ARTICLE pubs.acs.org/JAFC
Genetic Diversity of Thiamin and Folate in Primitive Cultivated and Wild Potato (Solanum) Species Aymeric Goyer* and Kortney Sweek Department of Botany and Plant Pathology, Hermiston Agricultural Research and Extension Center, Oregon State University, Hermiston, Oregon 97838, United States ABSTRACT: Biofortification of staple crops like potato via breeding is an attractive strategy to reduce human micronutrient deficiencies. A prerequisite is metabolic phenotyping of genetically diverse material which can potentially be used as parents in breeding programs. Thus, the natural genetic diversity of thiamin and folate contents was investigated in indigenous cultivated potatoes (Solanum tuberosum group Andigenum) and wild potato species (Solanum section Petota). Significant differences were found among clones and species. For about 50% of the clones there were variations in thiamin and folate contents between years. Genotypes which contained over 2-fold the thiamin and 4-fold the folate content compared to the modern variety Russet Burbank were identified and should be useful material to integrate in breeding programs which aim to enhance the nutritional value of potato. Primitive cultivars and wild species with widely different amounts of thiamin and folate will also be valuable tools to explore their respective metabolic regulation. KEYWORDS: thiamin, folate, potato tuber, Solanum tuberosum, Andigenum, wild potato species
’ INTRODUCTION Micronutrient deficiency is responsible for millions of deaths every year, especially among children, women, and the elderly of poor populations, and the death toll is very likely to increase as a result of the current economic and food crises.1,2 Several countries, including the United States, have implemented fortification of staple foods,3 leading to the eradication of severe micronutrient deficiencies. For instance, beriberi, a lethal disease due to severe thiamin deficiency, of which cases were occasionally reported as late as the 1930s, has mostly disappeared after the implementation of thiamin food fortification in 1942. Despite these measures, marginal nutritional deficiencies such as thiamin deficiency still commonly occur 4 and are often not identified because of a lack of typical symptoms, such as tachycardia, vomiting, or seizure.5 Meanwhile, many poor countries do not have the political will and the infrastructures necessary to implement food fortification, hence, micronutrient deficiencies remain common and severe in these regions. Biofortification of staple foods by breeding or biotechnology approaches is a low-cost, sustainable strategy for reducing micronutrient malnutrition occurrences.2 In particular, substantial research efforts have been made to increase vitamin contents of food crops by metabolic engineering. For instance, various projects have been aiming to increase folate content because of the deficiencies commonly occurring worldwide6,7 which are associated with the increased risk of neural tube defects (e.g., spina bifida), cardiovascular diseases, anemia, some types of cancer, and impairment in cognitive performance.8,9 Despite good success of these strategies, the general public has been rather reluctant to accept the introduction of transgenic foods in the market, thereby encouraging scientists to use other strategies more acceptable to the public, such as plant breeding. Largescale sequencing of plant genomes such as that of potato10 has facilitated breeding efforts by enabling faster and more targeted breeding strategies. While the costs associated with genome r 2011 American Chemical Society
sequencing have dropped and have enabled rapid progress, information on the natural genetic variation of micronutrients in crops remains scarce, thereby dramatically limiting advances toward nutritional enhancement. This is particularly true for the vitamins thiamin and folate, which are usually not detected or not identified in nontargeted metabolomics studies of potato.11,12 We have been exploring the natural genetic diversity of thiamin and folate contents in potato.13,14 Our previous studies have mainly focused on commercial potato varieties and advanced breeding lines. Although we found substantial variations in thiamin and folate contents (∼2.6-fold) in mature tubers, this group of germplasm represents only a small proportion of the overall genetic diversity of potatoes. Indeed, years of selection have tremendously decreased the gene pool of modern potato varieties. Indigenous cultivated (landraces) and wild (Solanum section Petota) potatoes from South and Central America, as well as the Southwestern United States, represent a tremendous genetic diversity and have been