As mentioned briefly above, several of the edible alliums are reproduced by vegetative means. Rakkyo, great-headed garlic and, in most instances, garlic are propagated from bulbs (cloves). All three species can produce inflorescences, but seed is not set and the inflorescences revert to bulbil or bulb production at various stages of development, except in the case of a few, recently discovered fertile garlic clones. Chinese chives flower and produce viable seed but normally, during egg cell formation, the chromosomes double and a complete set of 32 mother plant chromosomes enters the egg cell. There is, therefore, no fertilization and recombination, and the outcome is genetically equivalent to vegetative reproduction – this is termed apomixis. Hybridization occurs in about 10% of the offspring of crosses between cultivars of Chinese chives (Kojima et al., 1991), so a low percentage of normal fertilization and gene exchange can occur.
Pollination, although not usually resulting in fertilization, is necessary for seed production, since the nutritive seed endosperm tissue fails to form without pollination (Kojima and Kawaguchi, 1989).
Clones of garlic that can produce viable seed following normal meiosis, pollen formation and fertilization were discovered in the centre of origin of the crop in central Asia. Selection from these clones has resulted in improvements in fertility, seed yield and germination rate, and a reduction in seedling defects (Etoh and Simon, 2002). Using these clones, genetic studies on garlic are now possible (Zewdie et al., 2005). Many strains of shallot are also maintained vegetatively, but in recent years seed-propagated cultivars developed in Israel and The Netherlands have been of growing importance (Rabinowitch and Kamenetsky, 2002).
Despite the lack of genetic recombination in the vegetatively propagated crops, they still show great diversity between clones. For example, clones of garlic
exist which are adapted to bulb in virtually every climatic zone from Norway to the Equator. Selections also vary in clove size and colour according to local preference.
The scope for crop improvement has been restricted to the maintenance and multiplication of superior clones by vegetative means (Messiaen et al., 1993).
Random genetic changes can be obtained by inducing mutations in tissue culture and raising the resulting plantlets (Novak, 1990). Although not a genetic improvement, considerable increases in vigour can be achieved by eliminating viruses from these crops (see Chapter 5).
Onion, Japanese bunching onion, leeks and chives all produce fertile flowers and are normally predominantly cross-pollinated. These plants are, however, perfectly capable of self-pollination. In all these species the anthers of individual flowers ripen and shed their pollen before the stigmas are fully receptive (see Fig. 2.17b and c) – this is termed protandry (Currah, 1990). In wild alliums, which have only a few flowers per flower head, this is probably an effective barrier to self-pollination. However, the vegetable species can have up to 1000 individual flowers per umbel and, because the opening of different flowers may spread over 2 to 4 weeks, it is easy for pollen to fertilize the receptive stigma of a more advanced flower on the same flower head. Therefore, protandry offers only a partial barrier to self-pollination. Typically 75–90% of seeds result from cross-pollination in onion seed fields, and leeks are normally more than 80% cross-pollinated. In certain conditions, for example in the mesh cages used by plant breeders to isolate plants from stray insects carrying unwanted pollen, the degree of cross-pollination may decrease to only 23–56%
because bees and flies are often less active in these conditions.
In all these species the vigour and survival rate of seedlings derived from self-pollination (termed ‘selfs’) are much lower than from cross-pollination. In one study, 75% of crossed onion seed survived to produce bulbs after field sowing, whereas less than 50% of selfed seed did so (Currah and Ockenden, 1983). This is a manifestation of the ‘inbreeding depression’ typical of cross-pollinated or ‘outbreeding’ plant species. In a comparison of onions inbred by one generation of selfing with ordinary open-pollinated populations, the average bulb yield of several inbreds was only 64% of that of the open-pollinated populations, the mean maturity date was delayed by 12 days and thick-necked bulbs increased from 2 to 12% (Dowker and Fennell, 1981). After three generations of onion selfing, plant survival rates may be down to 50%, and low vigour results in only 70% of the survivors being capable of producing seeds (Jones and Mann, 1963). In a trial with leeks, mean plant weights were decreased by 35% by one generation of inbreeding and by 60% by two generations of selfing (Pink, 1992). Because of this severe inbreeding de-pression, seed derived wholly from cross-pollination has a higher survival and yield potential than seed from an open-pollinated seed crop, which always contains a proportion of selfs. One of the benefits of using hybrid cultivars (see below) is simply that they guarantee that all the seed sown derives from cross-pollination.
