Qualitative and quantitative genetic studies of Arabidopsis thaliana.

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Copyright 0 1991 by the Genetics Society of America

Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics

Edited

by

James

F.

Crow

and William

F. Dove

Qualitative and Quantitative Genetic Studies

of Arabidopsis thaliana

Bruce Griffing and Randall L.

Scholl

The Arabidopsis Biological Resource Center at O.S.U., College of Biological Sciences, Ohio State University, Columbus, Ohio 43210

G

ENETIC research involving Arabidopsis thaliana

dates back to the 1940s. A renewed interest has developed recently, however, due to recognition of the unique genomic properties of Arabidopsis, which make it an ideal system for molecular studies of flowering plants. This interest has increased to such a point that the international community of Arabidopsis geneticists organized a formal Arabidopsis thaliana

Genome Research Project (MEYEROWITZ et al. 1990). Their overall goal is “to understand the physiology, biochemistry, and growth and developmental proc- esses of a flowering plant at the molecular level, using Arabidopsis as an experimental model system.”

The plan for this project predicts, with some assur- ance, that “recent progress in the area of genome mapping and of sequencing of specific genes amply demonstrate that Arabidopsis will be as useful to plant biology, and possibly to higher eukaryotes, as E. coli

has been to microbiology in particular and to biology in general.” This is heady stuff to be focused on a small, useless (i.e., noncommercial) plant, but it indi- cates that many plant biologists worldwide have con- fidence that Arabidopsis is becoming the system of choice for flowering plants.

There have been many reviews of Arabidopsis genetic research over the years, including eight since

1985. Hence a similar review will not be presented but rather a different perspective will be given, with a different orientation of past work and future prog- noses. Here, Arabidopsis research is partitioned into a dichotomy based on the complexity of the genetic systems. The two categories are qualitative and guan- titatzue systems. The qualitative studies deal with genes of sufficiently large effect to be manipulated individ- ually. The quantitative genetic studies deal with variables controlled by genes at many loci which are, characteristically, strongly influenced by environmental fluctuations. Such a classification is somewhat ar- bitrary, and under certain conditions one system may

Genetics 129: 605-609 (November, 1991)

be converted into the other. Nevertheless, it is a useful classification for what follows.

At present, qualitative genes are the targets of molecular geneticists and quantitative genes have been largely ignored. Recently, however, interest in molecular analyses of quantitative systems and molecular approaches to plant improvement has increased. This is because essentially all important agronomic traits are controlled by quantitative systems, and the quantitative genes must be located in order to clone and manipulate them. How Arabidopsis might be used to help solve this complex problem will be discussed in the last section.

We shall present a brief account of qualitative studies and then a somewhat more detailed account of those quantitative studies that relate to arguments to be made regarding strategies for the molecular analysis of quantitative genes. The objectives of this perspective are to point out characteristics of Arabidopsis that make it ideally suited for qualitative and quantitative genetic analyses, and to present progress in these areas; to outline procedures for cloning qualitative genes and progress to date; and to suggest a procedure for cloning quantitative genes.

We also wish to recognize the contributions of one of the pioneers of Arabidopsis research, JOHN LAN- GRIDGE, whose initiatives provide the basis for the

overall theme of this perspective.

Qualitative genetics: Qualitative genetic studies are concerned with the induction, isolation, location and developmental study of individual mutations of large effect. Characteristics of Arabidopsis that facilitate qualitative studies are a rapid generation time (about 5 weeks), small plant size, many seeds per plant, many self-fertilized but cross-compatible ecotypes collected worldwide, a low chromosome number ( n = 5), and ease of isolating mutants.

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came Professor of Botany at the University of Frank- furt am Main, Germany. He was an enthusiastic pro- moter of Arabidopsis for the study of plant biology (LAIBACH 1943). According t o R ~ D E I (1974), however, Arabidopsis genetics was formally initiated by two graduate students, ERNA REINHOLZ (a student of LAIBACH in Germany) and LANGRIDGE (a student of DAVID CATCHESIDE in Australia). T h e following discussion centers on developments from these two initiatives.