used to introgress desirable traits such as disease resistance genes15 into modern varieties. Yet, compared to the potential exploitable diversity in exotic potato combined with the number of species, it has not been fully exploited. Introduction of such material in breeding programs will help to expand the gene pool, which has been greatly restricted. The purpose of this study was to determine the range of thiamin and folate concentrations in diverse primitive cultivars and wild potato species, and to identify material which could be used as parents in breeding programs for thiamin and folate enhancement in potato. Recent studies suggest that all modern potato varieties originate from breeding with Chilean landraces from the group Chilotanum of the species S. tuberosum.16,17 Received: September 14, 2011 Accepted: November 16, 2011 Revised: November 7, 2011 Published: November 16, 2011 13072
dx.doi.org/10.1021/jf203736e | J. Agric. Food Chem. 2011, 59, 13072–13080
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Table 1. Thiamin Concentrations (Mean ( SE) in Primitive and Modern Potato Varieties concn (ng g 1) 2010a
2009 PI no./variety
clone
species
FW
FW
1021 ( 48
1272 ( 30**
5461 ( 130
1369 ( 152*
6114 ( 679
Russet Burbank
Russet Burbank
tuberosum
Russet Norkotah
Russet Norkotah
tuberosum
Yukon Gold
Yukon Gold
tuberosum
209421
RN 057.08
andigena
DW b
833 ( 241
1439 ( 145**
5337 ( 558
1315 ( 95
810 ( 110
1420 ( 28
7437 ( 149
1387 ( 106
6937 ( 528
217448
RN 061.13
andigena
1308 ( 85
225667
RN 004.06
andigena
1776 ( 47
225667
RN 004.12
andigena
1461 ( 113
1309 ( 88
7272 ( 492
225673 225677
RN 005.05 RN 008.12
andigena andigena
1555 ( 126 1444 ( 35
1176 ( 81* 1680 ( 99*
5881 ( 403c 7030 ( 415
225686
RN 010.06
andigena
1346 ( 44
1544 ( 115
9081 ( 674
225688
RN 011.03
andigena
1489 ( 104
1336 ( 206
8244 ( 1275
225689
RN 012.01
andigena
1500 ( 107
225689
RN 012.02
andigena
1401 ( 382
1959 ( 102
9993 ( 522
225689
RN 012.05
andigena
1242 ( 51
2317 ( 160**
9817 ( 677
225689
RN 012.12
andigena
1830 ( 82
1915 ( 132
10462 ( 722
225693 225693
RN 013.04 RN 013.09
andigena andigena
1231 ( 253 688 ( 83
1057 ( 79 1248 ( 37**
8739 ( 656 9313 ( 273
225694
RN 014.15
andigena
1989 ( 150
1570 ( 328
9813 ( 2048
225705
RN 017.08
andigena
1372 ( 70
1501 ( 131
8435 ( 734
225710
RN 018.03
andigena
490 ( 91
720 ( 68
4587 ( 433
234007
RN 047.05
andigena
1493 ( 30
1453 ( 25
8913 ( 152
234015
RN 049.06
andigena
968 ( 91
280868
RN 065.09
andigena
1151 ( 71
1249 ( 39
5866 ( 184
280871 280921
RN 067.01 RN 068.08
andigena andigena
1513 ( 177 1518 ( 211
1422 ( 42 1511 ( 47
6803 ( 203 7441 ( 233
280952
RN 070.01
andigena
924 ( 34
281080
RN 090.05
andigena
1657 ( 152
281080
RN 090.06
andigena
965 ( 32
1470 ( 82
6590 ( 367
1538 ( 119**
7432 ( 573
1436 ( 140
7636 ( 743
292110
RN 044.10
andigena
1202 ( 95
310490
RN 002.03
andigena
1266 ( 121
320355
RN 027.01
andigena
981 ( 28
320370 320370
RN 031.01 RN 031.14
andigena andigena
1105 ( 177 910 ( 58
607 ( 108* 1509 ( 28**
5841 ( 1040 7736 ( 123
1765 ( 34**
7415 ( 142
2325 ( 216*
12567 ( 1169
320373
RN 033.01
andigena
1503 ( 33
320373
RN 033.02
andigena
1183 ( 210
320377
RN 035.01
andigena
1766 ( 90
320377
RN 035.06
andigena
2273 ( 107
320377
RN 035.08
andigena
2179 ( 143
1084 ( 62**
9765 ( 562
320379
RN 036.05
andigena
986 ( 201
986 ( 88
8653 ( 768
320379 320379
RN 036.06 RN 036.15
andigena andigena
1888 ( 80 1828 ( 127
1260 ( 61** 1166 ( 17**
6267 ( 304 6071 ( 88
320387
RN 039.05
andigena
320390
RN 041.06
andigena
1550 ( 204
1427 ( 135
16401 ( 1554
2043 ( 272
10753 ( 1432
320390
RN 041.09
andigena
1453 ( 132
1526 ( 124
7746 ( 630
320391
RN 042.04
andigena
1395 ( 44
1127 ( 82*
5964 ( 432
320391
RN 042.14
andigena
1408 ( 23
320391
RN 042.15
andigena
1467 ( 112
2205 ( 168**
13206 ( 1007
473260 473260
RN 104.02 RN 104.05
andigena andigena
974 ( 108 1227 ( 149
1685 ( 78** 2010 ( 46**
7912 ( 367 9394 ( 213
473260
RN 104.06
andigena
1136 ( 112
1561 ( 200
7689 ( 987
13073
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Table 1. Continued concn (ng g 1) 2010a
2009 PI no./variety 473260 473276
clone
species
RN 104.08 RN 105.01
andigena andigena
FW
FW
1492 ( 38 1154 ( 99
1788 ( 167 1377 ( 132
DW 8553 ( 798 6014 ( 578
473276
RN 105.12
andigena
1437 ( 18
1338 ( 53
6281 ( 248
473276
RN 105.14
andigena
1059 ( 67
1060 ( 31
4472 ( 131
1602 ( 82*
498171
RN 043.11
andigena
1397 ( 37
498310
RN 112.08
andigena
1361 ( 110
498310
RN 112.10
andigena
898 ( 60
707 ( 41**
7628 ( 389 2908 ( 167
a
Data for 2010 are shown on both fresh weight and dry weight bases. b Significant difference between years P < 0.01 (**) or P < 0.05 (*) as determined by Student's t test. c Dry matter content was not determined for this clone, therefore we assume 20% dry matter content.
