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Ploidy – 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 Vigour effects – heterotic boost from divergent parental genomes Redundancy – masking of recessive alleles Loss of self incompatability → asexual reproduction • 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 Comai (2005), Nature Reviews Genetics 6: 836-846 Alternation of Generations The sporophytic generation may be diploid (2n = 2x) or polyploid (2n = _x) 1 pair homologous chromosomes 0 sets of homoeologous chromosomes AA A A VAVA 2n = 2x = 14 30,000 genes 2 pairs of homologous chromosomes 2 sets of homoeologous chromosomes AABB A ABB VAVAVBVB 2n = 4x = 28 60,000 genes 3 pairs of homologous chromosomes 3 sets of homoeologous chromosomes AABBDD A ABBDD VAVAVBVBVDVD 2n = 6x = 42 90,000 genes Euploid • 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 gametophytice generation but x = 12 2n=2x=14 2n=4x=48 Aneuploid • 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 • Can cause stock production problems Polyploid • 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 • Poor pairing in AB F1 hybrid Restored by genome duplication Ancestral genomes pair • Multivalent formation in autopolyploids Laggard chromosomes Aneuploid gametes 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 homologues are formed into bivalents and meiosis can proceed normally Odd numbers (3x, 5x etc) tend to be sterile or abnormal • Banana 2n = 3x = 33; Sugar Beet 2n = 3x = 27 • 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 tetaploid with a diploid • Newly synthesized autopolyploids generally sterile Formation of multivalents disrupts meiosis Advantage in breeding some crops • Seedless water-melon 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) 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 All D 1T : 1S 1Q : 4T : 1D 1Q : 1T Quadriplex (AAAA) Quadriplex (Q) For more details see ‘Plant Breeding’ by Brown, Caligari & Campos All Q 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 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) • Generally behave like diploids due to bivalent pairing Homologues from each ancestral species pair even though genomes may be collinear The Evolution of Bread Wheat Hordeum spontaneum Wild barley 2n = 2x =14 AA=BB=DD 2n=2x=14 Hordeum vulgare Cultivated barley 2n = 2x =14 The Bread Wheat Genome Brassicas – The Triangle of U Wheat Pairing • Ph1 locus on 5B affects pairing in wheat (Sears & Riley) • Promotes homologue pairs • Blocks homoeologue pairs • Gene has been cloned • Cluster of cdk-like genes • Okadaic acid mimics Ph1 deletions • Can use Ph1 deletions to develop introgressions Griffiths et al., 2006 Nature For more information see https://www.jic.ac.uk/staff/graham-moore/Wheat_meiosis.htm Breeding Autoployploids • • • • • Species has a low basic chromosome number Economic part of the plant is the vegetative part Plant is cross-pollinated (allogamous) Plant has a perennial habit Plant has the ability to reproduce vegetatively DR Dewey 1980. Some applications and misapplication of induced polyploidy to plant breeding Polyploids - Non Bivalent Pairs • Homologous chromosomes pairing with autopolyploids • Homoeologous pairing in allopolyploids • Gametes will not all get the same number of chromosomes Levels of infertility • Promote bivalent pairing Polyploids – No Pairing • 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 Triticale an allo-hexa/octaploid • Wheat (durum or bread) Rye hybrid Bread Wheat AABBDD x Infertile F1 ABDR Fertile F1 AABBDDRR Rye RR 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 homoeologues 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 x Diploid Inbred AA Triploid F1 AAA Grow with Fertile Diploid to stimulate seedless fruit production Seedless Water Melons • The consumer benefits but breeding is more difficult and hence expensive Development of suitable tetraploids Selection against sterility and fruit abnormalities Select parents for reduced seed coats Reduced seed yield for seed company Grower devotes up to 33% field to 2x pollinator Haploids • Single basic set of chromosomes Maize – n=10; bread wheat – n=21; barley – n=7 • Haploid plants can be nurtured to grow and establish Tend to be smaller 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 sample at any stage in the selfing cycle Population size is critical • Can you produce enough to maintain genetic gain? • How big a population do you need to produce to separate out linkage effcts from pleiotropy? Doubled Haploidy • • • • • • • Production of ‘Instant’ Inbreds Shortens Breeding Cycle Makes Selection More Effective Can make Pure Stock Production Easier Pollination by Alien species Anther/Microspore Culture Best estimate of additive genetic variance 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 stress bi-cellular pollen mature pollen male gametophyte uni-cellular microspore: cell with restricted developmental potential embryogenesis embryogenic microspore totipotent cell embryo, sporophyte Androgenesis Induction Reprogramming of microspores towards sporophytic development Sucrose and nitrogen starvation Heat shock Ethanol Gamma irradiation Cold stress Colchicine treatment pH Separate or in combination Osmotic stress Hordeum bulbosum wide crosses Produce F1 from Desired Cross Emasculate 2-3 days before pollen shed Ensure plentiful supply of pollen from wide species (alien) 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 1012 days 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 Grow out plants and harvest seed from fertiles Anther Culture Healthy Donor Plants Essential (Use Growth Room) Harvest spikes when flag leaf sheath 1-5 cms Apply stress conditions Plate out anthers on induction medium Sub-culture if necessary Transfer to rooting medium Microspore Culture Isolate microspores at early to mid uninucleate stage Concentrate and plate out on medium Apply stress conditions, incubate Sub-culture and allow green plants to develop Grow out in Glasshouse Numbers of DH Cultivars Species Numbers Method Rice >100 Anther culture Anther, micospore culture & wide crossing Microspore culture, spontaneous DH lines Barley >100 Rapeseed >50 Wheat >20 Anther culture, wide crossing Pepper >10 F1 from DH parent(s) Asparagus >10 Female x DH supermale Tobacco >10 Microspore culture, anther culture Also: mustard, eggplant, melon, swede, triticale Doubled Haploids & Parental Development • 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 DH Problems • • • • • • Mechanisms poorly understood Responsible genes still to be discovered Doubling of haploid genome Albinism in cereals Legumes remain recalcitrant Requires specialist technicians and facilities