GENETICS
Compared with many crop species only a small number of ‘qualitative’ genes with easily visible effects have been described in onion. One example is a recessive gene which, when homozygous, results in dwarf seedstalks. In the heterozygous condition the seedstalks are of normal length because the allele for normal length is dominant over that for dwarf. Genetic analyses of onions are time-consuming because of the biennial generation time and the severe inbreeding depression, which means that it is difficult to produce and maintain a large number of near-homozygous inbred lines ideal for genetic linkage analysis. King et al. (1998) stated that just 17 morphological or disease-resistance gene loci had been described for onion, including those for: colours of bulbs, foliage, anthers and seedcoats; male fertility restoration in cytoplasmic male-sterility; pink-root resis-tance; ozone damage resisresis-tance; dwarf seedstalk; and four loci deleterious for chlorophyll.
The colour of onion skins is determined by the combined effect of a number of major genes, each of which has different alleles causing well-defined qualitative effects (El-Shafie and Davis, 1967). This is a good example of
‘epistasis’, an important genetic phenomenon where the interaction of several different genes determines the outcome, in this case of whether the onion bulb has a white, yellow or red skin. Many of these epistatic effects have now been explained in molecular terms. They provide elegant examples of how changes in chromosomal DNA sequences result in modifications of gene control, enzyme synthesis or enzyme function to modify a biosynthetic pathway and thereby the resulting plant (the ‘phenotype’). Five major genes affecting bulb colour were discovered by classic genetic inheritance studies. The interactions between these genes imply that they act sequentially along the biosynthetic pathway for anthocyanin pigments (see Fig 3.3; Kim et al., 2004b, 2005a;
Chapter 8, this volume).
First, this pathway depends on the basic colour factor or C gene, which has both dominant and recessive alleles. White-skinned onions lack the enzyme chalcone synthase (CHS), so that they do not produce any pigments of the anthocyanin pathway (see Fig 3.3). The C gene appears to be a regulatory gene that controls whether or not the genes coding for two CHS enzymes actually transcribe and initiate the process to synthesize the enzyme proteins (Kim et al., 2005b). Yellow onions from the USA have a deletion in the DNA of the gene that that transcribes for the enzyme dihydroflavonol 4-reductase (DFR) (see Fig 3.3;
Kim et al., 2004a). This mutation prevents the production of DFR and therefore blocks the pathway after dihydroquercetin, resulting in the accumulation of the yellow quercetin pigment without any red cyanidin.
Brazilian yellow onions do produce DFR and also the anthocyanidin synthase (ANS) protein (see Fig 3.3), but a point nucleotide mutation in the DNA sequence coding for ANS results in the substitution of the amino acid glycine by arginine at the corresponding point in the amino acid sequence of the enzyme.
Fig. 3.3. The anthocyanin pigments biosynthesis pathway in onions showing how the gene alleles that determine skin colour act at the enzyme level to determine which pigments are produced. The enzymes are abbreviated as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FLS, flavonol synthase; UFGT, UDP glucose-flavonoid 3-o-glucosyl transferase (annotated from Kim et al., 2004b. Courtesy of Molecular Genetics and Genomics).
The arginine substitution is close to an active binding site in the enzyme and this probably causes its inactivation, hence blocking anthocyanidin synthesis (Kim et al., 2005a). Crosses between US and Brazilian yellow onions result in some red onions, since some of the offspring inherit the alleles producing normal DFR from the Brazilian parent along with alleles for functioning ANS from the US parent, thereby allowing the biosynthetic pathway to cyanidin to function (see Fig 3.3).
This explains the long-recognized ‘complementary gene effect’, where the interaction of two gene loci could result in red-skinned offspring from crosses between two yellow-skinned parents (El Shafie and Davis, 1967).
