PLS221

Solanacea: TOMATO: Solanum lycopersicum syn. Lycopersicon esculentum

List of references (includes reading assignments)

Ode to the tomato by P. Neruda

Contents:

  • Origin and domestication
  • Suitability as a research organism
  • History of cytogenetics research: mutants, chromosomes, wild species and their collection.
  • Chromosome identification: pachytene analysis, variation in chromosome number: trisomics and monosomics
  • Trisomic analysis, centromere mapping, maximum equational segregation, double reduction.
  • Use of chromosomal aberrations in mapping: deficiencies and translocations
  • F2 linkage analysis: product-ratio method. Gene tagging and useful linkages for breeding
  • Genome organization, basics of QTLs
  • Wild species, wide hybridization, breeding and evolution
  • Self and unilateral incompatibility

Tomato slides

Charles M Rick, father of tomato genetics


The basic chromosome number of the genus is x=12, being all the known species diploid, 2n=2x=24.

History: The origins and early events in the domestication of this crop are obscure. We are reasonable certain of three aspects:

1) It originated in the New World, all the related wild species are native of the Andean region: Chile, Ecuador and Peru.

2) The tomato had reached a fairly advanced stage of domestication before being taken to Europe. Early types taken to Europe had large fruits (wild species have small fruits) and a good array of colors, shapes and sizes.

3) The most likely ancestor of the cultivated tomato is the wild cherry tomato technically known as L. esculentum var. cerasiforme. This is found growing in tropical and sub-tropical America.

Domestication: The weight of the data indicates that Mexico is the probable center of domestication of the tomato.

The first record of tomato in Europe is from 1554. The colonists brought the tomato back to the Americas.

The popularity of the tomato is due mostly to its appearance, taste and derivative products that can be obtained, more than for nutritional value, although it is a rich source of vitamins A and C, and antioxidants.

The main attributes of the tomato as a desirable research organism are:

1) Short life cycle (65 to 75 days (seed to seed).

2) Self pollinates but it is easy to hybridize (easy to emasculate, collect and store pollen).

3) Many seeds/pollination and per plant (50-350/ft; 10,000-20,000/ plant).

4) Large, recognizable chromosomes on the basis of heterochromatic landmarks.

5) Good array of wild relatives.

6) Regenerative plasticity by vegetative propagation and adequate tissue culture ability.

7) Transformation ability by Agrobacterium, amenable to molecular manipulation.

8) Genome replete of conventional and molecular markers and well developed linkage groups.

9) Plant structure allows detection of vast array of hereditary modification.

10) Most importantly, its basic diploid nature. Qualitative traits are monogenic. Relatively small genome, Genome size, 2.0 pg/2c, = 9.50 x 105Kb/1c(950Mbp). 2c (DNA content on unreplicated somatic cells, 1c on unreplicated gametes)

Cytogenetic Research: (Read Quiros 1991)

Cytogenetic research in the tomato is one of the most advanced. Today, more than 1200 mutants have been described in the tomato, and of these at least 400 have been located on their respective chromosomes, and their linkage relationships have been elucidated. An ultra dense map of over 1000 molecular markers, are available. (Solanacea

Genomics Network)

; International Tomato Sequencing Project; Lycopersicon Genomic Resources

:

Variation in chromosome number (List of tomato stocks, TGRC)

Ploidy: Variation resulting in the addition or subtraction of entire sets of chromosomes.

A. Euploidy. The most important deviant euploids reported in tomatoes are monoploids, triplods, and tetraploids.

B. Aneuploidy: Trisomics have been the most useful aneuploids generated in the tomato.

1. Primary trisomics: These are individuals with an additional normal chromosome. Trisomics arise at a high frequency in 3n x 2n crosses. Approximately 43% of the progeny resulting from this type of cross are primary trisomics.

2. Tertiary trisomics: Individuals with an additional, translocated chromosome consisting of two non-homologous arms.

3. Secondary trisomics: Individuals with an additional chromosome, which is an isochromosome.

4. Telotrisomics: Individuals with an extra chromosome, which consists of a single arm and the centromere.

5. Primary Monosomics: These are individuals with a missing chromosome. The general formula is 2n-1.

6. Tertiary monosomics. These are individuals where two non-homologous chromosomes are missing, but these are replaced by a tertiary (translocated) chromosome carrying two non-homologous arms of the missing chromosomes.

Cytology of the aneuploids: Each trisomic or monosomic type can be identified by diagnostic chromosomal associations during the meiotic prophase. These are described in the table below: (See tomato meiosis)
 

Primary trisomic

Pachytene and diakinesis/metaphase I: 12II+ 1I, 11II +1III

Secondary trisomic

Pachytene: Isochromosome paired with itself, triradial III 

Diakinesis/Met I: 11II +ring of III, 12II + 1I (mini ring).

Tertiary trisomic

Diakinesis/Met I: 10II + 1V (see photo)

Telo trisomic

Diakinesis/Met I: heteromorphic III, or tiny univalent

Primary Monosomic

Diakinesis/Met I: 11II + 1I

Tertiary Monosomic

Diakinesis/Met I: 10II + 1III, 10II + 3I 

Cytological identification of tomato chromosomes:

This is done in pollen mother cells (PMC) during pachytene, when chromosomes initiate their contraction. That is when the heterochromatic regions are clearly distinguished. These regions, along with centromere position and chromosome size are the main landmarks for chromosome identification. A handy key for the identification of chromosomes was created by Khush (TGC 13:12-14, 1963). The following criteria are used for pachytene identification:


Additional criteria: proportion of chromatic and achromatic regions, presence of specific knobs etc.