REINHOLZ (1 947) worked out details for producing mutants by seed irradiation. This and other mutagenic procedures generated hundreds of mutants and led to traditional qualitative studies. T h e kinds of mutants isolated and studied included morphological mutants affecting all plant parts, developmental (including homeotic) mutants, physiological (including phytohor- monal) mutants, and biochemical (including condi- tional lethal, herbicide-resistant, and photorespira- tory) mutants. Mutant descriptions are found in all Arabidopsis reviews, but those especially devoted to

mutant descriptions are MCKELVIE ( 1 962); KOORN-

NEEF et al. (1983), MEYEROWITZ and PRUITT (1984),

ESTELLE and SOMERVILLE (1986) and KOORNNEEF (1987). Tremendous effort has been expended to assign about 200 qualitative mutations to the five chromosomes. This mapping is proceeding rapidly, the latest report being KOORNNEEF (1 987).

As MEYEROWITZ (1987) pointed out, this approach to qualitative genetics is not unique to Arabidopsis but has been used with considerable success in other experimentally useful plants, such as maize and tomato. T h e properties of Arabidopsis permit these studies to be accomplished more quickly and effi- ciently, but no new techniques are introduced which are applicable only to Arabidopsis.

We turn now to the LANCRIDGE initiative, which

did introduce a technique unique to Arabidopsis. In his graduate studies, LANCRIDGE wanted to extend the BEADLE-TATUM analytical procedure from Neu- rospora to flowering plants. This required growing plants aseptically on well defined media through their entire life cycle. He searched the literature and dis- covered A. thaliana as described by LAIBACH in 1943. This plant proved ideal. Although LAIBACH had indi- cated that aseptic culture of Arabidopsis was possible, he gave no procedural details. Thus, it was up to

LANCRIDCE to develop procedures using standard test tubes and other containers suitable for large numbers of plants. These procedures were set out in his Ph.D. dissertation (1955a) and published in 1957, opening a new dimension for Arabidopsis research which was not available with any other flowering plant.

LANGRIDGE’S first paper (1955b) reported the dis-

covery of the first auxotrophic mutant in flowering plants. LANGRIDGE’S dream of extending the BEADLE-

TATUM technique to flowering plants had come true. This mutant involved thiamine synthesis, and it is interesting to note that the second mutant reported

by BEADLE-TATUM was also a thiamine auxotroph. Although an auxotrophic mutation in a flowering plant was of considerable interest, probably of more general interest were the pictures of a plant flowering in a test tube and of a Petri dish containing 45 plants! During the late 1950s, LANGRIDGE used the aseptic culture method to study mutants and a variety of other phenomena in Arabidopsis. Thus, the begin- nings of aseptic culture techniques were established, and this novel approach has been utilized in much Arabidopsis research involving the isolation of mutants by special screening methods and in studies of their nutritional requirements. As discussed in the next section, it has also been used in a novel way in quantitative genetic research.

Quantitative genetics: Quantitative genetic analyses deal with traits that are jointly determined by genes at many loci and are usually strongly influenced by environmental fluctuations. Only those quantitative studies combining the LANGRIDCE culture procedures with growth of plants in controlled environments will be considered here. These studies are unique to Arabidopsis and demonstrate its remarkable utility for quantitative studies in a flowering plant. This class of experimental procedures will be inte- grated with the molecular approach in the last section. In the late 1950s and early 1960s the excellent Canberra phytotron facilities (developed by the Divi- sion of Plant Industry, CSIRO) became operational. It soon became apparent that Arabidopsis culture is ideal for such a facility for several reasons. First, test- tube culture permits many plants (at least 1000) to be grown in a single growth cabinet. Second, rapid plant growth results in rapid experiments (15-20 days), which ensures economy of cabinet use and minimizes the probability of cabinet breakdown during an experiment.

Furthermore, test-tube culture ensures that each plant receives the same quantity of a defined nutrient medium. Growing a single plant in each tube elimi- nates competition between plants for limited nutrients. These two properties of test-tube culture min- imize environmental variation and thereby reduce the number of plants required for measuring a quantita-

tive variable. Growth cabinets also provide controlled and reproducible climates, producing exact and re-

producible plant responses for many environmental variables.

