telotrisomics as starting material for transformation with the intent of telomere-mediated truncation. Telotrisomics are extra chromosomes that consist of a chromosome arm that www.sciencedirect.com
results from misdivision of the centromere. A complete collection, for example, is available for rice. The concept is that with truncation of this extra chromosome, there will be no normal chromosome with a terminal deletion and thus the truncation event can be recovered and passed to the next generation. Such an approach is useful in species for which there are no supernumerary B chromosomes or for which there are no tetraploid varieties available — the two previous means of recovering truncated chromosomes. This approach was successful in producing engineered minichromosomes in rice [19]. It should be noted that truncated chromosomes could also be recovered by using diploid material as the target of transformation. Such a case was found in maize by using the cobombardment of the transforming construct and free telomeres [15]. The minichromosome recovered had an endogenous A centromere together with the added genes and was present in cells with the normal number of chromosomes. It is apparently the case that the free telomeres fractured the chromosomes to such an extent Current Opinion in Plant Biology 2014, 19:76–80
78 Physiology and metabolism
that both arms were removed. At the time of formation, one would anticipate that a monosomic condition would be generated for the fragmented chromosome. However, the recovered minichromosome was found in an otherwise diploid plant suggesting that any such monosomic state was selected against in competition with cells that regain two copies of the chromosome via nondisjunction, for example. Clearly, the exact mechanism cannot be reconstructed after the fact but the stated scenario seems reasonable.
background that contains one or more normal B chromosomes. Using this fact, engineered truncated mini B chromosomes were subjected to an accumulation regime [27]. Multiple copies could be recovered ranging up to 19. Thus, in addition to engineering high expression of genes on the minichromosome, it is possible in the case of those derived from B chromosomes that the output can be amplified further if gene silencing mechanisms can be avoided.
Behavior of small chromosomes With the production of truncated chromosomes or engineered minichromosomes now found in several plant species, future developments will focus on how to modify these chromosomes in vivo and add to them. Previous methods for modifying transgenes have relied on site-specific recombination systems. Such systems involve a recombinase that recognizes two target sites and catalyzes an exchange. The product of this reaction can either be an inversion of the DNA between the target sites or excision of the intervening DNA as a circle depending on the relative orientation. This technique has been used to remove selectable markers from transgene cassettes present in normal chromosomes [21–23]. Gaeta and colleagues used the Cre-lox system to demonstrate that the selectable marker on an engineered minichromosome could be removed in vivo. This demonstration served as a proof of concept that sequences could be removed in vivo from engineered minichromosomes.
Minichromosome copy number amplification Supernumerary or B chromosomes of maize [24] are a favored target for producing engineered minichromosomes because they are basically genetically inert causing little in the way of phenotypic consequence until their numbers approach about 15. They are not present in most inbred lines of maize and their absence has no impact on the phenotype just as their presence has little to none. Thus, their truncation can be easily recognized and the truncation events can be readily recovered. The normal B chromosome is maintained in populations because of its accumulation mechanism. This mechanism consists of nondisjunction at the second pollen mitosis [25] that produces the two maize sperm and the preferential fertilization of the egg as opposed to the polar nuclei by the sperm with two B chromosomes [26]. The centromere is the apparent target of nondisjunction but will only occur when the distal tip of the B chromosome is present in the same nucleus. Thus, the truncated B chromosomes lose the property of nondisjunction and subsequently behave as normal chromosomes — a useful circumstance for engineered minichromosomes. However, the truncated B chromosomes can be induced to undergo nondisjunction if they are present in a genetic Current Opinion in Plant Biology 2014, 19:76–80
Coping with the meiotic transmission of small chromosomes is an area for future development in the field. Small chromosomes often lack sister chromatid cohesion in meiosis I and thus the chromatids separate at this division whereas normal sized chromosomes do not [28,29]. This fact precludes a 100% transmission if a pair of minichromosomes is present because there will be microspores at the end of meiosis that will be missing a minichromosome becaue of sister separation in meiosis I and random distribution in meiosis II. Also small chromosomes often cannot find their pairing partners in meiosis, which is a fact that also militates against complete transmission from a pair of minichromosomes because they independently assort rather than segregate from each other. These considerations can potentially be overcome. One way would be to use a truncated B chromosome that is sufficiently long to find its pairing partner and to maintain sister chromatid cohesion. Truncated B’s of about half size or larger appear to fulfill these criteria [28,29]. A second potential means to overcome these issues would be to place a gametophytic selection on a minichromosome. If a plant has one such minichromosome and it was used as a male parent, then there would be an effective complete transmission to the next generation of the mini. Gametophytic selection might work via placing a normal copy of a nuclear male sterile gene on the minichromosome or alternatively using a gametophytically acting nuclear restorer of fertility for a cytoplasmic male sterility system such as cms-S in maize. It should be noted, however, that for crop species that have difficult breeding programs but use vegetative propagation for field planting such as sugarcane, cassava, banana, potato, and others, these issues are irrelevant. Breeding programs could select an optimal genotype carrying the desired minichromosome and then expand it for field propagation. The current state of the art needs no further adjustments for these types of crops.
