INTRODUCTION TO PLANT BREEDING

AGRONOMY 815 / COURSE NOTES

P. STEPHEN BAENZIGER, 338 Keim Hall, 472-1538

DEPARTMENT OF AGRONOMY / UNIVERSITY OF NEBRASKA

CHROMOSOME REMODELING

Refs. Morris, R. 1983.
Remodeling crop chromosomes. In Crop Breeding. D.R. Wood (ed) ASA-CSSA. p109-129
Fehr, Chapt. 4 & 5
Welsh, Chapts. 5 and 18
Poehlman, Chapt. 4
Briggs and Knowles, Chapts. 21, 22 & 23.

Terminology and ploidy concepts.

Basic gametic or haploid number of chromosomes of a plant = n.

The number of chromosomes present in somatic cells, regardless of the number of genomes = n.

Basic set of different chromosomes (x) possessed by a particular species = genome.

Euploidy = variation in numbers of basic sets of chromosomes (genomes). Diploids possess 2n chromosomes and x = n.

Many species possess more than two chromosome sets . . . .

Polyploids — 2 main classes:

3 or more doses of the same genome. Autoploids.
2 or more doses of each genome but 2 or more kinds genomes.Alloploids.

Polyploidy is common in nature. e.g., Chromosome numbers for some major crops:



                    Basic genome
                    number, x      2n     ploidy


corn                  10          20       2x
                                                  Diploid
barley (cultivated)    7          14       2x


alfalfa                8          32       4x

potato                12          48       4x   Tetraploid      

cotton (upland)       13          52       4x
                                             Alloploid
wheat (bread)          7          42       6x             Hexaploid

Amphidiploids — polyploids made up of different genomes but act like diploids in their chromosome pairing and movement.

Genetic ratios and segregation patterns.

In autoploids there are several doses of the same gene. Instead of 3 possible genotypes at a locus as i

n a diploid, e.g., AA, Aa or aa, there would be 5 possibilities in an autotetraploid — AAAA, AAAa, AAaa, Aaaa, aaaa.

Inheritance studies are difficult! See Allard, Chapt 30., also Busbice et al., pp 283 — 318 in Hansen (ed.) Alfalfa Science and Technology. ASA. Comment on chromatid (genes are independent or partially independent of the centromere) vs. Chromosome (genes segregate with the centromere) inheritance.

Manipulation of plant chromosomes to create desirable genotypes may involve:

Increases/decreases in chromosome (genome) number;

Combining genes (genomes) from two different species;

Transferring one or more genes from a wild relative to a crop plant.

Requirements for chromosome remodeling — lab & greenhouse space plus expertise in chromosome techniques.

In considering applications of the various approaches, need to evaluate objective sought, method/technique used, and degree of success attained.

Increasing chromosome number — autoploid, a number of examples have evolved under natural conditions.

Possible causes:

Unreduced gametes due to some abnormal behavior during meiosis.

Normal cell division may have failed to occur at one stage during development resulting in a tetraploid cell. If normal division followed, then a sector of tetraploid cells would be formed and if these included floral parts, the tetraploidy would be passed on to the next generation.

Chromosome doubling can be induced artificially through the use of colchicine (a natural substance from the Autumn crocus, discovered in 1930's to induce chromosome doubling during mitosis).
Colchicine binds to a protein, tubulin, that is necessary for assembling functional spindle fibers. Without a spindle the cell plate fails to form so that the chromosomes and their duplicates remain in the same cell.

Colchicine is usually applied to germinating seeds/seedlings, long enough to affect one cycle of cell division. Not all the cells will be dividing so the plant will end up with a mixture of diploid and tetraploid cells . . . must include cells that will form floral organs if the doubled condition is to be passed on to the progeny.

Why would we want to increase the ploidy level?

In some plant species a reasonable increase in numbers of genomes has been found to be accompanied by increased cell size and larger plant organs e.g., fruits, flowers. This increase in size however, has some detrimental side effects. Larger cells contain greater amounts of water and in some cases this leads to a greater sensitivity to low temperatures. Fertility problems due to irregular meiosis, also. Plus unexpected effects from interallelic interaction changes due to dosage of genes involved.

To improve chances of success in inducing polyploidy, use

Diploid with relatively low chromosome number.

A species that can be vegetatively as well as sexually propagated — means do not have to hybridize immediately and have multiple chances to hybridize.

Cross-pollinated species rather than a self-pollinated species. Greater variability due to heterozygosity (greater buffering).

Perennial forages possess most/all the desired characteristics. e.g., red clover . . . tetraploids have been quite successful, although still need further improvement.

Questions to ask if one wants to induce chromosome doubling in a certain crop:

What do I hope to gain by making a tetraploid crop?

What are the advantages and/or limitations of this crop for tetraploidy?