T h e remainder of this section briefly reviews Ara- bidopsis studies conducted in the Canberra phytotron. They are directly attributable to LANGRIDGE or his colleagues.

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Perspectives 607

possibility of a “chemical cure of climatic lesions” for sensitive crop plants. LANGRIDGE and GRIFFING (1 959)

put this hypothesis to test by growing 43 ecotypes of Arabidopsis at different temperature regimens. Eight ecotypes showed high temperature sensitivity and three of these were cured by appropriate supplements. Hence, the hypothesis was corroborated.

A quantitative design was added to the procedures for most of the remaining phytotron studies. This not only permitted estimation of genetic parameters associated with different environmental regimes, but also provided opportunities to explore genotype-environmental interactions.

The first such study (GRIFFING and LANGRIDGE

1963) compared phenotypic stability of an inbreeding species, Arabidopsis, with that of an outbreeding species, Drosophila. The study involved two parts. First, growth response was characterized at six different temperature regimens for each of 38 Arabidopsis ecotypes. Second, a quantitative genetic analysis was conducted with a diallel of five ecotypes and all possible hybrids grown at the six temperature regimens. T h e heterotic responses of Arabidopsis closely paral- leled those of Drosophila reared in similar environmental conditions, the differences being only in de- gree. About 5000 plants were included. In practical terms, Arabidopsis is the only plant that could be used for such a comprehensive study. Therefore, it is not surprising that SEWAU WRIGHT (1 977) described this work as “probably the most thoroughly controlled study of heterosis that has been made with a self- fertilizing plant.”

An even more massive study was conducted by PEDERSON (1968), who grew 10 ecotypes, 15 F2s and

15 double crosses at three different levels of four environmental variables (temperature, light intensity, moisture stress and nutrient concentration). This involved over 8,600 plants !

Another novel use of Arabidopsis culture was made

by BROCK (1970). He was interested in detecting variation in quantitatively inherited traits induced by thermal neutrons and gamma irradiation. This required comparing genotypic variation in irradiated and unirradiated populations. He pointed out that culturing Arabidopsis in growth cabinets controlled the environmental component of variation so well that small differences in genotypic variation were detecta- ble.

In 197 1, GRIFFING and ZSIROS examined the role of heterosis in the total stimulus pattern. Two ecotypes and their hybrid progeny were grown in different but quantitatively related environmental regimens. These included three temperatures, two nutritional levels and two planting densities (one and two plants per tube), and plants were harvested at four times. Different forms of heterosis were identified as

dependent on temperature regimes, nutrient levels, biotic environments, and developmental times. This type of experiment illustrates how the complexities of heterosis can be dissected into more meaningful parts. More recently, GRIFFING (1 989) conducted a simi- larly designed experiment involving competition induced by including more than one plant per tube, in order to test a genetic theory specifically designed to accommodate competition between plants. The Ara- bidopsis model system provided a successful test.

It is clear from the above that Arabidopsis culture in controlled-environment facilities provides a procedure that remains unique to this flowering plant. The system allows almost complete control over all major facets of plant growth: through appropriate genetic design, the genetic facet of plant growth is controlled; with test-tube culture, the composition and quantity of nutrients available to each plant is controlled; and using growth cabinets, the physical environment in which the plant grows is controlled. Thus, Arabidopsis culture in the phytotron facilitates the dissection of a

quantitative variable into qualitative subunits that may be detected by linkage studies, leading to the cloning and characterization of quantitative genes.

Molecular genetics: According to the Arabidopsis thaliana Genome Research Project, the immediate goal of molecular geneticists working with Arabidop- sis is “identification and characterization of the structure, function and regulation of Arabidopsis genes.” The first step is to clone genes, notably the develop- mentally important ones. However, this represents a substantial challenge and requires unique approaches. Arabidopsis, and the genetic techniques developed for it, lend themselves well.

For molecular genetic studies, the primary advan- tage of Arabidopsis over other flowering plants is the physical properties of its genome. That it has the smallest known genome among flowering plants was suggested as early as 1961 by SPARROW and EVANS and confirmed in MEYEROWITZ’S laboratory (LEU- TWILER, HOUGH-EVANS and MEYEROWITZ 1984;

PRUITT and MEYEROWITZ 1986). The reasons for a small genome include little repetitive DNA and, in some cases, simpler gene families.

