33个杨柳品种指纹图谱构建

doi:10.12302/j.issn.1000-2006.202008048

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (2): 35-42.doi: 10.12302/j.issn.1000-2006.202008048

33个杨柳品种指纹图谱构建

何旭东¹^,²(), 郑纪伟¹^,², 孙冲²^,³, 何开跃³, 王保松¹^,²^,^*()

1.江苏省林业科学研究院,江苏南京 211153
2.江苏省农业种质资源保护与利用平台柳树资源圃, 江苏南京 211153
3.南京林业大学生物与环境学院,江苏南京 210037

收稿日期:2020-08-28 接受日期:2020-09-25 出版日期:2021-03-30 发布日期:2021-04-09
通讯作者: 王保松
基金资助:
江苏省林业科学研究院自主科研项目(BM2018022-01);江苏省林业科学研究院青年基金项目(JAF-2016-01)

Construction of fingerprints for 33 varieties in Salicaceae

HE Xudong¹^,²(), ZHENG Jiwei¹^,², SUN Chong²^,³, HE Kaiyue³, WANG Baosong¹^,²^,^*()

1. Jiangsu Academy of Forestry, Nanjing 211153, China
2. Willow Nursery of the Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 211153, China
3. College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China

Received:2020-08-28 Accepted:2020-09-25 Online:2021-03-30 Published:2021-04-09
Contact: WANG Baosong

摘要/Abstract

摘要：

【目的】构建杨树与柳树优良品种的指纹图谱,对不同品种进行准确鉴定。【方法】利用柳树EST-SSR标记对33个杨树与柳树优良品种进行通用性检测及基因分型,通过核心引物间的组合构建品种指纹图谱。同时采用非加权组平均法进行聚类,分析各品种间亲缘关系。【结果】12个EST-SSR标记共扩增出97条等位片段,每个位点等位基因数5~13个不等,平均为8.1个。12个位点的多态信息含量(PIC)变化范围为0.637 2~0.834 8,平均为0.781 7。优选的3对核心引物中,SALeSSR0340与SALeSSR0346组合可完全区分12个杨树品种,而SALeSSR0259、SALeSSR0340与SALeSSR0346的组合可完全区分所有的33个品种。聚类分析显示33个品种间的遗传相似系数为0.68~0.96,并分成杨属与柳属两大类,与传统的杨柳科系统分类学一致。各品种间亲缘关系聚类结果与遗传背景相吻合。【结论】对33个杨柳树优良品种进行指纹图谱构建与亲缘关系分析,表明EST-SSR标记可有效反映品种间的差异,建立的基因分型体系准确、高效,可为杨树与柳树品种鉴定、保护与推广工作提供科学依据。

关键词: 杨树, 柳树, SSR标记, 指纹图谱, 亲缘关系, 基因分型

Abstract:

【Objective】Populus and Salix are members of Salicaceae family. They are dioecious catkin-bearing woody plants that propagate easily, grow fast, and serve as an important source of wood (for furniture), plywood, fiberboard, and pulp and paper industries. In recent years, numerous Populus and Salix varieties have been selected and widely used for afforestation. However, the morphological characteristics among the varieties of Populus and Salix are quite similar, making it difficult to popularize and protect them. To precisely identify different varieties of Salicaceae, a fingerprint for improved varieties of Populus and Salix was established in this study. 【Methods】A total of 38 pairs of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers with high polymorphism information content (PIC) derived from Salix in a previous study were selected randomly to evaluate the transferability of 33 varieties of Salicaceae, of which 12 were genotyped using the fluorescence dUTP method. The fingerprint of different varieties was constructed by the combination of the core primers. To elucidate the genetic relationship between the different varieties, the unweighted pair group method arithmetic average (UPGMA) was used for clustering analysis. 【Result】Among the 38 EST-SSR markers, transferability was 68.4% and 57.89% across all the Populus and Salix varieties, respectively. After genotyping, a total of 97 alleles were detected by 12 pairs of EST-SSR markers, and the number of alleles per locus ranged from 5 to 13, with an average of 8.1 alleles. The PIC of the 12 loci varied from 0.637 2 to 0.834 8, with an average of 0.781 7. Among the three optimal pairs of core primers, the combination of SALeSSR0340 and SALeSSR0346 completely distinguish 12 Populus varieties, and the combination of SALeSSR0259, SALeSSR0340, and SALeSSR0346 completely identified all the 33 varieties used in our study. Cluster analysis demonstrated that the similarity coefficient for the 33 improved varieties ranged from 0.68 to 0.96, and all the improved varieties could be separated into two clades, namely, the genera Populus and Salix, which consisted of the traditional taxonomy of Salicaceae. The clustering result of the genetic relationship of each variety also corresponded with the genetic background. 【Conclusion】In this study, the fingerprints of 33 improved varieties of Salicaceae were constructed and their genetic relationships were evaluated. The divergences between varieties could be effectively screened by the EST-SSR markers, and the genotyping system established in this study was also accurate and efficient. These results can provide scientific evidence for the identification, protection, and popularization of improved varieties of Populus and Salix.

