Following a cross between two parents carrying contrasting alleles at a range of gene loci, the inheritance of the alleles for each locus can be followed in subsequent generations. If two characteristics conditioned by two different genetic loci are inherited independently they are said to be ‘unlinked’, and the gene loci can be assumed to be located on different chromosomes. If, in contrast, the inheritance of the two characteristics shows some correlation and not just random association, their gene loci are said to be ‘linked’ and they can normally be assumed to be on the
same chromosome. The degree to which the two characteristics are inherited together, in other words the strength of the linkage, can be quantified.
Characteristics that rarely recombine can be assumed to be controlled by genes that are very close to each other along the same chromosome, so that their alleles are rarely resorted by crossing over during meiosis.
By quantifying the frequencies of recombination between linked gene loci for a number of characteristics it is possible to order the strength of the linkages between them and construct a ‘linkage map’ showing their relative positions along the chromosome (see Fig. 3.4). The chromosomes are the physical reality
Fig. 3.4. Genetic maps of alliums. (a) A linkage map of A. cepa (shallot) chromosome 4. The long codenames apply to DNA markers of the Amplified Fragment Length Polymorphism (AFLP) type that was used for map construction.
The locations of the genes coding for the enzymes alliinase, malate dehydrogenase (MDH) and phosphoglucomutase (PGM) are shown on the map (part of Fig. 2 of
van Heusden et al., 2000b. Courtesy of Theoretical and Applied Genetics). (b) The integrated physical and recombination map of A. fistulosum chromosome 8. The AFLP marker linkage map is on the right and the physical locations of the markers along the chromosome are shown on the left in terms of percentages of the total chromosome length (part of Fig. 3 of Khrustaleva et al., 2005. Courtesy of Genetics). (c) The physical distribution of recombination frequency along chromosome 8 of a recombinant hybrid between A. fistulosum and A. roylei. The black arrow indicates the position of the centromere (Fig. 5 of Khrustaleva et al., 2005. Courtesy of Genetics). (d) The density of occurrence of AFLP markers along a recombinant A. fistulosum A. roylei chromosome 8. Two sorts of markers are shown corresponding to two types of restriction enzyme combinations used to derive markers. The PstI/MseI markers probably correspond with areas of actively expressed genes (Fig. 7 of Khrustaleva et al., 2005. Courtesy of Genetics).
underlying a linkage map derived from inheritance statistics. Separate ‘linkage groups’ of characteristics that are inherited together, each corresponding to a different chromosome, should result from a linkage map based on many characteristics. The independent inheritance of characteristics determined by genes in different linkage groups reflects the random assortment of the two segregants from each chromosome going to each daughter cell during meiosis.
Considerable research effort is now being devoted to the development of genetic linkage maps for the edible alliums, particularly onion, as reviewed in McCallum (2007) and Havey et al. (In press).
The first genetic maps were based on the inheritance of easily observable characteristics but, as we have seen, there are relatively few of these known for alliums. Moreover, some of the observable characteristics, like disease resistance, need considerable effort to assess. Nowadays, genetic maps are based primarily on molecular ‘markers’, which can be found in large numbers and which can be rapidly detected using techniques of molecular biology. One of the useful outcomes of such work is that the inheritance in plant breeding lines of important properties that need much effort to assess – for example, disease resistance or the Ms genes for fertility restoration (see below; Gokce et al., 2002) – can be followed easily by using molecular markers closely linked to a desired trait to rapidly and cheaply detect its presence or absence in individual plants in a breeding programme. Often, such tests can be done at the seedling stage, so that only individuals retaining alleles for a desired trait need be grown on. This is termed
‘marker-assisted breeding’. Molecular markers used in the genetic mapping of alliums were described by Klaas and Friesen (2002). Briefly, molecular markers group into isoenzymes and DNA-based techniques. Isozymes are allelic variants of particular enzymes that can be distinguished and followed in inheritance studies (Shigyo et al., 1996).
DNA-based techniques break down into a number of subcategories. First there are techniques based on differences in the sizes of DNA fragments. These are generated by applying a succession of cleaving enzymes to the DNA.
Genotypes may differ in the resulting size of DNA fragments from a particular point along the chromosomes, and the inheritance of these differences in size fragments can be followed. Secondly there are techniques based on DNA sequence differences that vary between genotypes. For example, there may be sequence repeats, nucleotide omissions or nucleotide substitutions. These molecular variants that show up as detectable DNA differences at the same location along the homologous chromosomes are called polymorphisms. The inheritance of the different variants within a polymorphism can be followed, just as can the inheritance of different alleles for contrasting forms of a visible attribute like long or short flower stalks.
The first linkage map of onion was based on DNA polymorphisms and has 114 loci located over 12 linkage groups (King et al., 1998). It was derived by following the inheritance of the DNA markers in 58 third-generation families of controlled crosses and selfs derived from an original cross between an inbred
line from the low-pungency, low-soluble solid cv. ‘Ailsa Craig’ and an inbred from the higher-pungency and soluble solid cv. ‘Brigham Yellow Globe’. Alleles for male fertility restoration, a complementary gene for red bulb colour (see previous section) and the enzyme coding for alliinase (see Fig. 8.2) were located on the linkage map and associated with neighbouring DNA markers. A sub-sequent analysis of this cross followed the inheritance of DNA markers in parallel with differences in sugar and fructan (see Chapter 8) content deriving from the parent lines. Results showed chromosome regions on linkage groups A, D and E to be associated with the control of fructan content (Havey et al., 2004).
