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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (3): 330-340.doi: 10.3724/SP.J.1006.2020.94077

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Assessment of genetic diversity by using EST-SSR markers in Lupinus

Hong-Yan ZHANG1,5,*,Tao YANG1,*,Rong LIU1,*,Fang JIN2,Li-Ke ZHANG2,Hai-Tian YU3,Jin-Guo HU4,Feng YANG3,Dong WANG1,Yu-Hua HE3,*(),Xu-Xiao ZONG1,*()   

  1. 1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / The National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China
    2. National Agro-Tech Extension & Service Centre, Beijing 100125, China;
    3. Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Kunming 650205, Yunnan, China
    4. USDA-ARS Western Regional Plant Introduction Station (WRPIS), Pullman, WA 99164, USA
  • Received:2019-05-21 Accepted:2019-09-26 Online:2020-03-12 Published:2019-11-11
  • Contact: Hong-Yan ZHANG,Tao YANG,Rong LIU,Yu-Hua HE,Xu-Xiao ZONG E-mail:trbio@163.com;zongxuxiao@caas.cn
  • Supported by:
    This study was supported by the National Key Research and Development Program of China (2017YFE0105100);the Crop Germplasm Resources Protection(2019NWB036-07);the Agricultural Science and Technology Innovation Program (ASTIP) in CAAS, and the Fundamental Research Funds for Central Non-Profit of Institute of Crop Sciences, CAAS.(S2018XC04)

Abstract:

In order to explore the evolutionary relationship of Lupinus preliminarily as well as to excavate and utilize lupin resources from the “Old World” effectively, the genetic diversity among the species under Lupinus genus was analyzed. Ninety-five polymorphic pairs of EST-SSR markers developed based on the transcriptome of narrow-leaved lupin (Lupinus angustifolius L.) were used to scan 133 lupin accessions from 22 species. A total of 1318 alleles were detected with 13.87 alleles per locus on average, ranging from 3 to 37 alleles; the polymorphism information content (PIC) ranged from 0.39 to 0.91 with the mean value of 0.63; the genetic diversity ranged from 0.41 to 0.92 with the mean value of 0.78. This study showed evolutionary relations among the 22 species under Lupinus genus from the “Old World” and the “New World” based on Neighbor-Joining (NJ) method, which is consistent with previous studies. Moreover, seventy-seven lupin accessions of seven Lupinus species from the “Old World” were divided into 4 groups; there was no overlap of accession from different species contained in each identified group, detected by all the three analysis methods like population structure, cluster analysis based on UPGMA and principal component analysis (PCA).

Key words: EST-SSR, Lupinus genus, transcriptome, genetic diversity

Fig. 1

Geographic distribution of lupin accessions from different continents"

Fig. 2

Distribution of 95 pairs of EST-SSR on the chromosome of narrow-leafed lupine (Lupinus angustifolius)"

Table 1

Genetic parameters of 95 pairs of polymorphic markers"