Combining minichromosomes and haploid breeding Recent findings suggest that minichromosomes can be combined with haploid breeding to facilitate transgene transfer to many new varieties or to assess how a transgene behaves in a different genetic background. Haploid www.sciencedirect.com
Minichromosomes Birchler 79
breeding is now used extensively in maize to develop new genetic materials. Haploid inducer lines have been optimized from the original stock 6 line discovered by Coe [30]. When they are used as a male parent, the paternal chromosomes are lost in a fraction of the progeny producing maternal haploids. However, cytological and genetic evidence suggested that occasionally chromosomal material from the inducer lines is maintained and incorporated into the haploids that are doubled [31–33]. Recently, it was discovered that the B chromosome of maize when present in the inducer lines could be recovered in progeny maternal haploids as an extra chromosome [34]. Our laboratory has confirmed this result [M McCaw and JA Birchler, unpublished]. Given this result, it is likely that minichromosomes introgressed into inducer lines could be transferred to haploids. Using this procedure, it would then be possible to incorporate the minichromosome into new inbred lines much more quickly than by repeated backcrossing. Also, the mere ability to place engineered minichromosomes into haploids would allow the evaluation of the interaction of the included transgenes with various established or novel genomic constitutions. Haploid oat is produced by using maize pollen onto oat and a related type of phenomenon might be at work in those oat–maize addition lines that often result in a plant in which a haploid oat is carrying an extra maize chromosome [35]. Haploid inducer lines that function via modified centromeric histone exhibit the retention of chromosomes from the inducer at a certain frequency and thus they might be used in the same fashion with engineered minichromosomes [36].
In the future, new properties could be conferred to plants by the introduction of whole biochemical pathways. Such properties might include means to modify the use of chemical fertilizers, to develop insect, microbial, or viral resistances, to adapt crops to new field conditions, to improve nutritional qualities, and to increase yield among the many possibilities. This technology might also allow the use of plants as factories to generate foreign proteins or metabolites in mass quantities. In basic studies, chromosomes could be produced to specification to understand the parameters needed for normal chromosome structure, behavior, and the mechanism of recombination. Introduction of biochemical pathways onto engineered minichromosomes could facilitate the study of many aspects of metabolism. Adding regulatory factors could be used to study gene expression. In a broader sense, the development of new genetic engineering techniques will facilitate genomics studies in general.
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The next step in the development of effective synthetic chromosomes would be to have means to grow the chromosome to one’s own specifications. Techniques have been developed for the addition of new DNA into sites already present in the genome so it should be straightforward to adapt those methods to minichromosome vectors. With the ability to amend minichromosomes, a path could be envisioned by which there could be continuous additions to a chromosome. If it becomes possible to produce minichromosomes with large cargos or to recombine large cargos onto them [37], synthetic chromosomes could be grown in large increments. If gametophytic selection for enhanced transmission from one generation to the next could be incorporated into the minichromosome, a method to place the minichromosome into virtually all progeny would be established. Finally, the transfer of minichromosomes from a haploid inducer into numerous target lines will eliminate linkage drag and facilitate the placement of minichromosomes into many different varieties. The consolidation of these features into the same vectors should produce a minichromosome with high potential for routine and effective use. www.sciencedirect.com
Acknowledgements Research on this topic supported by NSF Plant Genome grant DBI 0701297 and IOS 1339198. The author thanks Nathan Swyers for producing Figure 1.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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