The document discusses gene duplication of alpha prolamin storage protein genes in maize. It finds that: 1) Maize underwent more extensive duplication and dispersal of alpha prolamin genes than sorghum after their lineages diverged. 2) Duplications of alpha prolamin genes in maize occurred both before and after the maize genome underwent allotetraploidization. 3) Different maize haplotypes have variable numbers of alpha prolamin gene copies and differences in intergenic regions due to transposable elements. This indicates the haplotypes diverged before modern breeding.

1. Short and long-distance placement of gene copies
in the maize genome
Mihai Miclaus, Jianhong Xu, and Joachim Messing
Rutgers University, Waksman Institute

2. •One of the most prominent features in plant genomes is the
duplication of genes.
•Synteny illustrates that copying of alpha prolamins genes occurred
before and after allotetraploidization.
•Here we compare haplotypes comprising copying events and their
chromosomal divergence.
•Genes that got more extensively duplicated in crop plants are
storage protein genes.
•We have investigated the copying events for the members of the alpha
prolamin gene families because of their variable copy number.
Background

3. • α-zeins (41)
• β-zeins (1)
• γ-zeins (3)
• δ-zeins (2)
• 19kD
• 22kD
Shewry and Halford 2002
• 10kDa
• 18kDa
• 16kD
• 27kD
• 50kD
• 15kD
Zeins (B73)
z1A1, z1A2 (9+3)
z1B (9)
z1D (5)
z1C1, z1C2 (14+1)
• α-kafirins (23)
• β- kafirins (1)
• γ- kafirins (2)
• δ- kafirins (1)
• k1A (2)
• k1C (20)
• k1D (1)
Kafirins (Btx623)
The prolamins

4. • Synteny between maize, sorghum, and rice shows that gamma prolamin
genes arose after the progenitor of rice split from the progenitor of
sorghum and maize.
50 mya
11.9 mya
4.8 mya
Oryza
Zea
Sorghum
Gamma prolamins in sorghum
and two maize chromosomes

5. •The alpha prolamin genes (19-kDa and 22-kDa) arose before the progenitors
of sorghum and maize split; maize underwent more extensive
gene copying and dispersal than sorghum did.
z – zein (maize)
k – kafirin (sorghum)
z1C1(azs22 2)
z1C1(azs22 16)
k1C1(k1C 1)
k1C18(k1C 18)
z1C1(azs22 13)
z1A2(z1A2 1)
z1A1(z1A1 6)
z1A1(z1A1 1)
z1B(z1B 4)
z1B(z1B 3)
k1A(k1A 2)
z1D(z1D 2)
k1D(k1D 1)
0.000.050.100.150.200.250.30
z1C1(azs22;2)
z1C2(azs22;16)
k1C(k1C;1)
k1C(k1C;18)
z1C1(azs22;13)
z1A1(z1A1;6)
z1A2(z1A2;1)
z1A1(z1A1;1)
z1B(z1B;4)
z1B(z1B;3)
k1A(k1A;2)
z1D(z1D;2)
k1D(k1D;1)
Allotetraploidization
(Ks)
Origin of alpha prolamins

6. 10 20 5030 6040 70 9080 100 110 120 130 140 150 160 170 200 kb19018010 20 5030 6040 70 9080 100 110 120 130 140 150 160 170
130’ 140’ 3010 4020 50 7060 80 90 kb150’ 370190 200 230210 240220 250 270260 280 290 300 310 320 330 340 350 360
The z1D cluster: where it all started…
Gyzma 1
Unknown REs
Xilon 1
Xilon 1
Huck 1 Zeon 1
Prem 1
Cinful 2
Cinful 2
Zeon 1
Huck 1
Zeon 1
Zeon 1
Opie 2
Opie 2
Ji 1
Zeon 1
Cinful 2
Pif 12
Cinful 2A
Tekay Gyzma 1
Opie 2 Prem 2 Dopia 4
Zeon 1 Solo
LTR
Xilon 1
Prem 2
Prem 2
Dopia 4
Ac9_ZM
Zeon 1
Prem 1
Cinful 2
Cinful 2
Huck 1 Opie 2
Zeon 1
Opie 2
Ji 1
Cinful 2
Solo LTR
Ds CACTA
B73
BSSS53
19 kDa Zein
Putative gene
LTR
Predicted gene
RE
TE
* ~83kb
• Genes collinear; two active and three inactive copies
• Large intergenic regions; REs and TEs after haplotypes split

7. 0.5 mya
The z1A1 cluster
…
10 4020 30 60 70 80 90 100 110 120 130 140 150kb50
10 4020 30 70 80 100 110 120 130 140 220kb50
B73
Prem 2 Cinful 2 Zeon 1 Ji 1 Zeon 1 Prem 2Zeon 1
Opie 2
Cinful 2
Prem 2 Cinful 2 Ji 1 Zeon 1 Xilon 1 Prem 2
Zeon 1
Prem 2
Solo-LTR
60 90
BSSS53
19 kDa Zein
Putative gene
LTR
Predicted gene
RE
*
• Youngest copy (0.5 mya) after haplotypes split
• Smaller intergenic regions; REs after haplotypes split

8. Haplotype variability
Locus Conserved Zeins Other genes REs TEs Intergenic
B73
(104kb) Z1A1
83% 5.7% n/a 57% n/a 37%
BSSS53
(107kb)
81% 4.7% n/a 72% n/a 21%
B73
(58kb) Z1A2
49% 3.6% n/a 51% n/a 45%
BSSS53
(10kb)
80% n/a n/a n/a n/a n/a
B73
(200kb) Z1B
99.5% 3.2% n/a 60% n/a 37%
BSSS53
(199kb)
100% 3.2% n/a 60% n/a 37%
B73
(111kb) Z1C1
75% 8.7% 0.7% 14% 4% 73%
BSSS53
(169kb)
50% 9% 0.3% 11% 5.3% 74%
B73/BSSS53
*single copy
Z1C2 99% n/a n/a n/a n/a n/a
B73
(220kb) Z1D
58% 1.4% 2.3% 58% 0.5% 38%
BSSS53
(220kb)
58% 1.4% 2.3% 44% 2.7% 50%

9. 1 2 3 4 5 6 7 8 9
z1D
10
z1A1
z1A2
z1C1
z1C2
4 5 6 7 8 9 10
Gene movement inside the maize genome
z1B
19 kDa zeins
22 kDa zeins

10. To sum up …
• Some donor copies are inactivated but still under selection; B73 has 41 and
BSSS53 has 47 copies; only 18 are expressed, some non-allelic.
• Gene duplications of prolamin genes are more extensive in maize than in
sorghum; many of them formed after allotetraploidization.
• Genes arising before allotetraploidization are lost on one of the maize
homoeologous regions.
• Genes are duplicated in dispersed and tandem locations, indicating two
possibly different copying mechanisms.
• Haplotypes arose before crop cultivation, indicating that breeding is based on
haplotypes that differ in intergenic regions and gene copies.

11. Acknowledgements
Moisés Cortés-Cruz
Galina Fuks
DOE