Therefore, in an attempt to capture as much genetic diversity as possible, we focused our screening on accessions which belong to the other cultivar group in S. tuberosum, the group Andigenum. This group comprises primitive cultivars which were previously classified in the groups Phureja (diploid), Stenotomum (diploid), and Andigena (tetraploid).16
’ MATERIALS AND METHODS Potato Material. Fifty-four clones from 33 accessions of primitive cultivars (S. tuberosum group Andigenum) and three modern potato varieties were grown in the fields at NRSP6, Sturgeon Bay, WI, in 2009 and 2010. Clones were planted in a screenhouse and grown for a few weeks before being transplanted to the field in early June. These were planted as plots of five, and each plot had a border plant at each end. Tubers were harvested mid-September. One to two tubers were harvested per plant from three plants per variety (one replicate represents one plant). When tubers weighed less than 5 g, more tubers were harvested per replicate. Tubers (skin-on) were washed with water, flash-frozen in liquid nitrogen, and either directly stored in a 80 °C freezer (2009) or freeze-dried before storage at 80 °C (2010). Freeze-drying samples was added to our procedure in 2010 to allow better comparison of vitamin contents among potato clones because moisture content varied quite significantly among clones (from 74 to 91% moisture). Moisture content was calculated by weight difference before and after freezedrying potato samples. Sixty-four accessions representing 25 wild potato species were grown under glass at NRSP6, Sturgeon Bay, WI. Seeds were sown on the first of February 2010. Four seedlings of each accession were transplanted into 15 cm pots containing Pro-Mix in the greenhouse on February 15th. Tubers were harvested from mid-June to mid-July, and stored in paper sacks for five months at 6 °C under 40 to 50% humidity before processing. The samples tested were bulks of tubers from the four seedlings. Chemicals and Reagents. Folates were from Schircks Laboratories (Jona, Switzerland). Rat plasma conjugase was from Pel-Freez (Rogers, AR) and was dialyzed before use as described.18 Difco thiamin assay medium LV (TAM-LV), Difco folic acid casei medium, and Lactobacilli Broth AOAC were from Becton, Dickinson, and Company (Sparks, MD). All other chemicals were from Sigma Chemical. Bacteria. Lyophilized cultures of Lactobacillus viridescens (ATCC 12706) and Lactobacillus rhamnosus (ATCC 7469) were obtained from the American Type Culture Collection (Manassas, VA). Stock cultures of L. viridescens were prepared by stab inoculation on Lactobacilli Broth supplemented with agar, and kept in duplicate in the refrigerator. Transfers were made at monthly intervals. Glycerol-cryoprotected cells of L. rhamnosus were prepared as described previously.19
Thiamin and Folate Extraction. Potato samples (either fresh or dry) were blended into fine powder in a Waring blender. For samples harvested in 2009, 0.5 g of fresh powdered material was used for extraction. For samples harvested in 2010, 0.1 g of dried powdered material was used for extraction. Thiamin was extracted by combining acid digestion and enzymatic hydrolysis as previously described.13 Potato samples were added to 10 mL of 0.1 N HCl and autoclaved for 15 min at 121 °C. After cooling at room temperature, pH was adjusted to 4.5 by adding 1 mL of 2 N Na-acetate. Two milliliters of taka-diastase (5 mg mL 1 in 0.2 N Na-acetate pH 4.5) was added to the samples. After 18 h at 37 °C, samples were transferred to 15 mL Falcon tubes, placed on ice, and centrifuged in a swinging bucket rotor at 2700g for 5 min at 4 °C to pellet debris. The supernatants were transferred to new Falcon tubes and volumes were adjusted to 13 mL with 0.2 N Na-acetate pH 4.5. Folates were extracted by a trienzyme treatment.14 Potato samples were homogenized in 15 mL Eppendorf tubes containing 10 mL of extraction buffer (50 mM HEPES/50 mM CHES, pH 7.85, containing 2% (w/v) sodium ascorbate and 10 mM 2-mercaptoethanol, deoxygenated by flushing with nitrogen), boiled for 10 min, and cooled immediately in ice. The homogenate was treated with protease (g14 units) for 2 h at 37 °C, boiled for 5 min, and cooled immediately in ice. The sample was then treated with α-amylase (g800 units) and rat plasma conjugase in large excess (0.5 mL) for 3 h at 37 °C, boiled for 5 min, and cooled immediately in ice. After centrifugation for 10 min at 3000g, the supernatant was transferred to a new tube. The residue was resuspended in 5 mL of extraction buffer, and recentrifuged for 10 min. The combined supernatants were adjusted to a 20 mL final volume with extraction buffer, flushed with nitrogen, frozen in liquid nitrogen, and stored at 80 °C