标记
Marker
等位基因
No. of allele
基因多样性
Gene diversity
PIC 标记
Marker
等位基因
No. of allele
基因多样性
Gene diversity
PIC
lup-est4467 22.00 0.88 0.87 lup-est6567 31.00 0.91 0.91
lup-est4113 17.00 0.80 0.78 lup-est2194 14.00 0.80 0.77
lup-est3140 14.00 0.73 0.68 lup-est4304 25.00 0.89 0.88
lup-est4349 10.00 0.65 0.62 lup-est3549 10.00 0.68 0.63
lup-est6531 7.00 0.42 0.39 lup-est2949 15.00 0.89 0.88
lup-est5958 11.00 0.73 0.69 lup-est2185 13.00 0.77 0.74
lup-est2267 17.00 0.88 0.86 lup-est3185 14.00 0.85 0.84
lup-est2076 16.00 0.82 0.80 lup-est2385 14.00 0.79 0.76
lup-est6513 16.00 0.85 0.84 lup-est3894 14.00 0.83 0.82
lup-est6558 19.00 0.91 0.90 lup-est1449 14.00 0.80 0.78
lup-est6258 16.00 0.86 0.85 lup-est2140 13.00 0.87 0.85
lup-est6104 17.00 0.71 0.69 lup-est3458 10.00 0.68 0.64
lup-est6049 11.00 0.73 0.69 lup-est6158 13.00 0.79 0.77
lup-est1985 12.00 0.76 0.73 lup-est2822 12.00 0.80 0.77
lup-est2531 30.00 0.89 0.88 lup-est5340 9.00 0.77 0.74
lup-est976 18.00 0.82 0.80 lup-est5440 12.00 0.73 0.69
lup-est2013 14.00 0.76 0.74 lup-est4549 18.00 0.87 0.86
lup-est840 7.00 0.73 0.68 lup-est5513 10.00 0.77 0.73
lup-est1640 12.00 0.76 0.73 lup-est1149 8.00 0.82 0.79
lup-est931 18.00 0.88 0.87 lup-est4576 7.00 0.70 0.65
lup-est1749 21.00 0.89 0.88 lup-est2694 18.00 0.87 0.86
lup-est2558 16.00 0.85 0.84 lup-est3494 12.00 0.85 0.83
lup-est458 9.00 0.72 0.67 lup-est158 7.00 0.79 0.75
lup-est4640 16.00 0.88 0.87 lup-est5049 10.00 0.80 0.77
lup-est5985 11.00 0.81 0.79 lup-est1058 8.00 0.68 0.64
lup-est2849 22.00 0.84 0.82 lup-est3994 21.00 0.91 0.90
lup-est3649 37.00 0.91 0.90 lup-est1594 12.00 0.80 0.78
lup-est2340 23.00 0.92 0.91 lup-est4322 16.00 0.85 0.84
lup-est4522 9.00 0.66 0.63 lup-est4185 12.00 0.83 0.81
lup-est5240 13.00 0.83 0.81 lup-est5749 7.00 0.70 0.64
lup-est2567 7.00 0.41 0.39 lup-est5667 9.00 0.64 0.60
lup-est58 10.00 0.73 0.69 lup-est3067 21.00 0.88 0.87
lup-est4458 12.00 0.86 0.84 lup-est1649 12.00 0.66 0.63
lup-est4131 9.00 0.72 0.67 lup-est5622 9.00 0.75 0.71
lup-est5740 13.00 0.63 0.58 lup-est3576 5.00 0.71 0.66
lup-est164 8.00 0.63 0.59 lup-est6213 9.00 0.78 0.75
lup-est6031 14.00 0.81 0.79 lup-est2358 11.00 0.88 0.87
lup-est922 13.00 0.82 0.80 lup-est2540 11.00 0.79 0.76
lup-est2313 20.00 0.84 0.83 lup-est4249 10.00 0.53 0.49
lup-est249 17.00 0.77 0.75 lup-est4949 13.00 0.67 0.61
lup-est2704 13.00 0.44 0.43 lup-est4158 14.00 0.80 0.77
lup-est1085 13.00 0.80 0.78 lup-est822 14.00 0.86 0.84
lup-est1622 19.00 0.81 0.79 lup-est3340 10.00 0.81 0.79
lup-est6504 9.00 0.67 0.61 lup-est1313 17.00 0.81 0.80
lup-est613 12.00 0.77 0.75 lup-est2404 12.00 0.80 0.78
lup-est449 12.00 0.69 0.66 lup-est713 25.00 0.80 0.77
lup-est4022 7.00 0.72 0.67 lup-est5967 13.00 0.80 0.78
lup-est5831 15.00 0.84 0.82 平均Mean 13.87 0.78 0.75

Fig. 3

Phylogenetic tree of 22 lupin species based on Nei’s genetic distances"

Fig. 4

UPGMA tree of the 7 lupin species from the “Old World” based on Nei’s genetic distances"

Table 2

Genetic identity and genetic distance among seven lupin species from the “Old World”"

