Genotyping of asparagus

(Asparagus officinalis L.) cultivars

with SSR markers

Yoko TAKEUCHI Kyushu University

Yumi SAKAGUCHI Fukuoka
Yukio OZAKI Japan
Hiroshi OKUBO
 Introduction
Molecular markers were reported in asparagus.
RAPD, AFLP, RFLP, allozyme etc

Problems
Dominant expression (RAPD, AFLP)
Low polymorphism (RFLP. allozyme)

SSR markers have been established from EST in

asparagus (Caruso et al., 2008).

Advantage Applicability of SSR as

Co-dominant expression genetic markers has
High polymorphism not been resolved.
 Experiments

1. Inheritance of SSR markers and

linkage arrangement of the loci

2. Genotyping of asparagus cultivars

with different ploidies
 Materials and Methods
Plant materials
Crossed parents and progenies

Table 1. Cross combinations for investigating the

segregation of SSR markers.

Cross Parentage
1 Hokkai100-5z × UC157-50
2 WC-103 × WC-98
3 Gold Schatz-1 × Hokkai100-3
z
Each number after cultivar names indicates the
individual plant number.
 Materials and Methods
Methods
DNA extraction with modified CTAB method

PCR amplification
PCR with SSR primer sets
(AG2, AG3, AG7, AG10, TC1 and TC7)

Genotyping
Genotyping by using the capillary sequencer
and GENESCAN

PCR and electrophoretic analysis of

Asp1-T7sp region
 Results
Table 2. Goodness-of-fit tests for segregation patterns at AG2, AG3, AG7, AG10, TC1 and TC7 loci.

Progeny genotype
SSR Cross Prospected genotype Expected ratio χ2 P
(Number of plants)
AG2 2 151/157×151/157 151/151 151/157 157/157 1: 2: 1 4.235 0.120
(23) (34) (11)
3 151/155×147/157 147/151 147/155 151/157 155/157 1: 1: 1: 1 0.870 0.833
(34) (30) (32) (27)
AG3 2 214/216×214/216 214/214 214/216 216/216 1: 2: 1 0.235 0.889
(18) (32) (18)
3 214/216×214/218 214/214 214/216 214/218 216/218 1: 1: 1: 1 6.138 0.105
AG2 (35) (39) (21) (28)
AG7 1 174/178×178/178 174/178 178/178 1: 1 0.480 0.488
Cross 2 (34) (41)
Segregation of the crossed progenies was not significantly
3 Progeny
174/178×172/174 172/174 genotype
172/178 174/174 174/178 1: 1: 1: 1 0.285 0.963
Prospected genotype (33) (31) (30) Expected ratio χ2
(29) P
different from Mendelian expectation at P=0.05.
AG10 1 (Number
177/183×177/177 177/177 of plants)
177/183 1: 1 0.000 1.000
Each SSR locus was governed by a single locus.
151/157×151/157 151/151 (37) 151/157(38) 157/157 1: 2: 1
0.120 4.235
3 177/183×177/177 177/177 177/183 1: 1 0.293 0.589
(23) (58)(34)(65) (11)
TC1 1 211/213×219/225 211/219 211/225 213/219 213/225 1: 1: 1: 1 3.240 0.356
(15) (21) (15) (24)
2 219/221×219/225 219/219 219/225 219/221 221/225 1: 1: 1: 1 6.824 0.078
(8) (22) (18) (20)
3 219/225×219/225 219/219 219/225 225/225 1: 2: 1 4.366 0.113
(40) (59) (24)
TC7 1 208/218×208/212 208/208 208/212 208/218 212/218 1: 1: 1: 1 1.107 0.775
(20) (19) (21) (15)
2 206/208×208/212 206/208 206/212 208/208 208/212 1: 1: 1: 1 0.824 0.844
(17) (15) (20) (16)
3 208/220×208/208 208/208 208/220 1: 1 0.130 0.718
(64) (59)
Table 3. Linkage tests for independent assortment for jointly
segregating SSR loci.
Gene loci AG2 AG3 AG7 AG10 TC1 TC7 Asp1-T7sp
AG2 N N N N N N
AG3 R N R N R
AG7 N N N N
AG10 R N ―
TC1 N N
TC7 N
Asp1-T7sp
N: No evidence of linkage.
R: Possible linkage pairs in all crosses investigated.
R: Segregation distortion was recognized, but there was no
evidence of the linkage.
―: Not investigated.
Linkage was recognized in ‘Asp1-T7sp, AG3’ with the
recombination fraction of 0.138 at 1% level.

AG3 locus is expected to locate on the L5 chromosomes.