until analysis. Microbiological Assay. Thiamin and folate concentrations were measured by using a microbiological assay employing L. viridescens or L. rhamnosus, respectively, and procedures were as described previously.13,14 Assays were performed on 96-well plates (Falcon microtiter plates). Wells contained growth medium supplemented with thiamin or folate standards, or potato extracts. After ∼18 h incubation at 30 or 37 °C for thiamin or folate assays, respectively, bacterial growth was measured at 630 nm on a BioTek Instrument EL 311 SX microplate autoreader (BioTek Instrument, Winooski, VT) and analyzed with the KCJr EIA application software. Results were calculated by reference to a standard curve using thiamin or 5-formyl-THF and were expressed as nanograms of thiamin or folic acid per gram of sample. For primitive cultivars harvested in 2010, data were converted from dry to fresh weight basis to allow comparison with 2009 data. Statistical Analysis. Student's t test was used to compare thiamin and folate concentrations in potato clones between 2009 and 2010. 13074
dx.doi.org/10.1021/jf203736e |J. Agric. Food Chem. 2011, 59, 13072–13080
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Table 2. Folate Concentrations (Mean ( SE) in Primitive and Modern Potato Varieties concn (ng g 1) 2010a
2009 PI no./variety
clone
species
FW
FW
DW
Russet Burbank
Russet Burbank
tuberosum
128 ( 4
152 ( 10*
651 ( 42
Russet Norkotah
Russet Norkotah
tuberosum
158 ( 19
189 ( 16
843 ( 70
Yukon Gold
Yukon Gold
tuberosum
72 ( 20
209421
RN 057.08
andigena
248 ( 12
243 ( 24
1272 ( 124
217448
RN 061.13
andigena
102 ( 10
88 ( 5
440 ( 27
225667
RN 004.06
andigena
72 ( 9
225667
RN 004.12
andigena
140 ( 5
81 ( 11**
452 ( 63
225673 225677
RN 005.05 RN 008.12
andigena andigena
65 ( 14 117 ( 21
136 ( 10** 123 ( 4
682 ( 51c 516 ( 17
225686
RN 010.06
andigena
91 ( 8
80 ( 5
473 ( 28
225688
RN 011.03
andigena
123 ( 14
155 ( 21
956 ( 127
225689
RN 012.01
andigena
118 ( 28
225689
RN 012.02
andigena
89 ( 25
238 ( 46*
1213 ( 234
225689
RN 012.05
andigena
73 ( 9
238 ( 50**
1007 ( 212
225689
RN 012.12
andigena
130 ( 14
321 ( 15**
1754 ( 83
225693 225693
RN 013.04 RN 013.09
andigena andigena
15 ( 1 119 ( 30
85 ( 22** 158 ( 53
706 ( 180 1179 ( 395
225694
RN 014.15
andigena
128 ( 14
107 ( 33
225705
RN 017.08
andigena
121 ( 38
184 ( 18
1035 ( 99
225710
RN 018.03
andigena
108 ( 16
329 ( 32**
2098 ( 203
234007
RN 047.05
andigena
200 ( 11
83 ( 11
931 ( 428
234015
RN 049.06
andigena
198 ( 8
220 ( 16
1484 ( 108
280868
RN 065.09
andigena
83 ( 35
301 ( 21**
1412 ( 80
280871 280921
RN 067.01 RN 068.08
andigena andigena
271 ( 72 195 ( 32
235 ( 14 264 ( 14
1122 ( 63 1302 ( 68
280952
RN 070.01
andigena
209 ( 16
281080
RN 090.05
andigena
129 ( 6
174 ( 8**
780 ( 38
281080
RN 090.06
andigena
221 ( 13
231 ( 11
1118 ( 52
292110
RN 044.10
andigena
232 ( 21
310490
RN 002.03
andigena
273 ( 12
320355
RN 027.01
andigena
165 ( 19
320370 320370
RN 031.01 RN 031.14
andigena andigena
110 ( 7 79 ( 11
190 ( 17**
973 ( 88
320373
RN 033.01
andigena
125 ( 7
182 ( 14**
763 ( 59
320373
RN 033.02
andigena
164 ( 28
320377
RN 035.01
andigena
165 ( 7
211 ( 8**
1142 ( 42
320377
RN 035.06
andigena
163 ( 8
320377
RN 035.08
andigena
154 ( 11
320379
RN 036.05
andigena
68 ( 10
102 ( 8*
892 ( 70
320379 320379
RN 036.06 RN 036.15
andigena andigena
96 ( 5 103 ( 12
98 ( 36 139 ( 46
487 ( 178 725 ( 241
102 ( 14
1176 ( 164
b
187 ( 23**
99 ( 14*
720 ( 90
668 ( 208
896 ( 127
320387
RN 039.05
andigena
320390
RN 041.06
andigena
61 ( 11
74 ( 1
388 ( 8
320390
RN 041.09
andigena
113 ( 11
228 ( 39*
1157 ( 198
320391
RN 042.04
andigena
105 ( 9
99 ( 7
524 ( 39
320391
RN 042.14
andigena
109 ( 21
320391
RN 042.15
andigena
131 ( 23
147 ( 12
881 ( 74
473260 473260
RN 104.02 RN 104.05
andigena andigena
306 ( 77 148 ( 45
416 ( 23 304 ( 35*
1955 ( 107 1422 ( 166
473260
RN 104.06
andigena
148 ( 16
222 ( 9**
1095 ( 45
13075
dx.doi.org/10.1021/jf203736e |J. Agric. Food Chem. 2011, 59, 13072–13080
Journal of Agricultural and Food Chemistry
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Table 2. Continued concn (ng g 1) 2010a
2009 clone
species
FW
FW
DW
473260 473276
PI no./variety
RN 104.08 RN 105.01
andigena andigena
134 ( 8 180 ( 8
83 ( 17* 153 ( 17
400 ( 82 670 ( 75
473276
RN 105.12
andigena
281 ( 26
187 ( 10**
473276
RN 105.14
andigena
205 ( 14
272 ( 39
1147 ( 163
498171
RN 043.11
andigena
112 ( 33
85 ( 17
403 ( 83
498310
RN 112.08
andigena
165 ( 6
171 ( 13
702 ( 55
498310
RN 112.10
andigena
232 ( 39
876 ( 47
a
Data for 2010 are shown on both fresh weight and dry weight bases. b Significant difference between years P < 0.01 (**) or P < 0.05 (*) as determined by Student's t test. c Dry matter content was not determined for this clone, therefore we assumed 20% dry matter content.
