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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (4): 577-586.doi: 10.3724/SP.J.1006.2021.04131

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

Molecular detection of G1 marker for orange rust resistance and analysis of candidate resistance WAK gene in sugarcane

WANG Heng-Bo(), CHEN Shu-Qi, GUO Jin-Long, QUE You-Xiong*()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / National sugarcane Engineering Technology Research, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2020-06-18 Accepted:2020-10-14 Online:2021-04-12 Published:2020-11-02
  • Contact: QUE You-Xiong E-mail:wanghengbo_0354@126.com;queyouxiong@126.com
  • Supported by:
    Program of Introducing International Super Agricultural Science and Technology (948 Program)(2014-S18);China Agricultural (Sugar Crop) Research System(CARS-17);Fujian Agriculture and Forestry University Science and Technology Development Special Fund(KFA18025A)

Abstract:

Sugarcane orange rust is an important fungal disease caused by Puccinia kuehnii Butler, which could lead to a reduction in sugarcane production and sugar content and cause serious losses to the sugarcane industry in worldwide. In this study, the molecular marker G1 was used to detect orange rust resistance genes in cultivars, ancestral species and the related genus of Saccharum in the world. The representative amplified bands were cloned, sequenced, functionally annotated, and clustered, and the origin and evolution of resistance genes was then analyzed. The results showed that 83 and 34 were detected with G1 marker, accounting for 66.9% and 67.4% in 124 Chinese and 46 foreign sugarcane cultivars, respectively. Among 34 sugarcane ancestral species and the related genus of Saccharum, 17 were detected by G1 marker, accounting for 50%, of which the highest percentage (100%) was in Saccharum spontaneum. Functional annotation revealed that G1 target gene encoded a wall-associated receptor-like kinase (WAK), and three proteins with high similarity were identified from the haploid proteome database of sugarcane cultivars. These proteins all contain the extracellular domain, transmembrane domain and intracellular domain with kinase activity of typical cell wall receptor structures. Phylogenetic analysis of nucleotide sequences clearly showed the origin and evolution of the candidate resistance WAK genes. Specifically, the WAK genes amplified by G1 marker could be divided into three groups. The first group is from S. spontaneum and Saccharum robustum. The second group is from S. robustum, Saccharum officinarum and Narenga porphyrooma. The third group is from S. spontaneum, S. robustum, Saccharum sinense and cultivars. These results provided an important support for breeding of sugarcane cultivars resistant to orange rust, and lay a foundation for further analysis of molecular mechanism of resistance genes.

Key words: detection, molecular markers, wall-associated receptor-like kinase, orange rust, sugarcane

Table S1

Origins of 170 sugarcane cultivars and their PCR amplification results using G1 marker"