C. Assignment of genes and linkage groups to chromosomes:

1. Trisomic analysis: Primary trisomics are most useful for assessing linkage groups or single genes to chromosomes. The rationale behind the trisomic test rests in the presence of 3 members instead of 2 for a given chromosome, which results in a F2 segregation deviating from the 3:1 disomic ratio.

Chromosomal segregation vs, maximum equational segregation (MES) requirements for double reduction.

2. Determination of centromere position and arm locations:  After assignment of a marker on a chromosome with a primary trisomic, other trisomics may be used to provide information regarding position of centromeres, arm location and orientation of linkage maps when a linkage group has been detected. For this purpose, secondary, tertiary, telo or complex trisomics can be used.

3.Mapping by induced deficiencies: Induced deficiencies by radiation have been very useful to determine the physical location of a gene on its chromosome by pseudo-dominance (Khush and Rick 1968). By this technique they were able to reveal the loci of 35 genes on 18arms of the 24 chromosomes present in the tomato genome.

For example: Irradiation of pollen from wild type plants was used to pollinate plants homozygous for recessive mutants br and au on chromosome 1. TheF1 plants displaying the mutant phenotypes had a deletion in the terminal segment of the short arm of chromosome 1.

4. F2 mapping with markers: With the development of all these cytogenetic stocks, it is seldom necessary to perform trisomic tests for the location of new genes in tomato. A large number of morphological mutants and molecular markers such as isozymes and more recently DNA restriction fragments, have been used to develop tester stocks for linkage analysis. These cover practically all 24 arms of the tomato genome. Morphological markers determining phenotypes identifiable at seedling stage have been used extensively for linkage analysis in the tomato for many years.

For example, by crossing the six following tester stocks to an unallocated mutant, it is possible to pinpoint the chromosome arm where the mutant is located

Translocations

Reciprocal translocations: These are mutual exchange of broken chromosome fragments between non-homologous chromosomes. For these to occur, two breaks have take place, one in each chromosome (see animation). A series of translocation stocks has been developed in the tomato. They have been an important source of tertiary trisomics by non-disjunction of multivalents formed in heterozygous individuals, resulting in n+1 spores (Khush and Rick 1967).

Gill et al (1983) developed a tester set of eight translocations involving all 12 chromosomes of the tomato by treatments of seed with thermal neutrons or ethyleneoxide. They can be used to identify a series of unknown translocations. Example:
 

 

group I

group I

group II

group II

group III

group III

group IV

group IV

 

Stocks

T1-3

T3-8

T2-10

T2-11

T4-7

T5-7

T6-12

T9-12

Outcome: unknown 

must have translocated

unk 1

6

2x4

6

6

2x4

2x4

2x4

2x4

chromosomes 1 and 2

unk 2

2x4

2x4

6

6

6

6

2x4

2x4

chromosomes 2 and 7

Mapping genes to translocation breakpoints

Based on the fact that heterozygous individuals for reciprocal translocation have a phenotype. They are semi-sterile (SS). A simple pollen count is used to determine level of fertility by squashing an anther in a drop of 2% acetocarmine in a microscope slide.

The test consists in crossing a homozygous translocation stock by a homozygous recessive individual for the gene to be located on one of the chromosomes involved in the translocation . The resulting double heterozygote (for the translocation and for the gene in question) is testcrossed to the homozygous recessive parent for the mutant. The progeny is scored for parental and recombinant phenotypes to determine whether SS and the mutant gene are linked or assort independently.

Genome organization and heterochromatin in the tomato:

According to the calculations of Paterson et al. (1996), 77% of the tomato genome consists of heterochromatin, and 23% of euchromatin. DNA density in heterochromatin is almost five times higher than in euchromatin. Of the total amount of DNA in the tomato genome, which is approximately 1 pg/1C, 0.77 pg are in the heterochromatin and 0.23 pg are in the euchromatin.

The tomato genome has almost 5 times more DNA content than Arabidopsis thaliana. However, since 77% of the tomato genome is heterochormatin, which is genetically inert, its effective genome size is 0.23 pg, which is equivalent to the genome size of Arabidopsis. Heterochromatin is formed mostly by repeated DNA sequences. The organization of these sequences in the genome varies, they may consist of short repeating units tandemly arrayed and clustered in specific regions of the chromosomes (telomeres, centromeres), or they may be interspersed with unique sequences and scattered through out the genome. Genes are located in long continuous stretches of euchromatin mostly at the end of the chromosomes.

Wang et al (2006) provide insight into euchromatin and pericentric heterochrom. In euchromatin, gene density of 1 gene/6.7 kb, similar to A. thaliana and rice. In heterochromatin gene density lower 10-100X. It is heavily populated of retrotransposons mostly Jinling Ty3/gypsy like. It spread in pericentric heterochromatin of cultivated and wild tomatoes probably recently, since it is not present in potato. 90% of genes are on euchromatin, which is approximately in 25 % of the genome.

Expected number of genes in euchromatin based on sequencing (Lukas et al 2009) is 40,000. A high quality draft of euchromatin sequence is expected to be available in 2010. So far 1/3 of the genome has been sequenced, BAC by BAC, which will be combined with shotgun sequencing. Transcription factor and disease resistance genes have been mapped and characterized.

The tomato genome is typical of a diploid, with little duplication. However, comparative genomics between Arabidopsis and Lycopersicon hints ancient polyploidy events for these species (Ku et al. 2000). It is estimated that only 10 to 15% of DNA is repeated in the tomato. Four major families of repeated DNA have been identified in the tomato (Ganal et al 1988). In situ hybridization techniques have been used to map physically some of these sequences in metaphase and pachytene chromosomes, especially for rDNA and telomeric sequences (Xu and Earle 1996).


II and III form less than 1% of the genome. I and II are tomato specific, III are detected in tomato and some potato species.