How much time (in terms of plant generations) and labor am I prepared to give in order to develop an adapted tetraploid crop?

Ref. Dewey, D.R. 1980. Some applications and misapplications of induced polyploidy in plant breeding. In W.H. Lewis (ed) Polyploidy: Biological Relevance. Plenum Publ. Corp.

Decreasing chromosome number (halving) — Haploidy.

In nature, normal fertilization may not take place but the egg cell may still divide and a 1n embryo develop. A low frequency of haploids can be found in many species. If this occurs in a diploid then the somatic cells will only possess the haploid number of chromosomes. In the case of a polyploid a polyhaploid may be formed.

Interest in haploidy:

Identification and evaluation of recessive alleles without the masking effect of dominants on homologous chromosomes;

Easier to study inheritance of characters in polyhaploids vs. polyploids;

Crossing with related wild species with fewer genomes may be easier with polyhaploid having the same number of genomes as the wild species (i. e., potatoes);

Doubled haploids result in completely homozygous diploids.

Generation of haploids. (Kasha, K.J. (ed) 1974. Haploids in Higher Plants. Advances and Potentials. Univ. Guelph.)

Androgenesis — haploid plants developed from anthers or pollen grains.

Wide crosses to stimulate parthenogenesis and chromosome elimination.

Genetic systems to induce haploidy (e.g., hap gene in barley, indeterminant gametophyte in maize).

Problem with haploidy — chromosome instability which can result in aneuploidy.

Potential of doubled haploids.

Corn experience not so promising. However, in barley Parks et al., found that doubled haploids were a very rapid method of producing usable populations for breeding and selection. Considerable efforts in canola (also called rapeseed), wheat, and rice.

Particularly useful in crops with long generation times (winter wheat, or biennials).

Parks et al., 1976. Field performance of doubled haploid barley lines in comparison with lines developed by the pedigree and single seed descent methods. Can. J. Pl. Sci. 56: 467-474.

Genetic Bridges — method by which genes are exchanged between plant sources or transferred in one direction from a wild species to a crop.

Largest bridge — transfer of genomes.

Smaller bridge — transfer of single chromosome.

Footbridge — transfer of small segment of a chromosome with valuable genes.

Combining genes from different species — Alloploid.

Triticale story (see Hulse & Spurgeon, 1974. Triticale. Sci. Amer. 231: 72-80.)

Objective — to combine the yield, protein and baking characteristics of wheat with the drought tolerance, adaptation to poor soils and lysine content of rye. (note  mostly quantitative traits.)

Triticale (Triticosecale spp.)  A new crop from the combined genomes of wheat and rye. The first fertile wheat x rye hybrid was obtained in 1888 by Rimpau in Germany.

tetraploid wheat x rye(pollen) 2n = 4x = 28 2n = 2x = 14 AABB RR colchicine ® ® ® F₁ haploids . . . embryo culture ABR primary triticale (note: has wheat cytoplasm) Select for genetic stability. triticale x wheat — chance cross in Mexico nursery triticale (with desirable height, day length insensitivity and inc. fertility)

Problem encountered with triticale:

Shriveled seeds — abnormal endosperm development caused by what appears to be nonessential, late replicating DNA at the tips of the rye chromosomes that seems to cause abnormalities in the nuclei (high ploidy numbers).

Other allopolyploids synthesized:
radish x cabbage Karpechenko, 1928. also, wheat x barley potato x tomato

ANEUPLOIDY — variation in chromosome number that represents the loss or gain of one or a few chromosomes, but not an entire genome.

Not as important in evolution as euploidy, but important in plant breeding and genetic studies.

DISOMIC . . . a chromosome present in the normal pair or 2n condition

MONOSOMIC . . . one chromosome missing (2n-1)

NULLISOMIC . . . both chromosomes of a pair missing (2n-2)

TRISOMIC . . . an extra chromosome present (2n+1)

TETRASOMIC . . 2 extra chromosomes present (2n+2) etc.

Aneuploids may arise spontaneously as a result of gametes that have received less than the normal number of chromosomes. e.g., Through partial nondisjunction (lack of separation) in one or a few chromosome pairs in Anaphase I — resulting gametes will have extra chromosomes, or be missing one. Also, abnormal pairing in triploids ® aneuploidy.

Aneuploidy — valuable in locating genes on specific chromosomes and in understanding gene functions.

Extensive studies in wheat, tomato, barley, rice, cotton, tobacco.

Trisomics, monosomics and nullisomics utilized to greatest extent.

e.g., Trisomic: Develop a trisomic for each chromosome pair. Cross variety with gene to be located successively with each trisomic and look for abnormal segregation. Chromosome on which the gene is located is identified from the cross to the trisomic in which the character is segregating for the trisomic ratio (anything but a disomic ration) rather than the normal diploid ratio.