物种名称
Species ID
L. atlanticus L. cosentinii
(沙质平原羽扇豆)
L. albus
(白羽扇豆)
L. angustifolius
(窄叶羽扇豆)
L. hispanicus L. luteus
(黄羽扇豆)
L. pilosus
L. atlanticus 0.663 0.235 0.167 0.187 0.197 0.561
L. cosentinii
(沙质平原羽扇豆)
0.411 0.199 0.158 0.187 0.167 0.556
L. albus
(白羽扇豆)
1.45 1.613 0.244 0.205 0.263 0.254
L. angustifolius
(窄叶羽扇豆)
1.789 1.845 1.412 0.196 0.254 0.223
L. hispanicus 1.676 1.679 1.586 1.628 0.487 0.213
L. luteus
(黄羽扇豆)
1.625 1.792 1.336 1.372 0.719 0.218
L. pilosus 0.578 0.586 1.369 1.502 1.547 1.524

Fig. 5

Genetic diversity of the 77 lupin accessions from the “Old World” (a) the scatter plot of mean ln P(D) from 1 to 10; (b) ln (ΔK) values plotted from 1 to 10; (c) structure of the 77 accessions based on Structure software when k = 4; (d) NJ tree of the 77 accessions based on Nei’s genetic distances; (e) principal component analysis of the 77 accessions based on 95 EST-SSR markers."

Table 3

Analysis of genetic differentiation among seven Lupinus species by AMOVA"

变异来源
Source
自由度
df
方差
SS
均方差
MS
Est. Var. % P-value
组群内Within pops 6 6584.338 1097.39 108.0748 80.00 0.01
组群间Among pops 70 1852.636 26.466 26.466 20.00 0.01

Table 4

Analysis of genetic differentiation (Fst) among seven lupin species"

物种名称
Species ID
L. atlanticus L. cosentinii
(沙质平原羽扇豆)
L. albus
(白羽扇豆)
L. angustifolius
(窄叶羽扇豆)
L. hispanicus L. luteus
(黄羽扇豆)
L. pilosus
L. atlanticus 0
L. cosentinii
(沙质平原羽扇豆)
0.475 0
L. albus
(白羽扇豆)
0.617 0.661 0
L. angustifolius
(窄叶羽扇豆)
0.614 0.639 0.546 0
L. hispanicus 0.688 0.714 0.636 0.602 0
L. luteus
(黄羽扇豆)
0.707 0.743 0.635 0.597 0.573 0
L. pilosus 0.320 0.341 0.470 0.476 0.535 0.531 0

Supplement table 1

Origin information of 133 lupin accessions used in this study"