Figure 1. Frequency of primitive and modern cultivars within thiamin concentration brackets. One-way analysis of variance (ANOVA) was carried out to compare the means of thiamin and folate concentrations among clones and among species. All statistical analyses were carried out using Statgraphics Centurion XVI.
’ RESULTS AND DISCUSSION We investigated the genetic variation of thiamin and folate in 54 primitive cultivated potatoes (S. tuberosum group Andigenum) grown in 2009 and 2010 (Tables 1 and 2). There were significant differences in thiamin and folate concentrations among clones from the same harvest as determined by ANOVA (P < 0.01 at the 95% confidence level). Thiamin and folate concentrations ranged from 490 to 2325 ng g 1 FW and from 15 to 416 ng g 1 FW, respectively (or 2908 to 16401 ng g 1 DW for thiamin and 388 to 2098 ng g 1 DW for folate in 2010). The majority of the clones had thiamin and folate concentrations in the ranges of 1200 1600 and 100 200 ng g 1 FW, respectively, in both years (Figures 1 and 2). Common commercial varieties Russet Burbank, Russet Norkotah, and Yukon Gold were among this majority of clones. For 46% of the clones, there were statistically significant differences in thiamin and folate concentrations between years (Tables 1 and 2), suggesting low genetic stability of these clones. Of particular interest are clones which have
Figure 2. Frequency of primitive and modern cultivars within folate concentration brackets.
relatively high thiamin or folate content compared to modern varieties and are stable over the years. The clone RN 035.01 (PI 320377) is a good example: in both years it contained over 13076
dx.doi.org/10.1021/jf203736e |J. Agric. Food Chem. 2011, 59, 13072–13080
Journal of Agricultural and Food Chemistry Table 3. Thiamin and Folate Concentrations (ng g PI no.
species
500047
S. acaule subsp. aemulans
243510
S. bulbocastanum
ARTICLE 1
DW ( SE) in Wild Potato species thiamin 10065 ( 37 4800 ( 273
folate
% dry matter
803 ( 12
19
815 ( 24
23
545751
S. bulbocastanum
4830 ( 304
767 ( 0
21
545964
S. boliviense
5884 ( 189
423 ( 22
30
597736
S. boliviense
4317 ( 209
3031 ( 46
28
265863
S. bukasovii
7146 ( 100
413 ( 56
17
197760
S. chacoense subsp. chacoense
6265 ( 0
347 ( 33
30
275139 320293
S. chacoense subsp. chacoense S. chacoense subsp. chacoense
4318 ( 328 5820 ( 0
207 ( 22
23 29
472837
S. commersonii subsp. commersonii
6621 ( 32
570 ( 12
24
473411
S. commersonii subsp. commersonii
5415 ( 342
260 ( 11
34
558050
S. commersonii subsp. commersonii
6784 ( 65
498232
S. demissum
7113 ( 133
457 ( 34
26 22
34
265579
S. gourlayi subsp. gourlayi
7858 ( 546
962 ( 25
473011
S. gourlayi subsp. gourlayi
8093 ( 34
864 ( 25
20
473062 251065
S. gourlayi subsp. gourlayi S. hjertingii
6784 ( 65 4710 ( 363
593 ( 35 780 ( 36
21 29
283103
S. hjertingii
5043 ( 153
1090 ( 103
26
545715
S. hjertingii
6653 ( 65
1179 ( 25
26
265867
S. infundibuliforme
6787 ( 392
614 ( 25
23
458324
S. infundibuliforme
5105 ( 277
520 ( 40
23
472894
S. infundibuliforme
6010 ( 0
1273 ( 0
23
275262
S. jamesii
6365 ( 418
1199 ( 25
42
458425 592422
S. jamesii S. jamesii
7717 ( 135 6300 ( 353
1085 ( 30 644 ( 45
33 40 23
472923
S. kurtzianum
4049 ( 177
505 ( 25
472941
S. kurtzianum
3754 ( 117
673 ( 25
23
498359
S. kurtzianum
4798 ( 91
485 ( 5
26
500041
S. microdontum
5199 ( 370
1704 ( 5
22
265873
S. megistacrolobum
5243 ( 31
817 ( 0
11
473133
S. megistacrolobum
5855 ( 0
648 ( 40
16
498383 498130
S. megistacrolobum S. okadae
5118 ( 348 5953 ( 32
564 ( 5 1015 ( 30
20 21
1099 ( 5
26
435079
S. oplocense
5467 ( 128
473185
S. oplocense
3727 ( 128
881 ( 84
29
473190
S. oplocense
2896 ( 58
527 ( 14
23
184774
S. pinnatisectum
6055 ( 460
986 ( 65
40
275236
S. pinnatisectum
4397 ( 61
513 ( 13
42
347766
S. pinnatisectum
7193 ( 0
865 ( 42
37
310953 473369
S. raphanifolium S. raphanifolium
6618 ( 369 7332 ( 207
494 ( 7 520 ( 33
27 28 22
473370
S. raphanifolium
6282 ( 33
686 ( 0
230464
S. sanctae-rosae
5470 ( 385
475 ( 52
25
205407
S. spegazzinii
5188 ( 667
417 ( 20
28
472978
S. spegazzinii
5563 ( 32
406 ( 10
22
500053
S. spegazzinii
6417 ( 366
1054 ( 16
18
473504
S. sparsipilum
5725 ( 130
503 ( 4
30
545912 597687
S. sparsipilum S. sparsipilum
5563 ( 32 6418 ( 366
853 ( 13 1207 ( 35
31 20
636406
S. stoloniferuma
6583 ( 201
1312 ( 14
29
641031
S. stoloniferuma
5953 ( 164
789 ( 13
24
643994
S. stoloniferuma
4520 ( 62
597 ( 18
26
558447
S. stoloniferumb
5520 ( 66
507 ( 12
27
558451
S. stoloniferumb
5126 ( 65
801 ( 0
26
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Table 3. Continued PI no.