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and

Although chromosome walking can be applied to other plant species, its most efficient use would be with Arabidopsis because of the small genome. The procedure requires dense restriction fragment length polymorphism (RFLP) maps which are now under construction, the marker total for these maps being about 300.

The development of yeast artificial chromosome (YAC) libraries, with very large insert sizes, should greatly enhance the efficiency of cloning Arabidopsis genes (GRILL and SOMERVILLE 1991). Once a gene has been associated with a RFLP marker, it should be found within one or a few YAC-sized steps. It is also feasible to develop a complete physical map of over- lapping YAC clones within a few years. Then mapping a mutation should place it within a small set of YAC clones.

Another approach to gene cloning in Arabidopsis is random tagging of genes. Several methods are possible, the one already developed being insertion of T-

DNA by Agrobacterium transformation (FELDMANN

et al. 1989). Thousands of tagged lines have been identified and several genes have been cloned and characterized. Yet another cloning method being developed uses subtractive hybridization procedures with deletion mutants (STRAUS and AUSUBEL 1990).

These approaches have moved Arabidopsis to the forefront of plant molecular biology and biotechnol- ogy. Any or all of them may be possible with other plant species. However, the special properties of Ar- abidopsis make it the clear choice for such research. Aseptic growth now represents a major tool for effi- ciently isolating interesting mutants in Arabidopsis. Hence, the contributions of pioneers in this field, especially LANCRIDGE’S development of the genetic system, should be remembered as Arabidopsis emerges as a model for genetical research with flowering plants.

Synthesis: Consider now the actual and potential results from applying molecular analyses to the qualitative and quantitative genetics of Arabidopsis.

Qualitative genetics: Several cloning methods have been applied. The Arabidopsis thaliana Genome Re- search Project Report reveals that about 60 qualitative genes have been cloned. Thus, the Project is well underway with this class of genes. While problems remain in isolating and characterizing qualitative genes, including the development of gene replace- ment techniques and more efficient expression systems, progress toward the major goal is rapid.

Quantitative genetics: No quantitative genes have been cloned, so that the following discussion is purely speculative. Let us assume that chromosome walking is the most plausible method. In this case, the first step is to map quantitative trait loci (QTLs) to their approximate chromosomal positions.

Attempts to map QTLs go back to the early 1920s,

with marginal success. However, a recent resurgence

of interest has developed among molecular geneticists interested in plant improvement. For example, PAT-

TERSON et al. (1988) reported on the use of a complete

RFLP tomato linkage map to locate 15 QTLs to their approximate chromosomal positions. However, with present techniques available for tomato, the final step, exactly pinpointing the location of these genes so that they can be cloned, remains elusive. This problem holds for all agronomic crops.

Because of its unique genomic properties, a way may exist to clone quantitative genes in Arabidopsis. The procedure with the greatest chance of success would be as follows. First, grow all plants in a regime using test-tube culture in growth cabinets, thus mini- mizing environmental variation and enhancing dissection of the quantitative system into individual genes. Next, roughly locate the quantitative genes of larger effect using a dense RFLP map. Then chromosome- walk with YAC clones to pinpoint their locations. Applying techniques such as complementation testing, identify and clone the quantitative gene. Lastly, use molecular techniques to characterize the genes and their main functions and interactions.

Finally, the practical aspect of Arabidopsis would be realized by making DNA probes of quantitative genes and using them to locate homologous genes in any agronomic crop. For example, heterosis could be examined. In the section on quantitative genetic systems, we suggested that heterosis in Arabidopsis was similar to that in a crossbreeding species. Therefore, assuming that Arabidopsis heterosis is of a general type, it could be subjected to molecular analysis in Arabidopsis and then, through appropriate DNA probing, the analyses could be transferred to any agronomic crop. We also suggested that heterosis could be dissected into more meaningful parts. In this case, its molecular analyses in Arabidopsis could provide the means for similar analyses in crop species. In this way, the molecular basis of heterosis, which has been so elusive in the past, may finally come to be understood.

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