Accession Species Continents Longitude (E) Latitude (N)
PI 284712a,c L. arbustus North America -121.45 38.57
PI 284714a L. arbustus North America -121.45 38.57
PI 232569a,c L. argenteus North America -77.03 38.88
PI 504372a L. argenteus North America -111.87 40.77
PI 504374a L. argenteus North America -123.03 44.93
PI 577284a L. argenteus North America -116.22 43.62
PI 477930a L. argenteus North America -123.03 44.93
PI 477931a L. argenteus North America -123.03 44.93
PI 477936a L. argenteus North America -123.03 44.93
PI 504330a L. argenteus North America -77.03 38.88
PI 384612a,b,c L. atlanticus Africa -6.85 34.03
PI 384613a,b L. atlanticus Africa -6.85 34.03
W6 17965a,b L. atlanticus Africa -6.85 34.03
PI 284715a,c L. concinnus North America -121.45 38.57
PI 284716a L. concinnus North America -121.45 38.57
PI 660715a,b,c L. cosentinii Oceania 149.13 -35.30
W6 17967a,b L. cosentinii Europe -9.08 38.70
PI 185099a,c L. elegans North America -99.15 19.47
W6 15613a L. elegans North America -121.45 38.57
PI 344647a,c L. garfieldensis North America -77.03 38.88
PI 344648a L. garfieldensis North America -77.03 38.88
PI 477932a,c L. lepidus North America -123.03 44.93
PI 477934a L. lepidus North America -123.03 44.93
PI 504325a L. lepidus North America -123.03 44.93
PI 504391a,c L. leucophyllus North America -77.03 38.88
PI 504397a L. leucophyllus North America -123.03 44.93
PI 504398a L. leucophyllus North America -123.03 44.93
PI 504399a L. leucophyllus North America -123.03 44.93
PI 504400a L. leucophyllus North America -123.03 44.93
PI 602371a,c L. mexicanus Europe 19.25 47.43
PI 615395a L. mexicanus North America -121.45 38.57
PI 615398a L. mexicanus North America -121.45 38.57
PI 615399a L. mexicanus North America -121.45 38.57
W6 14749a L. mexicanus Europe 19.25 47.43
PI 602373a,c L. microcarpus North America -121.45 38.57
PI 602376a L. microcarpus North America -121.45 38.57
PI 344664a,c L. polyphyllus North America -116.22 43.62
PI 442515a L. polyphyllus Europe 4.35 50.85
PI 477122a L. polyphyllus Europe 13.03 52.52
PI 504411a L. polyphyllus North America -116.22 43.62
PI 504412a L. polyphyllus North America -77.03 38.88
PI 504413a L. polyphyllus North America -77.03 38.88
PI 504414a L. polyphyllus North America -123.03 44.93
PI 452129a,c L. rivularis North America -123.03 44.93
PI 660700a L. rivularis North America -123.03 44.93
PI 660701a L. rivularis North America -123.03 44.93
PI 660720a L. rivularis North America -123.03 44.93
PI 284726a L. stiversii North America -121.45 38.57
PI 602374a,c L. stiversii North America -121.45 38.57
PI 284728a,c L. succulentus North America -121.45 38.57
PI 577290a L. succulentus North America -77.03 38.88
PI 615405a,b,c L. albus North America -92.32 34.73
PI 615406a,b L. albus North America -92.32 34.73
PI 615409b L. albus North America -84.42 33.77
PI 660685b L. albus Europe -6.97 38.88
PI 660711b L. albus Europe -16.23 28.45
PI 660712b L. albus Africa 36.85 7.38
PI 660713b L. albus Asia 36.32 33.05
PI 660714b L. albus Africa 31.28 30.00
PI 660721a,b L. albus Europe 2.03 48.85
PI 660722b L. albus Europe 2.03 48.85
PI 660723b L. albus North America -86.03 32.37