编号
No.
品种
Cultivar
来源
Origin
G1标记
Amplification result
备注
Note
1 赣南 99-591 Gannan 99-591 中国江西 Jiangxi, China - S. hybrid
2 四川 89-103 Sichuan 89-103 中国四川 Sichuan, China + S. hybrid
3 内江 00-118 Neijiang 00-118 中国四川 Sichuan, China + S. hybrid
4 内江 03-218 Neijiang 03-218 中国四川 Sichuan, China - S. hybrid
5 内江 92-44 Neijiang 92-44 中国四川 Sichuan, China + S. hybrid
6 内江 90-112 Neijiang 90-112 中国四川 Sichuan, China + S. hybrid
7 四川 73-219 Sichuan 73-219 中国四川 Sichuan, China + S. hybrid
8 新台糖 27号 ROC27 中国台湾 Taiwan, China - S. hybrid
9 新台糖 1号 ROC 1 中国台湾 Taiwan, China + S. hybrid
10 新台糖 16号 ROC 16 中国台湾 Taiwan, China + S. hybrid
11 新台糖 11号 ROC11 中国台湾 Taiwan, China + S. hybrid
12 新台糖 26号 ROC 26 中国台湾 Taiwan, China + S. hybrid
13 新台糖 95-0432 ROC 95-0432 中国台湾 Taiwan, China + S. hybrid
14 新台糖 95-8899 ROC 95-8899 中国台湾 Taiwan, China + S. hybrid
15 新台糖 20号 ROC 20 中国台湾 Taiwan, China + S. hybrid
16 新台糖 21号 ROC 21 中国台湾 Taiwan, China + S. hybrid
17 新台糖 23号 ROC 23 中国台湾 Taiwan, China - S. hybrid
18 新台糖 24号 ROC 24 中国台湾 Taiwan, China - S. hybrid
19 新台糖 10号 ROC10 中国台湾 Taiwan, China - S. hybrid
20 PT 43-52 中国台湾 Taiwan, China + S. hybrid
21 F108 中国台湾 Taiwan, China - S. hybrid
22 新台糖 22号 ROC 22 中国台湾Taiwan, China + S. hybrid
23 崖城 05-179 Yacheng 05-179 中国海南Hainan, China + S. hybrid
24 崖城 71-374 Yacheng 71-374 中国海南Hainan, China + S. hybrid
25 崖城 93-26 Yacheng 93-26 中国海南Hainan, China - S. hybrid
26 崖城 94-30 Yacheng 94-30 中国海南Hainan, China + S. hybrid
27 崖城 94-46 Yacheng 94-46 中国海南Hainan, China + S. hybrid
28 崖城 97-46 Yacheng 97-46 中国海南Hainan, China - S. hybrid
29 崖城 98-11 Yacheng 98-11 中国海南Hainan, China - S. hybrid
30 崖城 98-2 Yacheng 98-2 中国海南Hainan, China + S. hybrid
31 崖城 96-66 Yacheng 96-66 中国海南Hainan, China - S. hybrid
32 崖城 01-76 Yacheng 01-76 中国海南Hainan, China + S. hybrid
33 崖城 96-4 Yacheng 96-4 中国海南Hainan, China - S. hybrid
34 崖城 99-6 Yacheng 99-6 中国海南Hainan, China + S. hybrid
35 崖城 96-24 Yacheng 96-24 中国海南Hainan, China + S. hybrid
36 福农 02-3924 Funong 02-3924 中国福建Fujian, China - S. hybrid
37 福农 04-3504 Funong 04-3504 中国福建Fujian, China + S. hybrid
38 福农 99-20169 Funong 99-20169 中国福建Fujian, China - S. hybrid
39 福农 38号 Funong 38 中国福建Fujian, China + S. hybrid
40 福农 39号 Funong 39 中国福建Fujian, China - S. hybrid
41 福农 41号 Funong 41 中国福建Fujian, China + S. hybrid
42 闽糖 96-261 Mintang 96-261 中国福建Fujian, China + S. hybrid
43 农林 8 Nonglin 8 中国福建Fujian, China + S. hybrid
44 华南65-21 Huanan 65-21 中国广东Guangdong, China - S. hybrid
45 桂糖 94-119 Guitang 94-119 中国广西Guangxi, China - S. hybrid
46 桂糖 94-38 Guitang 94-38 中国广西Guangxi, China - S. hybrid
47 桂糖 96-211 Guitang 96-211 中国广西Guangxi, China + S. hybrid
48 柳城 01-1137 Liucheng 01-1137 中国广西Guangxi, China + S. hybrid
49 柳城 04-382 Liucheng 04-382 中国广西Guangxi, China + S. hybrid
50 柳城 05-129 Liucheng 05-129 中国广西Guangxi, China + S. hybrid
51 桂糖 02-761 Guitang 02-761 中国广西Guangxi, China + S. hybrid
52 桂糖 73-167 Guitang 73-167 中国广西Guangxi, China + S. hybrid