a
species b
thiamin
folate
% dry matter
4640 ( 96 4544 ( 64
795 ( 6 950 ( 59
24 27
1143 ( 41
27
586947 275228
S. stoloniferum S. stoloniferumc
498033
S. stoloniferumc
6324 ( 0
473243
S. berthaultii
5493 ( 628
614 ( 37
22
473336
S. berthaultii
4834 ( 161
1160 ( 74
20
161173
S. verrucosum
7259 ( 35
666 ( 15
27
275255
S. verrucosum
6602 ( 412
577 ( 30
28
498062
S. verrucosum
6771 ( 173
674 ( 53
26
230468 500062
S. vernei subsp. vernei S. vernei subsp. vernei
8633 ( 111 9853 ( 351
1567 ( 8 1665 ( 54
18 19
Previously “fendleri”. b Previously “polytrichon”. c Previously “papita”.
1.7 times more thiamin on a fresh weight basis than Russet Burbank, a commonly grown russet variety, and over 2.3 times more thiamin than Russet Burbank on a dry weight basis. Because potato clones had dry matter content varying from 9 to 26%, comparing metabolite contents among clones on a dry weight basis is a better indication of the quantitative variation present among clones for these vitamins. For instance, although the clone RN 035.08 (PI 320377) had low thiamin content relative to Russet Burbank on a fresh weight basis in 2010, it had ∼1.8 more thiamin on a dry weight basis. The clone RN 035.06 (PI 320377) contained 2.2 times more thiamin than Russet Burbank on a fresh weight basis. Unfortunately, this clone did not produce tubers in 2010 and we were not able to determine whether this clone had high thiamin content over multiple years. Nevertheless, these clones had higher thiamin concentrations than any of those reported in modern potato varieties,13 and although data from additional years would be necessary to fully assess the stability of these clones, this accession (PI 320377) seems to be a promising material to integrate in breeding programs for thiamin enhancement. There were other interesting clones, such as RN 039.05 (PI 320387) which had the highest thiamin concentrations, containing three times more thiamin than Russet Burbank. The clone RN 104.02 (PI 473260) had the highest folate content with 2.4 and 2.7 times more folate than Russet Burbank in 2009 and 2010, respectively, on a fresh weight basis, and 3 times more on a dry weight basis. There was no statistically significant difference in mean folate concentrations in RN 104.02 between the two years, suggesting that this clone may be stable for folate content. Segregating clones from the same accession also had relatively high folate concentrations in 2010, except for RN 104.08, but had much lower amounts in 2009 on a fresh weight basis. Therefore, the clone RN 104.02 could be useful for folate enhancement, and caution should be taken in selecting appropriate clones among this accession. We extended our screening to 25 wild potato species comprising 64 different accessions (Table 3). There were significant differences in thiamin and folate concentrations among clones and among species as determined by ANOVA (P < 0.01 at the 95% confidence level). S. vernei subsp. vernei and S. acaule subsp. aemulans had the highest thiamin concentrations and were significantly different from all other species (Fisher’s LSD at the 95% confidence level). S. boliviense accession 597736 had the highest folate concentrations with 3031 ng g 1 DW, exceeding any folate concentrations that we have so far found in potato,20 but the other S. boliviense accession had a much lower amount, suggesting variability within species. Interestingly, in
addition to being relatively high for thiamin, S. vernei subsp. vernei also had relatively high folate concentrations. Overall, thiamin concentrations ranged from 2896 to 10065 ng g 1 DW, while folate concentrations ranged from 207 to 3031 ng g 1 DW. Although multiple year or location data will be necessary to determine the genetic stability of these clones, folate concentrations reported here in four accessions (PI 243510, 472923, 184774, and 205407) were very close to those we previously reported for these same accessions grown under different environmental conditions,20 suggesting that genetically stable clones could be identified. This study shows that systematic screening of indigenous landraces and wild potato species is a valuable approach in order to extend the limits of thiamin and folate concentrations beyond those found in modern cultivars. We identified clones which contained over 2-fold the amount of thiamin and 4-fold the amount of folate in Russet Burbank, a potato variety largely grown in North America. Based on thiamin and folate concentrations found in RN 039.05 (PI 320387) and S. boliviense (PI 597736) (16401 and 3031 ng g 1 DW, respectively), one can estimate that introgression of quantitative trait loci associated with the high