PI 660724b L. albus Asia 35.93 31.95
PI 660726a,b L. albus Oceania 149.13 -35.30
PI 666380b L. albus Europe -4.03 39.86
PI 666381b L. albus Europe -6.94 37.29
PI 666382b L. albus Europe -4.03 39.86
PI 666385b L. albus Oceania 149.13 -35.30
PI 666386b L. albus Africa 3.22 36.70
W6 15551b L. albus Europe 2.03 48.85
W6 19548a,b L. albus South America -70.07 -33.43
W6 39795a,b L. albus Europe 13.03 52.52
W6 39796a,b L. albus Europe 13.03 52.52
W6 39797b L. albus Europe 13.03 52.52
W6 39798b L. albus Europe 13.03 52.52
W6 39800a,b L. albus Europe 13.03 52.52
W6 39801a,b L. albus Europe -9.08 38.70
W6 39802a,b L. albus Asia 35.22 31.78
PI 168527b,c L. angustifolius Europe -9.08 38.70
PI 180708b L. angustifolius Europe 13.03 52.52
PI 208682b L. angustifolius Africa 3.22 36.70
PI 255472b L. angustifolius Europe 20.47 44.82
PI 274821a,b L. angustifolius Europe 21.00 52.25
PI 274811a,b L. angustifolius Europe 21.00 52.25
PI 274812b L. angustifolius Europe 21.00 52.25
PI 274813b L. angustifolius Europe 21.00 52.25
PI 274814a,b L. angustifolius Europe 21.00 52.25
PI 274815a,b L. angustifolius Europe 21.00 52.25
PI 274817b L. angustifolius Europe 21.00 52.25
PI 274819a,b L. angustifolius Europe 21.00 52.25
PI 274820b L. angustifolius Europe 21.00 52.25
w6 39768b L. angustifolius Europe 13.03 52.52
w6 39769a,b L. angustifolius Europe 13.03 52.52
w6 39771b L. angustifolius Europe 13.03 52.52
w6 39772a,b L. angustifolius Europe -3.70 40.43
w6 39775a,b L. angustifolius Europe 13.03 52.52
w6 39776a,b L. angustifolius Europe 13.03 52.52
w6 39777a,b L. angustifolius Europe 13.03 52.52
PI 384554b,c L. hispanicus Europe -9.08 38.70
PI 384555b L. hispanicus Europe -9.08 38.70
PI 384556a,b L. hispanicus Europe -9.08 38.70
PI 384558a,b L. hispanicus Europe -9.08 38.70
PI 384559b L. hispanicus Europe -9.08 38.70
PI 384560a,b L. hispanicus Europe -9.08 38.70
PI 384561a,b L. hispanicus Europe -9.08 38.70
PI 384562b L. hispanicus Europe -9.08 38.70
PI 168539a,b,c L. luteus Europe -9.08 38.70
PI 168544b L. luteus Europe -9.08 38.70
PI 168546a,b L. luteus Europe -9.08 38.70
PI 189159b L. luteus Europe 4.87 52.35
PI 224493a,b L. luteus Europe 4.87 52.35
PI 289168a,b L. luteus Europe 19.25 47.43
PI 255475a,b L. luteus Europe 20.47 44.82
PI 266298b L. luteus Europe 12.99 55.61
PI 206508a,c L. mutabilis South America -76.92 -12.10
PI 432332a L. mutabilis South America -76.92 -2.10
PI 457966a L. mutabilis South America -71.98 -13.52
PI 457967a L. mutabilis Africa -6.85 34.03
PI 457972a L. mutabilis South America -76.92 -12.10
PI 249758a,b,c L. pilosus Europe 24.02 35.51
PI 338647b L. pilosus Africa -6.85 34.03
PI 491183b L. pilosus Asia 32.90 40.03
PI 602369b L. pilosus Asia 37.81 28.49
PI 602375a,b L. pilosus Asia 35.22 31.78
PI 664108a,b L. pilosus Europe 19.25 47.43
PI 664109b L. pilosus Europe 12.57 55.72
PI 664110b L. pilosus Asia 35.22 31.78
PI 664111b L. pilosus Europe -9.08 38.70
PI 344687a,c L. sericeus North America -116.22 43.62
PI 344694a L. sericeus North America -116.22 43.62
PI 452488a L. sericeus North America -112.03 46.58
PI 477950a L. sericeus North America -77.03 38.88
PI 477952a L. sericeus North America -77.03 38.88