53 柳城 03-182 Liucheng 03-182 中国广西Guangxi, China + S. hybrid
54 桂糖 03-591 Guitang 03-591 中国广西Guangxi, China + S. hybrid
55 桂糖 03-1156 Guitang 03-1156 中国广西Guangxi, China - S. hybrid
56 桂糖 03-1403 Guitang 03-1403 中国广西Guangxi, China + S. hybrid
57 桂糖 03-2357 Guitang 03-2357 中国广西Guangxi, China - S. hybrid
58 桂糖 04-1007 Guitang 04-1007 中国广西Guangxi, China + S. hybrid
59 桂糖 08-278 Guitang 08-278 中国广西Guangxi, China - S. hybrid
60 桂糖 02-390 Guitang 02-390 中国广西Guangxi, China + S. hybrid
61 桂糖 05-2605 Guitang 05-2605 中国广西Guangxi, China - S. hybrid
62 桂糖 05-2743 Guitang 05-2743 中国广西Guangxi, China + S. hybrid
63 桂糖 05-322 Guitang 05-322 中国广西Guangxi, China + S. hybrid
64 桂糖 05-3661 Guitang 05-3661 中国广西Guangxi, China + S. hybrid
65 桂糖 05-375 Guitang 05-375 中国广西Guangxi, China + S. hybrid
66 桂糖 05-378 Guitang 05-378 中国广西Guangxi, China + S. hybrid
67 桂糖 05-3846 Guitang 05-3846 中国广西Guangxi, China - S. hybrid
68 桂糖 08-297 Guitang 08-297 中国广西Guangxi, China + S. hybrid
69 桂糖 35号 Guitang 35 中国广西Guangxi, China - S. hybrid
70 桂糖 05-827 Guitang 05-827 中国广西Guangxi, China + S. hybrid
71 桂糖 07-713 Guitang 07-713 中国广西Guangxi, China - S. hybrid
72 桂糖 02-1247 Guitang 02-1247 中国广西Guangxi, China + S. hybrid
73 桂糖 02-208 Guitang 02-208 中国广西Guangxi, China - S. hybrid
74 桂糖 02-619 Guitang 02-619 中国广西Guangxi, China + S. hybrid
75 桂糖 00-245 Guitang 00-245 中国广西Guangxi, China + S. hybrid
76 桂糖 96-211 Guitang 96-211 中国广西Guangxi, China + S. hybrid
77 桂糖 00-122 Guitang 00-122 中国广西Guangxi, China - S. hybrid
78 桂糖 02-351 Guitang 02-351 中国广西Guangxi, China - S. hybrid
79 桂糖 09-03 Guitang 09-03 中国广西Guangxi, China + S. hybrid
80 桂糖 96-167 Guitang 96-167 中国广西Guangxi, China + S. hybrid
81 桂糖 97-69 Guitang 97-69 中国广西Guangxi, China - S. hybrid
82 桂糖 00-173 Guitang 00-173 中国广西Guangxi, China - S. hybrid
83 桂糖 02-467 Guitang 02-467 中国广西Guangxi, China + S. hybrid
84 桂糖 29号 Guitang 29 中国广西Guangxi, China + S. hybrid
85 桂糖 30号 Guitang 30 中国广西Guangxi, China - S. hybrid
86 桂糖 31号 Guitang 31 中国广西Guangxi, China - S. hybrid
87 桂糖 92-66 Guitang 92-66 中国广西Guangxi, China + S. hybrid
88 桂糖 00-257 Guitang 00-257 中国广西Guangxi, China + S. hybrid
89 桂糖 94-119 Guitang 94-119 中国广西Guangxi, China - S. hybrid
90 桂糖35号 Guitang 35 中国广西Guangxi, China + S. hybrid
91 柳城 03-1137 Liucheng 03-1137 中国广西Guangxi, China + S. hybrid
92 德蔗 93-88 Dezhe 93-88 中国云南Yunnan, China + S. hybrid
93 云蔗 02-2332 Yunzhe 02-2332 中国云南Yunnan, China - S. hybrid
94 云蔗 02-2540 Yunzhe 02-2540 中国云南Yunnan, China + S. hybrid
95 云蔗 02-588 Yunzhe 02-588 中国云南Yunnan, China + S. hybrid
96 云蔗 03-194 Yunzhe 03-194 中国云南Yunnan, China + S. hybrid
97 云蔗 04-214 Yunzhe 04-214 中国云南Yunnan, China + S. hybrid
98 云蔗 05-51 Yunzhe 05-51 中国云南Yunnan, China + S. hybrid
99 云蔗 06-407 Yunzhe 06-407 中国云南Yunnan, China + S. hybrid
100 云蔗 94-343 Yunzhe 94-343 中国云南Yunnan, China - S. hybrid
101 云南 65-225 Yunzhe 65-225 中国云南Yunnan, China + S. hybrid