Monosomics — viable in polyploid species where the loss of a chromosome is balanced by homologous or partially homologous chromosomes from other genomes. In a cross to the critical monosomic, the chromosome can be identified in the F₁ if the variety carries the recessive allele, or in the F₂ if the variety carries the dominant allele. — Utilized in wheat, tobacco, cotton and other crops. e.g., wheat . . . the 21 possible monosomics for hexaploid wheat were established in the variety Chinese Spring. Through monosomic analysis a single dominant gene for resistance to races A and B of the Hessian fly was located on chromosome 5A of wheat.

SUBSTITUTION LINES

Substitution of single chromosomes from a donor variety into a variety (done using monosomic chromosome lines in recipient variety) has been a useful method of analyzing quantitative characters where distinct segregation ratios are not observed.

e.g., Identification of the chromosomes with loci contributing to high protein content in wheat. Chromosomes from a high protein wheat variety are transferred by backcrossing into a set of wheat monosomics. Effect of each chromosome from the donor variety on protein content is identified.

SUBSTITUTING CHROMOSOMES (Introgression)

When genomes of crop species are combined with whole genomes of related wild species, undesirable genes often accompany the desirable genes we wish to introduce. One solution is to eliminate all chromosomes except the one containing the desirable trait.

e.g., Tobacco2n = 48( S & T genomes)

Objective: Transfer dominant gene for resistance to Tobacco Mosaic Virus (TMV) from diploid species Nicotiana glutinosa L.

N. tabacum x N. glutinosa SSTT GG b.c. to eliminate all N. glutinosa F₁ ¬ ¬ ¬ colchicine chromosomes except STG the one carrying SSTTGG x SSTT

resistance to TMV. SSTTG . . . In first b.c. G chromosome unpaired at meiosis and lagged in movement so many not included in daughter nuclei.

From second backcross a tobacco line obtained with 2n = 48, & bred true for TMV resistance. SSTT(G) x SSTT Had substituted chromosome with TMV resistance from glutinosa for tabacum chromosome. ® ® ® ® ® ® ® 23tabacum : 1 glutinosa chm. pairs chm. pair
= SUBSTITUTION LINE

With chromosome transfer, one may still have the problem of unwanted linked genes.
CHROMOSOME SEGMENT TRANSFER

Induced translocation method:

X-ray radiation to induce break in alien chromosome followed by fusion of desired segment with broken end of crop chromosome. e.g., Sears' program (1950's) for transferring leaf rust resistance from to wheat (involves a bridging cross via tetraploid wheat).

Induced cross over method:

Create conditions that bring about pairing between alien chromosome and crop chromosome at meiosis. Hopefully, crossover occurs in desirable region leaving the undesirable genes on the alien chromosome. Genomic relationships are critical to using this method. The relationship between the new genome and one of the crop plant genomes must be closer that the genomic relationships within the crop plant, (e.g., wheat).

e.g., Transfer of dominant Yr gene for Yellow (stripe) rust resistance to wheat from Aegilops comosum. Riley et al., 1968.

Induced translocation method. Sears, E.R. 1956. The transfer of leaf-rust resistance from Aegilops umbellulata to wheat. p. 1-22 In Symp. on Genetics and Plant Breeding. Brookhaven.

A. umbellulata x T. dicoccoides (emmer) ABB Sterile hybrid ABC colchicine T. aestivum (wheat) x Amphidiploid AABBDD AABBCC Hybrid AABBCD x Wheat ABBDD Some gametes: ABD + few C chromosomes ABD Select AABBDD + restistance chromosome (from C) Wheat x Irradiated pollen AABBDD

Select resistant AABBDD bearing R gene on small translocated segment. Note: critical element is using the male gamete which does not tolerate aneuploid pollen as a filter so that resistant plants have translocation chromosomes and not addition chromosomes.

Induced crossover method — Riley, Chapman and Johnson. 1968.

The incorporation of alien disease resistance in wheat by genetic interference with the regulation of meiotic chromosome synapses. Genet. Res. 12:199-219.

T. aestivum x A. comosum 2n = 42 2n = 14 AABBDD MM(Yr) b.c. F₁ ® ® ® colchicine ABDM—————AABBDDMM x AABBDD Amphidiploid Wheat parent After second b.c. selected for resistance to yellow rust.
44 chromosome addition line x A. speltoides (all 42 chromosomes + 1 pair of 2n = 14 2M comosum chromosomes with Yr gene) Ph suppressed pairing permitted with related wheat chm. T. aestivum ¬ ¬ ¬ ¬ 29 chromosome hybrid 21 T.aestivum b.c.'s 7 A.speltiodes ¯ self 1 A.comosum Rust resistant line 'Compair'