thiamin or folate phenotypes into modern potato varieties would give thiamin and folate concentrations of 3280 and 606 ng g 1 FW, respectively, assuming a 20% dry matter content (which is the average dry matter content found in mature tubers of most modern varieties). This means that such cooked high-thiamin potatoes (assuming an 86% retention of thiamin after oven-baking, the most destructive cooking method reported for thiamin13) would provide 56, 38, and 15% of the recommended daily allowance for thiamin (1.2 mg for healthy male adults) in Europe, North America, and Asia, respectively, where per capita consumption of potato in 2005 was 87.8, 60.0, and 23.9 kg, respectively (FAOSTAT). Similarly, such cooked highfolate potatoes (assuming an average 80% retention of folate after cooking21) would provide 28.8, 20.0, and 7.9% of the recommended daily allowance for folate (400 μg for healthy male adults) in Europe, North America, and Asia, respectively. By comparison, a baked russet potato commonly consumed in the U.S. provides 9.2% and 11.0% of the recommended daily allowance of thiamin and folate, respectively, according to the USDA Nutrient Database SR23, based on a 164 g daily consumption (North American average in 2005). Therefore, these clones should be useful in breeding programs aiming to enhance the nutritional value of potato. The development of new potato varieties with increased thiamin and folate concentrations will likely benefit the health status of consumers who do not or cannot follow dietary guidelines. 13078
dx.doi.org/10.1021/jf203736e |J. Agric. Food Chem. 2011, 59, 13072–13080
Journal of Agricultural and Food Chemistry Future studies should focus on screening more populations within promising species to identify clones with potentially even higher thiamin or folate contents than those reported here. Natural genetic diversity has been successfully used to explore metabolism regulation,22 26 and the materials reported in this paper could be useful tools for such investigations. Unveiling the regulation of these vitamins for which very little is currently understood6 will certainly provide useful information to biofortifying food crops. Also, additional replications will be necessary to determine the genetic stability of these clones, as large differences in thiamin or folate contents between years were sometimes noted. These materials might be great tools for study of, and eventual management of, the environment stimuli that increase thiamin and folate.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 541 567 8321. Fax: 541 567 2240. E-mail: aymeric.
[email protected]. Funding Sources
This work was supported by funding from the Agricultural Research Foundation and the Potato Commissions of Oregon, Washington, and Idaho.
’ ACKNOWLEDGMENT The authors would like to thank Dr. John Bamberg from the U.S. Potato Genebank for selecting and providing potato tubers, and Dr. Kathleen Haynes for critical reading of the manuscript. ’ ABBREVIATIONS USED FW, fresh weight; DW, dry weight ’ REFERENCES (1) Christian, P. Impact of the Economic Crisis and Increase in Food Prices on Child Mortality: Exploring Nutritional Pathways. J. Nutr. 2010, 140, 177S–181S. (2) Bouis, H. E. Plant breeding: a new tool for fighting micronutrient malnutrition. J. Nutr. 2002, 132, 491S–494S. (3) Backstrand, J. R. The history and future of food fortification in the United States: a public health perspective. Nutr. Rev. 2002, 60, 15–26. (4) Harper, C. Thiamine (vitamin B1) deficiency and associated brain damage is still common throughout the world and prevention is simple and safe!. Eur. J. Neurol. 2006, 13, 1078–82. (5) Suskind, D. L. Nutritional Deficiencies During Normal Growth. Pediatr. Clin. N. Am. 2009, 56, 1035–1053. (6) Hanson, A. D.; Gregory, J. F. Folate Biosynthesis, Turnover, and Transport in Plants. In Annual Review of Plant Biology; Merchant, S. S., Briggs, W. R., Ort, D., Eds.; Annual Reviews: Palo Alto, 2011; Vol. 62, pp 105 125. (7) Storozhenko, S.; Rabanel, S.; Zhang, G. F.; Rebeille, F.; Lambert, W.; Van Der Straeten, D. Folate enhancement in staple crops by metabolic engineering. Trends Food Sci. Technol. 2005, 16, 271–281. (8) Bailey, L. B.; Rampersaud, G. C.; Kauwell, G. P. Folic acid supplements and fortification affect the risk for neural tube defects, vascular disease and cancer: evolving science. J. Nutr. 2003, 133, 1961S– 1968S. (9) Ramos, M. I.; Allen, L. H.; Mungas, D. M.; Jagust, W. J.; Haan, M. N.; Green, R.; Miller, J. W. Low folate status is associated with impaired cognitive function and dementia in the Sacramento Area Latino Study on Aging. Am. J. Clin. Nutr. 2005, 82, 1346–1352.