Another gene variant found in some unusual gold-coloured onions involved a small DNA change that resulted in the production of inactive chalcone isomerase (CHI). The consequent block in flavonoid biosynthesis resulted in the accumulation of the bright yellow pigment chalcone (see Fig 3.3; Kim et al., 2004b). Another allele produces low levels of ANS (see Fig. 3.3), resulting in less cyanidin production and leading to pink rather than red bulbs. This is thought to be a result of changes in a regulatory sequence of DNA adjacent to the sequence actually coding for the ANS protein (Kim et al., 2005a).
The influence of qualitative genes with discrete major effects is often enhanced or reduced by a number of minor modifier genes with a quantitative influence. This is so for the major gene for seed stalk length described above, and also for the gene that confers resistance to pink root disease in onion. Twenty qualitative genes identified in edible alliums were listed by Rabinowitch (1988).
In more recent years, with application of molecular genetics to the alliums, gene loci are being identified more rapidly. These include genetic loci for the enzymes affecting bulb colour discussed above, for several sulfur-metabolism enzymes involved in the synthesis of flavour compounds and for a key enzyme in fructan biosynthesis (McCallum et al., 2005a, b, 2006; Chapter 8, this volume). The DNA sequences of the genes that code several of these enzymes have been determined, and this information is helping to clarify the structure and function of the enzymes (Shaw et al., 2005). A genetic locus for mildew resistance has been identified from wild alliums and is being introduced into onion (see Breeding for Disease and Pest Resistance, below). Shigyo and his colleagues have reported genetic loci for ten enzymes of carbohydrate and amino acid metabolism, using different variants of the enzymes (isozymes) for the genetic analyses and to locate on which chromosome the loci occur (Shigyo et al., 1996;
van Heusden et al., 2000a).
In addition to the identification of genes for individual discrete traits or enzymes, groupings of genes or polygenic regions controlling various aspects of biochemistry have been located to particular chromosomes or genetic linkage regions. These include the regions coding for fructans (Havey et al., 2004), flavour compounds (Galmarini et al., 2001), sugar content in leaves (Tran Thi Minh Hang et al., 2004) and flavonoids and anthocyanins in leaf sheaths and some of the enzymes involved in their synthesis (Shigyo et al., 1997; Masuzaki et al., 2006). In addition there are 2608 published sequences of DNA from onion
that express as RNA in roots, shoots or callus and which have correspondences with genes of known function in other organisms (Kuhl, et al., 2004; see Genomics, below).
The majority of traits, including most of those important for crop productivity, are controlled by the combined effects of a number of genes that influence the trait, each of which has a similar, small, ‘quantitative’ influence.
For example, yield, maturity date and ease of bolting are each conditioned by the additive effects of several genes. Thus, crosses between extreme types for maturity date give hybrids intermediate to the parents but, from the hybrids a continuous range, rather than a few distinct classes, of maturity dates are derived in later generations. The aim of breeding is to combine together in the complement of genes, or ‘genotype’ of a variety, alleles that are favourable for desired quantitative traits. For example, the genotype should contain all the favourable alleles for the genes that determine high yield either homozygously or, if these alleles are dominant over complementary alleles conditioning lower yields, the presence of the favourable dominant as a heterozygote is equally good.
A high level of heterozygosity exists in onion populations sustained by generations of random outcrossing. This allows deleterious recessive alleles to perpetuate in the genetic pool. Self-pollination results in homozygosity for a high proportion of these recessive alleles, thus manifesting their effect and causing the inbreeding depression described above. Prominent among the deleterious recessives are those causing chlorophyll deficiency. In one survey 20–30% of plants were heterozygous for a chlorophyll deficiency recessive allele and approximately 20 such gene loci were estimated to be present in onion populations (Berninger and Buret, 1967).
Because leeks are tetraploid and therefore carry four alleles for each gene, there is even more scope for deleterious recessive alleles to be carried in heterozygous genotypes than with a diploid such as onion. Between seven and 14 different chlorophyll deficiency genes have been found in leeks and, in an open-pollinated population, it was estimated that more than 60% of plants were carrying two copies of such deficiency alleles in one or more such genes (Berninger and Buret, 1967). Leeks are subject to severe inbreeding depression.