Polyploidy – so many options

Impacts of Ploidy Changes Changes in chromosome number and structure can have major health impacts e.g. trisomy 21 Polyploidy in cultivated and domesticated plants is widespread and of evolutionary and economic importance

Polyploidy – Pros and Cons Advantages Vigor effects – heterotic boost from divergent parental genomes Redundancy – masking of recessive alleles Buffering capacity Disadvantages Changes in cell structure & shape – doubling genome content increases cell volume Problems in cell division – mitosis and meiosis Changes in gene expression Epigenetic instability

Alternation of Generations The sporophytic generation may be diploid (2n = 2x) or polyploid (2n = _x) VAVA VAVAVBVB VAVAVBVBVDVD 2n = 2x = 14 30,000 genes 2n = 4x = 28 60,000 genes 2n = 6x = 42 90,000 genes 1 pair homologous chromosomes 0 sets of homoeologous chromosomes AA 2 pairs of homologous chromosomes 2 sets of homoeologous chromosomes AABB 3 pairs of homologous chromosomes 3 sets of homoeologous chromosomes AABBDD A A B B A A A A B B D D

Euploidy An organism with an exact multiple of a basic chromosome number (x) Can be diploid (2x), triploid (3x), tetraploid (4x) ….. Barley in the sporophytic generation is 2n = 14 n = 7 in the gametophtyic generation The base number (x) = 7 = n for a diploid Potato in the sporophytic generation is 2n = 48 n = 24 in the gametophytic generation but x = 12 2n=2x=14 2n=4x=48

Aneuploidy Not euploid – more or less chromosomes than a multiple of the basic number Monosomic – loss of a chromosome, (2n-1) Trisomic – addition of a chromosome, (2n+1) Of value in genetic studies

Polyploidy More than two basic sets of chromosomes Autopolyploidy – 3 or more copies of each chromosome in the basic number Allopolyploidy – 2 or more copies of ancestral genomes giving 4 or more copies of the basic number

Polyploid Formation Genome duplication Failure of spindle fibers in meiosis or mitosis Must lead to balance in order to achieve euploidy and long-term viability Imbalanced gametes = aneuploidy

Polyploids - Bivalent Pairing for viability Trivalent + of homologous chromosome pairing in autopolyploids = sterility Homoeologous pairing in allopolyploids = sterility If non-bivalent pairing, gametes will not all get the same number of chromosomes Must have bivalent pairing for fertility: polyploids behaving as diploids

Autopolyploids Three or more homologues for each chromosome in the basic number Even numbers (4x, 6x etc.) can be fertile Potato 2n = 4x = 48; Alfalfa 2n = 4x = 32 Pairs of homologs = bivalents and normal meiosis Odd numbers (3x, 5x, …) = sterile or abnormal Banana 2n = 3x = 33 The complete chromosome complement cannot form into pairs and normal meiosis is disrupted

New Autopolyploids Can be synthesized by the use of colchicine to double the chromosome complement Colchicine interferes with spindle formation in cell division A 2n homozygous cell undergoes replication of each chromosome during S phase of mitosis giving 2 copies of each No spindle at Anaphase and all can migrate to the same cell to give a homozygous tetraploid

New Autopolyploids Can also create triploids by crossing related tetraploid with a diploid Newly synthesized autopolyploids generally sterile Formation of multivalents disrupts meiosis Advantage in breeding some crops Seedless watermelon 2n = 3x =33

Genetics & Breeding of Autopolyploids Potentially very complex as up to 4 copies of an allele at each gene can be present Nulliplex, simplex, duplex, triplex, quadriplex ….. Cross Nulliplex (N) Simplex (S) Duplex (D) Triplex (T) Quadriplex (Q) Nulliplex (aaaa) All N Simplex (Aaaa) 1S : 1N 1D : 2S : 1N Duplex (AAaa) 1D : 4S : 1N 1T:5D:5S:1N 1Q:8T:18D: 8S:1N Triplex (AAAa) 1D : 1S 1T : 2D: 1S 1Q:5T:5D:1S 1Q : 2T : 1D Quadriplex (AAAA) All D 1T : 1S 1Q : 4T : 1D 1Q : 1T All Q

It’s all Bananas Cultivated bananas derived from diploid species Musa acuminata (A) and Musa balbisiana (B) Most edibles are triploids with genomes of AAA (desert), AAB (plantains), and ABB (Cooking) Irregular pairing means bananas are seedless Good for the consumer but problematic for the breeder and maintainer Evidence of pairing between homoeologous chromosomes from A and B genomes 90% desert bananas are cv. Cavendish

Sequencing to the rescue? Previous breeding efforts have looked at mutation Now major effort resulted in sequencing a wild Musa acuminata genome (AA) http://banana-genome.cirad.fr/

Seedless Watermelons An infertile triploid created from 4x and 2x parents Tetraploid Inbred AAAA Diploid Inbred AA x Triploid F1 AAA Grow with Fertile Diploid to stimulate seedless fruit production

Seedless Watermelons The consumer benefits but breeding is more difficult and hence expensive Development of suitable tetraploids Selection against sterility and fruit abnormalities Reduced seed yield for seed company Grower devotes up to 33% field to 2x pollinator

Allopolyploids An individual with chromosome sets from two or more different but related species Interspecific hybridization followed by chromosome doubling Spontaneous (natural forms) Colchicine (synthesized forms) Behave like diploids due to bivalent pairing Homologs within each ancestral species pair even though homoeologous genomes may be collinear