102 粤农 73-204 Yuenong 73-204 中国广东Guangdong, China + S. hybrid
103 粤甘 34号 Yuegan 34 中国广东Guangdong, China - S. hybrid
104 粤甘 16号 Yuegan 16 中国广东Guangdong, China + S. hybrid
105 粤农 73-204 Yuenong 73-204 中国广东Guangdong, China - S. hybrid
106 粤农 86-295 Yuenong 86-295 中国广东Guangdong, China - S. hybrid
107 粤糖 00-236 Yuetang 00-236 中国广东Guangdong, China + S. hybrid
108 粤糖 55 Yuetang 55 中国广东Guangdong, China + S. hybrid
109 粤糖 83-271 Yuetang 83-271 中国广东Guangdong, China + S. hybrid
110 粤糖 93-159 Yuetang 93-159 中国广东Guangdong, China + S. hybrid
111 粤糖 94-128 Yuetang 94-128 中国广东Guangdong, China + S. hybrid
112 粤糖 96-86 Yuetang 96-86 中国广东Guangdong, China + S. hybrid
113 湛蔗 19号 Zhanzhe 19 中国广东Guangdong, China + S. hybrid
114 湛蔗 22号 Zhanzhe 22 中国广东Guangdong, China + S. hybrid
115 湛蔗 24号 Zhanzhe 24 中国广东Guangdong, China - S. hybrid
116 湛蔗 74-141 Zhanzhe 74-141 中国广东Guangdong, China + S. hybrid
117 粤甘 24号 Yuegan 24 中国广东Guangdong, China + S. hybrid
118 粤糖 89-240 Yuetang 89-240 中国广东Guangdong, China + S. hybrid
119 湛蔗 92-126 Zhanzhe 92-126 中国广东Guangdong, China - S. hybrid
120 湛蔗 26号 Zhanzhe 26 中国广东Guangdong, China + S. hybrid
121 湛蔗 80-101 Zhanzhe 80-101 中国广东Guangdong, China + S. hybrid
122 粤糖 00-319 Guitang00-319 中国广东Guangdong, China + S. hybrid
123 粤糖 60号 Yuetang 60 中国广东Guangdong, China - S. hybrid
124 科5 Ke 5 中国广东Guangdong, China + S. hybrid
125 FR94-280 法国 France + S. hybrid
126 FR99-49 法国 France - S. hybrid
127 RB83-5089 巴西 Brazil + S. hybrid
128 Q208 澳大利亚 Australia - S. hybrid
129 Q117 澳大利亚 Australia - S. hybrid
130 Q200 澳大利亚 Australia + S. hybrid
131 Q209 澳大利亚 Australia + S. hybrid
132 Co1001 印度 India + S. hybrid
133 Co281 印度 India + S. hybrid
134 Co290 印度 India + S. hybrid
135 PINDAR 印度 India - S. hybrid
136 Co419 印度 India + S. hybrid
137 POJ2878 印度尼西亚 Indonesia - S. hybrid
138 NCO376 南非 South Africa + S. hybrid
139 NCO310 南非 South Africa + S. hybrid
140 CP67-412 美国 USA + S. hybrid
141 CP72-1210 美国 USA + S. hybrid
142 CP72-2086 美国 USA - S. hybrid
143 CP72-330 美国 USA + S. hybrid
144 CP72-3591 美国 USA + S. hybrid
145 CP82-1592 美国 USA + S. hybrid
146 CP84-1198 美国 USA - S. hybrid
147 CP85-1308 美国 USA + S. hybrid
148 CP86-1633 美国 USA + S. hybrid
149 CP88-1762 美国 USA + S. hybrid
150 CP89-1509 美国 USA + S. hybrid
151 CP89-2143 美国 USA - S. hybrid
152 CP92-1213 美国 USA + S. hybrid
153 CP94-1100 美国 USA + S. hybrid
154 CP94-1340 美国 USA + S. hybrid
155 CP96-1602 美国 USA + S. hybrid
156 H32-8560 美国 USA + S. hybrid
157 HoCP91-555 美国 USA + S. hybrid
158 HoCP92-624 美国 USA - S. hybrid
159 HoCP93-746 美国 USA + S. hybrid
160 HoCP95-988 美国 USA - S. hybrid
161 HoCP94-806 美国 USA + S. hybrid
162 HoCP95-950 美国 USA - S. hybrid
163 CP80-1277 美国 USA + S. hybrid
164 CP57-641 美国 USA + S. hybrid
165 CP92-1666 美国 USA - S. hybrid
166 CP93-182 美国 USA - S. hybrid
167 CP49-50 美国 USA + S. hybrid
168 CP67-412 美国 USA + S. hybrid
169 CP28-11 美国 USA - S. hybrid
170 R570 法国 France - S. hybrid