ARTICLE
(10) Xu, X.; Pan, S. K.; Cheng, S. F.; Zhang, B.; Mu, D. S.; Ni, P. X.; Zhang, G. Y.; Yang, S.; Li, R. Q.; Wang, J.; Orjeda, G.; Guzman, F.; Torres, M.; Lozano, R.; Ponce, O.; Martinez, D.; De la Cruz, G.; Chakrabarti, S. K.; Patil, V. U.; Skryabin, K. G.; Kuznetsov, B. B.; Ravin, N. V.; Kolganova, T. V.; Beletsky, A. V.; Mardanov, A. V.; Di Genova, A.; Bolser, D. M.; Martin, D. M. A.; Li, G. C.; Yang, Y.; Kuang, H. H.; Hu, Q.; Xiong, X. Y.; Bishop, G. J.; Sagredo, B.; Mejia, N.; Zagorski, W.; Gromadka, R.; Gawor, J.; Szczesny, P.; Huang, S. W.; Zhang, Z. H.; Liang, C. B.; He, J.; Li, Y.; He, Y.; Xu, J. F.; Zhang, Y. J.; Xie, B. Y.; Du, Y. C.; Qu, D. Y.; Bonierbale, M.; Ghislain, M.; Herrera, M. D.; Giuliano, G.; Pietrella, M.; Perrotta, G.; Facella, P.; O’Brien, K.; Feingold, S. E.; Barreiro, L. E.; Massa, G. A.; Diambra, L.; Whitty, B. R.; Vaillancourt, B.; Lin, H. N.; Massa, A.; Geoffroy, M.; Lundback, S.; DellaPenna, D.; Buell, C. R.; Sharma, S. K.; Marshall, D. F.; Waugh, R.; Bryan, G. J.; Destefanis, M.; Nagy, I.; Milbourne, D.; Thomson, S. J.; Fiers, M.; Jacobs, J. M. E.; Nielsen, K. L.; Sonderkaer, M.; Iovene, M.; Torres, G. A.; Jiang, J. M.; Veilleux, R. E.; Bachem, C. W. B.; de Boer, J.; Borm, T.; Kloosterman, B.; van Eck, H.; Datema, E.; Hekkert, B. T. L.; Goverse, A.; van Ham, R.; Visser, R. G. F. Potato Genome Sequencing, C. Genome sequence and analysis of the tuber crop potato. Nature 2011, 475, 189–U94. (11) Dobson, G.; Shepherd, T.; Verrall, S. R.; Conner, S.; McNicol, J. W.; Ramsay, G.; Shepherd, L. V. T.; Davies, H. V.; Stewart, D. Phytochemical Diversity in Tubers of Potato Cultivars and Landraces Using a GC-MS Metabolomics Approach. J. Agric. Food Chem. 2008, 56, 10280–10291. (12) Dobson, G.; Shepherd, T.; Verrall, S. R.; Griffiths, W. D.; Ramsay, G.; McNicol, J. W.; Davies, H. V.; Stewart, D. A Metabolomics Study of Cultivated Potato (Solanum tuberosum) Groups Andigena, Phureja, Stenotomum, and Tuberosum Using Gas ChromatographyMass Spectrometry. J. Agric. Food Chem. 2010, 58, 1214–1223. (13) Goyer, A.; Haynes, K. G. Vitamin B1 content in potato: Effect of genotype, tuber enlargement, and storage, and estimation of stability and broad-sense heritability. Am. J. Potato Res 2011, 88, 374–385. (14) Goyer, A.; Navarre, D. A. Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay. J. Agric. Food Chem. 2007, 55, 3523–3528. (15) Ottoman, R. J.; Hane, D. C.; Brown, C. R.; Yilma, S.; James, S. R.; Mosley, A. R.; Crosslin, J. M.; Vales, M. I. Validation and Implementation of Marker-Assisted Selection (MAS) for PVY Resistance (Rygene) in a Tetraploid Potato Breeding Program. Am. J. Potato Res. 2009, 86, 304–314. (16) Spooner, D. M.; Nunez, J.; Trujillo, G.; Herrera, M. D.; Guzman, F.; Ghislain, M. Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19398–19403. (17) Ovchinnikova, A.; Krylova, E.; Gavrilenko, T.; Smekalova, T.; Zhuk, M.; Knapp, S.; Spooner, D. M. Taxonomy of cultivated potatoes (Solanum section Petota: Solanaceae). Bot. J. Linn. Soc 2011, 165, 107–155. (18) Pfeiffer, C. M.; Rogers, L. M.; Gregory, J. F., III. Determination of folate in cereal-grain food products using trienzyme extraction and combined affinity and reversed-phase liquid chromatography. J. Agric. Food Chem 1997, 45, 407–413. (19) Horne, D. W.; Patterson, D. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin. Chem. 1988, 34, 2357–9. (20) Goyer, A.; Navarre, D. A. Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay. J. Agric. Food Chem. 2007, 55, 3523–8. (21) Navarre, D. A.; Goyer, A.; Shakya, R. Nutritional value of potatoes: Vitamin, Phytonutrient and Mineral Content. In Advances in potato chemistry and technology; Singh, J., Kaur, L., Eds.; Elsevier Inc.: 2009; pp 395 424. (22) Fernie, A.; Klee, H. The use of natural genetic diversity in the understanding of metabolic organization and regulation. Front. Plant Physiol. 2011, 2, 59. (23) Alonso-Blanco, C.; Aarts, M. G. M.; Bentsink, L.; Keurentjes, J. J. B.; Reymond, M.; Vreugdenhil, D.; Koornneef, M. What Has Natural 13079
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ARTICLE
Variation Taught Us about Plant Development, Physiology, and Adaptation? Plant Cell 2009, 21, 1877–1896. (24) Bentsink, L.; Yuan, K.; Koornneef, M.; Vreugdenhil, D. The genetics of phytate and phosphate accumulation in seeds and leaves of Arabidopsis thaliana, using natural variation. Theor. Appl. Genet. 2003, 106, 1234–1243. (25) Gilliland, L. U.; Magallanes-Lundback, M.; Hemming, C.; Supplee, A.; Koornneef, M.; Bentsink, L.; Dellapenna, D. Genetic basis for natural variation in seed vitamin E levels in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18834–18841. (26) Calenge, F.; Saliba-Colombani, V.; Mahieu, S.; Loudet, O.; Daniel-Vedele, F.; Krapp, A. Natural variation for carbohydrate content in Arabidopsis. Interaction with complex traits dissected by quantitative genetics. Plant Physiol. 2006, 141, 1630–1643.
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