Incipient allopolyploids – sterile unless there is doubling Interspecific hybrids have just one copy of each genome AA x BB → AB Haploid number of chromosomes from each species Gametes get the wrong number of chromosomes and hence infertility Use colchicine to double the chromosome complement

Polyploidy in the Triticeae Sporophytic generation Gametophytic generation Ploidy Level Genome Formula 2n = 14 n = 7 2x (diploid) e.g. Emmer wheat 2n = 2x = 14 2n = 28 n = 14 4x (tetraploid) e.g. Durum wheat 2n = 4x = 28 2n = 42 n = 21 6x (hexaploid) e.g. Bread wheat 2n =6x = 42

The Evolution of Bread Wheat (and barley) Hordeum spontaneum Wild barley 2n = 2x =14 AA=BB=DD 2n=2x=14 Hordeum vulgare Cultivated barley 2n = 2x =14

The Bread Wheat Genome

For more information see Wheat Pairing Ph1 locus on 5B affects pairing in wheat Promotes homologous pairing Blocks homoeologous pairing Gene has been cloned For more information see https://www.jic.ac.uk/staff/graham-moore/Wheat_meiosis.htm

Triticale an allo-hexa/octaploid Wheat (durum or bread) Rye hybrid Bread Wheat AABBDD Rye RR x Infertile F1 ABDR Fertile F1 AABBDDRR

Brassicas – The Triangle of U (Woo Jang-choon = Nagahara U)

Haploids Single basic set of chromosomes Maize – n=10; bread wheat – n=21; barley – n=7 Haploid plants can be nurtured to grow Only have the basic chromosome content (n) so are infertile – meiotic irregularities

Doubled Haploids Doubling the haploid chromosome content gives two exact copies No heterozygotes – “instant inbred lines” Sample pollen or egg cells from F1 plants A random sample of all the possible products of the first round of segregation from meiosis Shorten the breeding cycle Immortal genetic populations for research Can make doubled haploids at any stage in the selfing process (e.g. F1, F2, F3)

Doubled Haploids Production of ‘Instant’ Inbreds Shortens Breeding Cycle Makes Selection More Effective Can make Pure Seed Production Easier How? Pollination by alien species Anther/Microspore Culture Haploid inducing genes

Doubled Haploidy Time Line 1921: Natural production of haploids in Datura stramonium observed. Followed by Nicotiana tabacum (1924) 1952: Doubled haploid, inbred maize lines produced. Selected parthenogenic haploids and chromosome doubling 1964: Haploid plants from Datura innoxia by anther culture 1970: Haploid production in barley via wide crossing 1978/79: First doubled haploid cultivar: “Mingo” barley Currently: Routine technique in breeding many cereal and vegetable crops

Events in Androgenesis maturation bi-cellular pollen mature pollen male gametophyte stress uni-cellular microspore: embryogenesis embryo, sporophyte embryogenic microspore cell with restricted developmental potential Diverting the developmental pathway of the microspore towards embryos rather than pollen A. Touraev: Vienna Biocenter, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria alisher.touraev@univie.ac.at totipotent cell

Androgenesis Induction Reprogramming of microspores towards sporophytic development Heat shock Sucrose and nitrogen starvation Cold stress Colchicine treatment Re-programming of the microspore. A. Touraev: Vienna Biocenter, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria alisher.touraev@univie.ac.at Gamma irradiation Ethanol pH Osmotic stress Separate or in combination

Hordeum bulbosum wide crosses Produce F1 from Desired Cross Emasculate 2-3 days before pollen shed Ensure plentiful supply of pollen from wild relative Dust alien pollen onto open emasculated flowers Apply hormonal spray to pollinated spike (can repeat 2-3 days later) Bag pollinated spike and leave for 10-12 days The Hordeum bulbosum method to produce doubled haploid barley. P. Devaux: Florimond Desprez, Biotechnology Laboratory, P.O. Box 41, 59242 Cappelle en Pevel,e, France. pierre.devaux@florimond-desprez.fr

Hordeum bulbosum wide crosses Rescue developing embryos from spike pollinated with alien pollen Grow on in special rooting medium Once plants established, trim roots and treat with colchicine The Hordeum bulbosum method to produce doubled haploid barley. P. Devaux: Florimond Desprez, Biotechnology Laboratory, P.O. Box 41, 59242 Cappelle en Pevel,e, France. pierre.devaux@florimond-desprez.fr Grow out plants and harvest seed from fertile plants

Anther Culture Healthy Donor Plants Harvest spikes Apply stress conditions Plate out anthers on induction medium Sub-culture steps Spontaneous doubling Transfer to greenhouse Field http://barleyworld.org/sites/default/files/2014_dh_poster.pdf

Numbers of DH Cultivars Species Numbers Method Rice >100 Anther culture Barley Anther, micospore culture & wide crossing Rapeseed >50 Microspore culture, spontaneous DH lines Wheat >20 Anther culture, wide crossing Pepper >10 F1 from DH parent(s) Asparagus Female x DH supermale Tobacco Microspore culture, anther culture Cultivars derived through doubled haploidy B.P. Forster: Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. bforst@scri.sari.ac.uk Also: mustard, eggplant, melon, triticale

Doubled haploids & inbred line development in maize Hybrid maize (corn) is a major crop worldwide Hybrids derived from intermating inbred lines Inbred line development key to hybrid breeding Accelerate inbred line development means hybrid development also accelerated In vitro production of doubled haploids Anther or microspore culture In vivo production of doubled haploids Haploid inducer lines either as male or female Induction at >1% haploid lines; morphological marker for identification Possibly arise through defective sperm cell enabling fertilization but chromosomes eliminated