 Experiments

1. Inheritance of SSR markers and

linkage arrangement of the loci

2. Genotyping of asparagus cultivars

with different ploidies
 Materials and Methods

Plant materials
Diploid: ‘Zenyu953’
‘Welcome’ (WC)

Triploid: ‘Hiroshima Green’ (HIG)

Tetraploid: ‘Purple Welcome’ (PWC)

Methods
Same as experiment 1
 Results
Zenyu953-1 151bp
(151/155)z 155bp

HIG-3f 157bp
(151/157/157) 151bp

PWC-3f (147/149/149/151)
147bp 151bp

149bp

PWC-1m 157bp
(149/157/157/157)
149bp

Figure 1. Peak patterns in AG2 region. z Prospected genotype.

 Table 4. SSR genotypes in asparagus cultivars.
Table 4. SSR genotypes in asparagus
AG2 AG3 AG7 AG10
Individual
cultivars.
147 149 151 155 157 212 214 216 218 172 174 176 178 159 161 163 177 183 (bp)
diploid
AG2
z
Individual 1
Zenyu953-1 1 1 1 2 2
Zenyu953-2 147
1 1 149 151 1 155
1 157 (bp) 2 2
Zenyu953-3 1 1 1 1 2 2
diploid
Zenyu953-4 1 1 1 1 2 2
Zenyu953-5 1 1 1z 1 2 2
WC-1fZenyu953-1 1 1 11 1 1 2 2
WC-2f 1 1 2 2 2
WC-3f Zenyu953-2 1 1 2 1 1 2 2
WC-1m
WC-2m
Zenyu953-3 1
1
1
1
1 1
2
1 1 151/157
2
2
× 155/155 2
2
triploidZenyu953-4 1 1
HIG-1f 1 2 2 1 2 1 1 2
HIG-2fZenyu953-5 1 2 2 11 1 2 1 1 2
HIG-3f 1 2 2 1 2 1 1 2
HIG-1mWC-1f 1 2 11 1 1 1 2 1 1 2
HIG-2mWC-2f
tetraploid
1 2 12 1 1 2 1 1 2

PWC-1fWC-3f 1 2 1 1 4 1 2 151/151 2 ×3 157/157

1
PWC-2f 1 3 4 2 1 1 4
PWC-3fWC-1m 1 2 1 1 4 1 1 2 1 3 1
PWC-1m 1 3 4 3 1 1 3
PWC-2m
WC-2m 1 3 4
1 1 4 2 1 1
z
z
Individual
Individual gene numbergene number in each
in each region.

region.genotypes
Parental
Parental genotypes and genetic
of thepurity
diploidof the diploid cultivars
cultivars
 Table 4. SSR genotypes in asparagus cultivars.

TC1 TC7
Individual
211 217 219 221 223 225 227 229 194 196 206 208 212 216 218 228 (bp)
diploid
Zenyu953-1 1z 1 2
Zenyu953-2 1 1 2
Zenyu953-3 2 1 1
Zenyu953-4 2 1 1
Zenyu953-5 1 1 2
WC-1f 1 1 2
WC-2f 1 1 1 1
WC-3f 1 1 1 1
WC-1m 1 1 2
WC-2m 1 1 1 1
triploid
HIG-1f 1 1 1 1 1 1
HIG-2f 2 1 1 1 1
HIG-3f 1 1 1 2 1
HIG-1m 2 1 1 1 1
HIG-2m 1 1 1 2 1
tetraploid
PWC-1f 1 1 2 1 2 1
PWC-2f 2 1 1 3 1
PWC-3f 2 1 1 3 1
PWC-1m 2 1 1 1 1 1 1
PWC-2m 1 1 2 1 2 1
z
Individual gene number in each region.

Genotyping of triploid and tetraploid cultivars in all SSR regions

34 alleles in six loci
Alleles per locus ranged from four to eight
 151bp AG2
157bp (151/151/157) z

216bp
AG3 214bp
(214/216/218) 218bp

AG7 174bp
(174/174/178) 178bp

AG10 177bp
(177/177/183)
183bp

TC1
(219/219/225) 219bp
225bp

Figure 2. Peak patterns in five SSR regions in 07M-61 (a progeny

of Gold Schatz × Hokkai100). z Prospected genotype.
 (A)2x

Number of nuclei

100 200 300 400

(B)3x

100 200 300 400

Fluorescent intensity
Figure 3. Flow cytometric histogram patterns of Hokkai 100 (A)
and 07M-61 (a progeny of Gold Schatz × Hokkai100) (B).
 Conclusion

 SSR markers are useful for hybrid test.

 AG3 of SSR is considered to be on the

L5 chromosomes where sex
determination locus locates.

 SSR genotypes of different polyploid

cultivars were distinguishable.
 Acknowledgement

Dr. Mikeal L. Roose

University of California

for providing us the information of SSR markers

Thank you very much!