The 23rd Session of the International Poplar Commission The Mechanism of 2n Pollen Formatiom in Populus ? euramericana and P. ? popularis Speaker Jin-feng Zhang (Jennifer) zjf@bjfu. edu. cn Beijing Forestry University P. R. China OUTLINE 1 Introduction 2 Materials and Methods 3 Results and Analysis 4 Discussion 1 Introduction 1. 1 Polyploid breeding is an important part in poplar breeding ? ? Triploid white poplar trees Triploid and aneuploid hybrids in Populus trichocarpa ? P. deltoides ? Triploidy were found in the cultivar poplar clones in section Aigeiros Introduction 1. 2 Mechanisms of 2n gamete formation in plant ? ? ? ? ? Premeiotic doubling Omission of the first or second meiotic division Abnormal spindle Abnormal cytokinesis Nuclear fusion FDR ( first division restitution ) : Contains non-sister chromatids SDR ( second division restitution): Contains two sister chromatids 1 Introduction 1. 3 The objective of this study ? Poplar can produce 2n gamete naturally or by artificial induction Elucidation of the cytological mechanisms of 2n gamete formation has been seldom in poplar.

To detect and elucidate the mechanisms of 2n pollen formation in diploid poplar Results from this research may offer a more effective method for polyploid breeding in poplar in section Aigeiros. ? ? ? 2 Materials and Methods 2. 1 Plant materials ? Populus ? euramericana Four male : EA1, EA2, EA3 and EA4 One female: A ? P. ? popularis The offspring of (P. simonii ? (P. nigra var pyramidalis + Salix matsudana mixed pollen) ) One Male: P ? The crosses A ? EA1, A ? EA2, A ? EA3, A ? EA4, A? P 2 Materials and Methods 2. 2 Microsporogenesis observation 2. 3 Flow cytometry analyses 2. 4. Chromosome counting 2. 5. SSR analysis 3. Results and Analysis . 1 Cytological determination on 2n pollen formation 10 11 5 6 8 9 Table 1 The expected and observed rate of 2n pollen grains Sporads Code of poplar Dyad Triad Tetrad Total Expected rate of 2n pollen % Observed rate of 2n pollen % ? 2 EA1 EA2 EA3 EA4 P 539 163 689 2189 308 341 1818 682 4093 484 6629 5528 6354 1691 7308 7509 7509 7725 7973 8100 4. 96 7. 69 7. 14 36. 17 3. 15 0. 03 0. 09 10. 08 29. 41 2. 35 26. 056** ** Indicated significant difference between expected rate of 2n pollen from sporads sample and the observed rate of 2n pollen from pollen sample at P< 0. 01. The percentage were converted to arcsine data before ? 2 test. . Results and Analysis 3. 2. Detection of polyploid offspring of 2n pollen Diploid 61# Diploid 61# Triploid 65# Triploid 65# 0 50 Channels (FL2-A- 100 150 1. 27) 200 250 Diploid 61# Diploid 61# Triploid 73# Triploid 73# 0 50 Channels (FL2-A- 100 150 1. 27) 200 Diploid 61# ??? 61#+ 61# Diploid ??? 75# 75# Tetraploid Tetraploid 75# 0 50 Channels (FL2-A- 100 150 1. 27) 200 250 3. Results and Analysis 3. 3. SSR determination on mechanism of 2n pollen formation M > + M EA4> A EA4 4x 75# 2x 2x 2x 4x 2x 2x 75# > 4x M EA4 + 75# 2x 2x 2x A M > + 4x 2x 2x 2x EA4 A 75# (a) Primer: 14: M > + EA4 A (b) Primer: 41 4x 2x 2x 2x (c) Primer: 47 75# + 4x 2x 2x 2x EA4 A 75# M (d) Primer: 68 (e) Primer: 105 Table 2 Segregation of alleles at loci where the male Populus ? euramericana. (Dode) Guinier parent EA4 is heterozygous Code 14 41 47 68 105 SSR primer GCPM_2453-1 GCPM_3345-1 GCPM_3559-1 GCPM_432-1 ORPM_29 locus 1 2 3 4 5 6 EA4(>) AB AB AB AB A0 B0 A(+) CDE AC B A C0 DE 75#(4x) ABCDE ABC AB AB A0C0 B0DE 421(2x) 422(2x) 423(2x) ADE AC B A C0 BE ADE AC AB A C0 BE ADE AC AB A C0 BE These letters do not necessarily correspond to discrete alleles(e. g. the “E”band for GCPM_2453-1 may be the non specific amplification ) and ORPM_29 primer detects two loci, 0 means a null allele. . Results and Analysis 3. 3. SSR determination on mechanism of 2n pollen formation M > P + 3x 3x 2x 2x 2x A 65# 73# M > + 3x 3x 2x P A 65# 73# 2x 2x (a) Primer: 13 M > P + A 3x 3x 2x 65# 73# 2x 2x (b) Primer: 68 (c) Primer: 105 Table 3 Segregation of alleles at loci where the male P. ? popularis parent P is haterozygous Code 14 68 105 SSR primer GCPM_2453-1 GCPM_432-1 ORPM_29 4 AB BD AD AD BB AB AB locus 1 2 3 P(>) AB AB 00 A(+) CAD C C0 65#(3x) CAB AC C0 73#(3x) 321(2x) CAB BC C0 AD AC 00 322(2x) 323(2x) AD AC C0 AD AC 00

These letters do not necessarily correspond to discrete alleles(e. g. the “C”band for GCPM_24531 may be the non specific amplification) and the ORPM_29 primer detects two loci, 0 means a null allele. 4. Discussion 4. 1 Mechanisms of 2n pollen formation 4. 2 The biological reason for high percentage of 2n pollen 4. 3 The formation of 2n female gametes in poplars of section Aigeiros 4. 4 Polyploidy identification using molecular markers 4. 5 Implications of polyploidy for genetic research and tree breeding