Supplement table 2

Characteristics of 95 pairs of polymorphic primers"

引物
Primer
重复单元
Repeat motif
正向引物
Forward sequence (5°-3°)
正向引物
Reverse sequence (5°-3°)
退火温度
Tm (℃)
lup-est6104 (TTG)4 CTCAAGTTTTGGCTTCATGC CTCAAGTGCATTCATGGAAGT 54
lup-est4304 (TC)6 AGGGGTTTTGAAGAGAAAGG AGACAGTGATAGCTCAGTGGG 54
lup-est2704 (GA)5 TCAAGCCCTTCACTCAAAAC CCCCCTTTCTAAAAATCCAA 54
lup-est2313 (TC)4 GTGACAATGGCTCTTCAACC CCTTGTGCAAAAGTCCAAAT 54
lup-est713 (TAA)4 CAGCCATTAAAGCTTTTGGA AGAAAACACCAAAAGGGACC 54
lup-est613 (TTG)5 TTCCACCCATCATTGAAATC GGAGCAACAACTACACATCCA 54
lup-est1313 (TTA)5 GAAAACCACCACCACTTTTG GGGGGTAGTGCTAAGAAAGG 54
lup-est4458 (CAA)3 CAGAATCACACCCCAATCTC ATCCCAGGCCATTGACTAAT 54
lup-est5958 (AAC)4 CAACGGGGTCATTGAATTAG GTTTGTGACGGGTTAAGGTG 54
lup-est3458 (CAC)4 GACGACTCCAATGACACTCC GATGAGGGTTTCGAAGAGGT 54
lup-est1058 (GGT)4 TTTATCGGGAAGCATGTGTT ATAGGTGAGCCTGGGACTTC 54
lup-est2558 (CT)9 AGACCCACCAAACAATGAAA ATCCCGAGGCCAAATCTA 54
lup-est158 (CTT)3 TGGTTGTTGGGTCTAATAGCA TGAGGAGAACCATCATTCAAG 54
lup-est6558 (ATG)6 TGTTGATGCCAGAAACATTG GCCTTCCCTTCTCTACAACC 54
lup-est4949 (TGA)5 CTCAATCCCCTAGTGACCCT GGTAATTCCAAACTTGCCCT 54
lup-est1649 (TC)6 CAATCTTCTTCACTCTACTCCTCA CAAGTCTGAAATCCTTGCGT 54
lup-est1449 (TTG)6 GGTTTCCATCATAAGCATCC TCAGCACACTAGCAAGTACCC 54
lup-est5740 (AAT)5 ACATGTGCATGTTGAATTGC ATTTCGAGGACCAACAATGA 54
lup-est2540 (CAC)4 GAATCATTGAACCGTGATGG GAAAGGGTTCTCTGCTTCGT 54
lup-est4549 (TTG)4 AACCCCATTTTAAGCAAGGT CCACTCTTCCTCAGAAAAATCA 54
lup-est2949 (CAA)5 ATCAATGGCACAAGCAATG AACCAGTAGCAACAAGACCAA 54
lup-est3140 (CTT)4 CATTCAACCAAAACCCATGT ACACGCAGTTTGAAGAGAGC 54
lup-est2849 (TTC)5 CGATGGTCCATTGATATAGAAG AGACCTTCCGAAGTGAGACA 54
lup-est1149 (TGT)4 GCAATGTTGAGCTTCCAAAG CTTTCTTGGTGTTGGAGGAA 54
lup-est4640 (TTG)7 CCTTACCATTGCATCAGTCC GGTATAACCCCTTTTCAGCC 54
lup-est6031 (AAC)4 AAATCTAAGCCCATGGAACC CCCTGCTCTTCTCAAAGTCA 54
lup-est840 (ATC)5 CAACTTTTCAACGGGTTCAC GCAGAAGGGTAAAATACGCA 54
lup-est931 (GAA)4 CAATTTCACCCTCTTCGCTA CTTTAGCAACATCGAAGGGA 54
lup-est6531 (CCT)4 CAAGGTTTGCTTCGTAGGAG AGGGAGGAAATTGGATGTGT 54
lup-est1985 (TGC)4 AAGTGATGTCCTGAGCTTGC GCTCCACAGATGACACAACA 54