Another genetic map based on a cross between onion and the wild species A. roylei discriminated 262 DNA markers on the onion genome into eight linkage groups (van Heusden et al., 2000b). A gene for alliinase and the location of a gene for resistance to downy mildew disease derived from A. roylei were located by this map. Linkage groups in this map were later assigned to the individual physical chromosomes of A. cepa (see Fig. 3.4a; van Heusden et al., 2000a). This was done using molecular markers for A. cepa that could be located in the A. cepa A. roylei linkage map and also in chromosomes derived from A.
cepa in a set of eight lines of bunching onion, A. fistulosum, each of which contained an extra different chromosome from shallot (A. cepa) (Shigyo et al., 1996). These lines, termed ‘monosomic addition lines’, are the key to translating the linkage map into a ‘physical map’ located on the eight chromosomes of A. cepa (see Fig. 3.1). Analysis of the biochemical effects of different monosomic additions has shown that a group for flavonoid and anthocyanin biosynthesis is on chromosome 5A (Shigyo et al., 1997), and genes for the production of sugars in leaves on chromosomes 2A and 8A (Tran Thi Minh Hang et al., 2004).
A further refinement has been to compare ‘genetic distances’ between DNA markers – as shown by linkage maps – with the physical distance between markers along the chromosomes by direct visualization and position measure-ment of meiotic crossing over, using a refined staining technique, in parallel with the recombination mapping of markers (see Fig. 3.4; Khrustaleva et al., 2005).
Recombination frequency varied in different regions along the chromosome, indicating that the ‘genetic distances’ between markers correspond to variable physical distances along the chromosome (see Fig. 3.4c). The pattern of variation of recombination frequency was different to that found for cereal crops.
DNA sequence repeat markers that can be amplified by the polymerase chain reaction (PCR) have been developed for onion (McCallum et al., 2005b).
Polymorphisms for these markers have been detected both between and within onion breeding lines. Onion linkage maps derived from different original parent crosses have been aligned using markers of this type to locate common points on both maps (McCallum et al., 2005b). New families of inbred lines from crosses between parents with contrasting properties have been developed, so that these markers can be exploited to map the genes underlying these contrasts. Families
with more lines than the 60 or so produced so far are needed for finer-scale mapping to detect more closely linked genetic effects (McCallum et al., 2005b).
The development of male-fertile, seed-reproduced garlic has enabled the first genetic linkage map for garlic to be derived (Zewdie et al., 2005). The genetic markers used were developed from expressed DNA sequences from onion (Kuhl et al., 2004). A gene locus for fertile seed production was located on the linkage map.
Molecular markers are useful for surveys of genetic diversity both within and between allium species (Klaas and Friesen, 2002; McCallum et al., 2005b).
Markers are already finding practical applications within the onion industry;
for example, markers from chloroplast DNA characteristic of male-sterile cytoplasm are widely used to quickly classify cytoplasm in the development of maintainer and male-sterile lines during the breeding of onion hybrids (see below) and for quality control (i.e. confirming genetic identities) in breeding and hybrid-onion seed production (Havey, 2002; Jakse et al., 2005).
GENOMICS
The collection of DNA sequences and, for some organisms, the sequencing of all the genetic material, has made possible the comparison of DNA sequences within and between taxonomic groups (Havey et al., in press). The function of a DNA sequence can be tentatively inferred from knowledge on what enzyme or other protein it, or similar sequences, code for in other species. Taxonomic groups showing high similarity in their arrangement of DNA subsequences along the chromosomes are said to show high synteny or colinearity.
The study of genomes investigating how they function as a whole system in controlling growth, development and adaptation – and also to clarify the phylogenetic relationships between species – is a central concern of current biological research. Such genomic studies in the alliums are in their infancy, but a library has been created of 11,008 DNA sequences derived by sequencing DNA complementary to the RNA isolated from bulb, root, leaf and callus tissue of onion (Kuhl et al., 2004). The sequence information is freely available online at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=onion.
Since they are derived from RNA found in the tissues, these are the DNA sequences that are actively transcribing for RNA and hence they are termed
‘expressed sequence tags’ (EST). They should encompass the active genes in their respective tissues. Sixty per cent of these sequences matched proteins from other organisms, of which nearly 24% could be tentatively assigned to some gene functionality, mostly involved with metabolism. EST markers show-ing similarities to EST markers from rice were selected and linkage mapped using the lines derived from the ‘Ailsa Craig’ ‘Brigham Yellow Globe’ cross mentioned above under linkage maps. Physical locations for these markers on the chromosome were determined using the monosomic addition lines also
mentioned above. A comparison of the ordering of these ESTs in onion with that known for rice showed little similarity (i.e. scant colinearity). These results suggest that genomic information from the grass family crops (Order Poales), of which there is a vast amount, may not provide appropriate genomic models for crops in the Asparagales Order – e.g. alliums (Martin et al., 2005).
This makes it necessary to develop more genomic information for these plants. In view of the large genome and long generation time for onions and other vegetable alliums, a species in the Asparagales with a smaller genome and short life cycle is likely to be chosen as a ‘model’ for developing a detailed genetic map applicable to the Order. The expected large measure of colinearity of genes between the genomes of different species within an order would facilitate the application of genomic information from such a ‘model’ species to the crop species. As information is published on allium genetic sequences by research groups worldwide, it is being collated at the above web site so that genomic information is accumulating in a coordinated and collaborative way (Havey et al., in press).