Table S2

Origins of 34 germplasms from Saccharum and the related genus and their PCR amplification results using G1 marker"

编号
No.
品种
Name
来源
Origin
G1标记
Amplification result
备注
Note
1 贵州 78-2-12 Guizhou 78-2-12 中国贵州 Guizhou, China + Erianthus arundinaceus
2 云南 95-19 Yunnan 95-19 中国云南 Yunnan, China - Erianthus rockii
3 广西 79-8 Guangxi 79-8 中国广西 Guangxi, China - Miscanthus floridulus
4 云南 95-35 Yunnan 95-35 中国云南 Yunnan, China - Miscanthus sinensis
5 广西 89-13 Guangxi 89-13 中国广西 Guangxi, China + Narenga porphyrooma
6 广东 64号 Guangdong 64 中国广东 Guangdong, China - Narenga porphyrooma
7 HATUNI 印度 India + S. barberi
8 Katha 印度 India - S. barberi
9 Nangas 印度 India - S. barberi
10 LA-purple 印度尼西亚 Indonesia - S. officinarum
11 Badila 印度尼西亚 Indonesia - S. officinarum
12 路达仕 Loethers 美国 USA - S. officinarum
13 黑车里本 Black cheribon 美国 USA - S. officinarum
14 克里斯塔林娜 Crystalina 美国 USA - S. officinarum
15 云南大野Yunnandaye 中国云南 Yunnan, China - S. robustum
16 福建大野Fujiandaye 中国福建 Fujian, China + S. robustum
17 51NG3 印度尼西亚 Indonesia - S. robustum
18 57NG208 印度尼西亚 Indonesia - S. robustum
19 NG77004 印度尼西亚 Indonesia - S. robustum
20 MOL-6081 美国 USA + S. robustum
21 桂林竹蔗 Guilinzhuzhe 中国广西 Guangxi, China + S. sinense
22 Youba 中国云南 Yunnan, China - S. sinense
23 松溪竹蔗Songxizhuzhe 中国福建 Fujian, China - S. sinense
24 贵州 78-1-11 Guizhou 78-1-11 中国贵州 Guizhou, China + S. spontaneum
25 贵州 78-2-28 Guizhou 78-2-28 中国贵州 Guizhou, China + S. spontaneum
26 四川 79-1-26 Sichuan79-1-26 中国四川 Sichuan, China + S. spontaneum
27 四川 79-2-11 Sichuan 79-2-11 中国四川 Sichuan, China + S. spontaneum
28 云南 75-2-11 Yunnan 75-2-11 中国云南 Yunnan, China + S. spontaneum
29 广东 21号 Guangdong 21 中国广东 Guangdong, China + S. spontaneum
30 广东 30号 Guangdong 30 中国广东 Guangdong, China + S. spontaneum
31 福建 89-1-1 Fujian 89-1-1 中国福建 Fujian, China + S. spontaneum
32 福建 89-1-16 Fujian 89-1-16 中国福建 Fujian, China + S. spontaneum
33 印度1号 Yindu 1 印度 India + S. spontaneum
34 印度2号 Yindu 2 印度 India + S. spontaneum

Fig. 1

Electrophoretic patterns of G1 primers amplified in some Saccharum hybrid 1: HoCP 94-806; 2: Gannan 99-591; 3: Yuetang 00-236; 4: Guitang 05-827; 5: Guitang 00-257; 6: Yacheng 97-46; 7: ZZ 80-101; 8: CP 67-412; 9: Guitang 05-322; 10: Yacheng 94-46; 11: Guitang 08-278; 12: Guitang 00-245; 13: CP 89-1509; 14: Guitang 05-3846; 15: Guitang 29; 16: Yacheng 93-26; 17: CP 72-2086; 18: Yacheng 94-30; 19: CP 92-1213; 20: HoCP 92-624; 21: Guitang 05-2605; 22: Yacheng 96-24; 23: ZZ 22; 24: Yacheng 99-6; M:100 bp DNA ladder."

Fig. 2

Electrophoretic patterns of G1 markers amplified in Saccharum genus 1: Guizhou 78-2-12; 2: Guangxi 89-13; 3: Guangdong 64; 4: Yindu 1; 5: Guangxi 79-8; 6: LA-purple; 7: Loethers; 8: Yunnan 95-19; 9: Katha; 10: Yunnan 95-35; 11: Guilinzhuzhe; 12: Sichuan 78-2-11; 13: Sichuan 79-1-26; 14: Guizhou 78-2-28; 15: Guizhou 78-1-11; 16: Guangdong 30; 17:Guangdong 21; 18: Fujian 89-1-16; 19: Fujian 89-1-1; 20: Fujiandaye; 21: NG77-004; 22: 57NG208; 23: Yunnandaye; 24: 51NG3; M: 100 bp DNA ladder."

Fig. 3

Phylogenetic tree analysis of amplified sequences from Saccharum genus using G1 marker"

Fig. 4

Schematic diagram of the domain composition of ScWAK2 protein ED: extracellular domain; TM: transmembrane region; ID: intracellular domain; EGF2: EGF2-like region; EGF-Ca: calcium-binding EGF-like domain."

Fig. 5

Multiple sequences alignment of WAK protein from Arabidopsis, rice, sorghum, and sugarcane EGF1: EGF1-like region; EGF2: calcium-binding EGF-like domain; TM: transmembrane region; ATP-B: ATP-binding region; KAS: kinase active site are underlined."

Fig. 6

Phylogenetic analysis and motif prediction of WAK protein from sugarcane and other species"

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