lup-est4185 (GTG)5 GCAACAGTCACCTTGCATTA CCCTTAAACTCCACTTCAACC 54
lup-est3185 (CAA)5 CTCAACCCAACAAAACAACA GCACTGCAATCAGAAGTGAA 54
lup-est6567 (CTA)5 CCAACTCCAGTCACTGCTCT GCTCTCAAATGGGGTTGTAG 54
lup-est2567 (TAG)6 AGTGGCCAGGATAGTGTTGA CAAACTCCACCTCATTGTCC 54
lup-est5667 (GGA)4 CAAGGGTAAGTACCCGATCC GAGAGATGAGTGAAGCACAGAA 54
lup-est2076 (GGT)3 ATAGTTGCTGTAACTCGGCG CTGTCCTCCTCCTCCTTCTC 54
lup-est4467 (ACA)4 TGCATTCATGGCTTCACTTA TTGTGACAACTTGGAGGGAT 54
lup-est976 (GAA)4 GCTTAAGGGAGGAGCAAGAT GCTTCTTGTGCTGCTCTTTC 54
lup-est1622 (CAC)4 GTACCCCTGATGATGCTGAC TGGTGGATTGTCTGATGATG 54
lup-est4022 (GAA)4 TCTACTTCCTTCACAAAACGC CAAAAGGTAACGAAGCTCCA 54
lup-est822 (ATA)4 CATATTCAAGGCTTTGGTGG TTTGGGGCAAGTAGAAATTG 54
lup-est4522 (ATG)5 AGGTAACGTTGATTCGTGGA CACCAACAACACCACTACCA 54
lup-est2822 (CCT)4 AGACCAAGAGAGTGGAGGCT CACTCTCTCAGTAGGAGGCG 54
lup-est6213 (AAG)5 GGCGTGGGTTAAGCTTATGT ACCCCACCTTCTTCAATCAT 54
lup-est2694 (ATC)6 GGGGCTGCTTCATCTTATTT CGCCTCAATTCAAGGATATG 54
lup-est1594 (ATC)6 GAATGTTCCAGTTGGTGGAG ATCATAACCCATTGACCCCT 54
lup-est3894 (TC)6 GAAACCCAACCAATTGTTCA AACCAACATTCTCCACTTGC 54
lup-est2013 (ATG)4 CTATGGAAGATGCGCAGAAT CCCACTGAATCTGACTTGCT 54
lup-est6504 (ACC)4 TGCCTTCTATTGTCACATGC AGATTCTGACTAGCCCCACC 54
lup-est2404 (TGG)4 AAGCACCACCTTCTGTGCTA TCACTTTGGTTCCACTAGCC 54
lup-est6158 (AAC)4 ACCAAAGAAAACAAAACCCC GGACGTCCTTGTGCTGTTAC 54
lup-est458 (CGC)4 AGAGAAGAAACCGGAGGAAA GCATGAACACTTTCAGCACA 54
lup-est4158 (GTT)4 AGAGAGACCAGTGGGAGGAT AAAACCTGTTCGGCTTCTTT 54
lup-est6049 (TGG)5 CTGGCAGCATGGTTTTTAAG AAATGGACTTGTCCCAAACC 54
lup-est58 (GA)7 TCCAGCAGATCCGTTAATCT TCAGATCGCACTCTCTGTGA 54
lup-est2358 (TTG)4 GGTTGATGCAGCAGAAAAGT ACAACTTGCAAATCCACCAT 54
lup-est5049 (AAC)4 TCAGTTAAATCCAACGCCAC GGAATTCTGCGAAGGTATCA 54
lup-est5749 (TGC)4 TGTCTCGATGGTTCAGCATA GCCTTAGATGCCCATCTTCT 54
lup-est6258 (AAT)5 GCTGAAATTGTAGCAGCCAT TGGACCTTGTTTTTCAATGC 54
lup-est3340 (ATG)7 AAGGTTCAGGACTTGTTGAAGA ATCAACCTGTGACCCAGAAA 54
lup-est449 (CAG)6 AGCTCCTCCCAATGCTTATT ATGTTCCAGAGAAATGCCAG 54
lup-est3649 (ACA)4 ACTCAAGTCCAACAATCCCA CACCTTAAACGTGAGAAAGCA 54
lup-est2340 (CTA)5 AAACCCAGCAAATTCACAAG TCTGGAACTCCATTCTTTGC 54
lup-est5440 (TAT)4 GGCTTTTCTTCCCATTGTTT TTTCAGAACAACGTGTCCAA 54
lup-est4349 (AAC)5 AAGAAATGCCCTAGCTGGTT TCTTGTTGCTCTTCCTTTGG 54
lup-est3549 (CT)6 CATGTGCACTCCGTTTATCA GACCTTGTTGTGGGTATTGC 54
lup-est249 (ATG)4 ACAGGTCCATGGAAAACAGA GGTGATGAATTGGGATCTGA 54
lup-est5340 (CCT)5 CGAGAAGCGATCGACATAAT CGACACAGCAGAAGTTGTGA 54
lup-est4249 (CTT)6 AGCTTTGATTTTTGACTGCG TCGTAGAAGTGGATACCCCA 54
lup-est2140 (CAA)4 AGGAACAATCTCAAGCCACA GAGATTATGCTGCCACTGCT 54
lup-est1749 (TGC)3 GCGTAGCTGCTCTCATTTTC AGCTCCACATTCATCCTCAG 54
lup-est5240 (AAC)3 GCAAACTCATTCCCTTCAAA CCTCATCAACGACACCTTCT 54
lup-est1640 (TTC)4 TTAGGGCATTGAACCTTTTG CCACCGTCGTAAGTAACACC 54
lup-est5831 (CTT)5 CACAGACTCAACCTTGGCTT TACTGGCTCCAGATCAAACC 54
lup-est4131 (GTT)6 CAAGCCCCTTAACAAGTCCT CTGGCCAGAGCTGGTAAATA 54
lup-est2531 (TC)6 CACGTTGGGTTTCTTTCATT GAGAGCTTTGCACGTTGATT 54
lup-est2185 (CCA)4 CACTCTTCCAAACGCAAGTT AGCTTCTTTAGGGTTCGGAG 54
lup-est5985 (GAA)5 CCCCAATCTCAGAAATCAAA CAAGTCAGCTTGGGAAAGAA 54
lup-est4576 (ATT)6 CCATATAGCTGCTTCCTCCA AATGCACTTCTCCACCACTC 54
lup-est1085 (CCA)4 AACCTATCTGCTGCTGATGC AGTTCTGCATGCAATCTGGT 54
lup-est2385 (ATG)4 CATGAAGAAGAAGCCAAGGA TTCACCACTTTCATCTTCAGC 54
lup-est3067 (TCA)4 CAAAGATGCTCATCAAGAAGC TAGGGGTTGTTCTTTGTCCA 54
lup-est2267 (GTT)5 AGCCAAGTAGGTGCTGTTTG TCACCACCTCTCTCAAAACC 54
lup-est3576 (CAC)4 CTGGGAATTGAGGTTTGTTG ACTGAAGACGGTGCTGATTC 54
lup-est5967 (AAC)4 AAATCACAATCACCACAGCA TGCATTAGGGAGAAAGTTGG 54
lup-est922 (TTC)4 AGTAGGGTGGAGATGGAAGG GCTTCTCCATGCTCTCTTCA 54
lup-est5622 (ACC)5 TGCTCCACTTTGTTCTTTCC TTGGCTATGGCAGTTTGAAT 54
lup-est6513 (CAA)5 ACCCTTCACAGACATCTCCA TAAGAGACAACATTGGGGGA 54
lup-est4113 (TGG)5 GGTGTTGTTGGAGAGAAGGA TGGTTTTGTTGGCTGAGATT 54
lup-est5513 (ACT)4 AATTCACACGTGGAGGATTC GAGGATCCAAGTTTTGTGGA 54
lup-est4322 (TCT)4 CTGTGCTGGACTTGGTATCC CTTTGCATATTGGTTTGCCT 54
lup-est3494 (CTT)6 TAGCACTGGTTTTCATTGCC CCTGGTGAACCTGATTGAAG 54
lup-est3994 (ACT)4 GGAAACTCCACAAAACCCTT TGTGATACACTGGAATCCCC 54
lup-est2194 (AAT)6 GTGATCAAGATGTGGCAGTG GTTTGTTGTTGGTGCAATGA 54
lup-est164 (CAA)3 AACAAGATTTGGGAGGTTCC CCATCCGGAAGACATATTCA 54
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