Key words: Populus spp., Salix spp., SSR marker, fingerprint, genetic relationship, genotyping

中图分类号:

何旭东,郑纪伟,孙冲,等. 33个杨柳品种指纹图谱构建[J]. 南京林业大学学报（自然科学版）, 2021, 45(2): 35-42.

HE Xudong, ZHENG Jiwei, SUN Chong, HE Kaiyue, WANG Baosong. Construction of fingerprints for 33 varieties in Salicaceae[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(2): 35-42.DOI: 10.12302/j.issn.1000-2006.202008048.

图/表 6

表1

试验材料"

编号 code	品种 variety	遗传背景 genetic background
1	‘苏柳172’ Salix × jiangsuensis ‘J172’	(垂柳×白柳)×旱柳 (S. babylonica × S. alba)× S. matsudana
2	‘苏柳795’ S. × jiangsuensis ‘J795’	旱柳×白柳 S. matsudana × S. alba
3	‘苏柳799’ S. × jiangsuensis ‘J799’	旱柳×白柳 S. matsudana × S. alba
4	‘苏柳932’ S. × jiangsuensis ‘J932’	垂柳×白柳 S. babylonica × S. alba
5	‘苏柳1010’ S. × jiangsuensis ‘J1010’	垂柳×白柳 S. babylonica × S. alba
6	‘苏柳1011’ S. × jiangsuensis ‘J1011’	垂柳×白柳 S. babylonica × S. alba
7	‘苏柳52-2’ S. × jiangsuensis ‘J52-2’	簸箕柳×银柳 S. suchowensis × S. argyracea
8	‘苏柳8-26’ S. × jiangsuensis ‘J8-26’	簸箕柳×杞柳 S. suchowensis ×S. integra
9	‘苏柳9-6’ S. × jiangsuensis ‘J9-6’	杞柳×簸箕柳 S. integra × S. suchowensis
10	‘苏柳887’ S. × jiangsuensis ‘J887’	吐兰柳×银柳 S. turanica × S. argyracea
11	‘苏柳1050’ S. × jiangsuensis ‘J1050’	簸箕柳×银柳 S.suchowensis × S. argyracea
12	‘花叶柳’ S. integra ‘Tu Zhongyu’	杞柳 S. integra
13	‘竹柳’ ‘Zhuliu’	未知 Unknown
14	‘SV1’	杂交子代 Salix hybrid
15	‘SX61’	龙江柳 S. sachalinensis
16	‘S25’	钻石柳 S. eriocephala
17	‘SX64’	宫部氏柳 S. miyabeana
18	‘SX67’	宫部氏柳 S. miyabeana
19	‘Fish Creek’	欧洲红皮柳 S. purpurea
20	‘Onondaga’	欧洲红皮柳 S. purpurea
21	‘Otisco’	蒿柳 × 宫部氏柳 S. viminalis × S. miyabeana
22	‘南林95’杨 Populus × euramericana ‘Nanlin 95’	‘I-69’杨× ‘I-45’ P. deltoides cl. ‘Lux’ ×(P. × euramericana ‘I-45’)
23	‘南林895’杨 P. ×euramericana ‘Nanlin 895’	‘I-69’× ‘I-45’ P. deltoides cl. ‘Lux’ ×(P. × euramericana ‘I-45’)
24	‘I-69’杨 P. deltoides ‘I-69’	美洲黑杨 P. deltoides
25	‘I-72’杨 P. × euramericana ‘I-72’	欧美杨 P. × euramericana
26	‘35’杨 P. deltoides ‘Dvina’	美洲黑杨 P. deltoides
27	‘苏杨7号’ P. deltoides ‘Suyang-7’	‘I-69’杨实生苗 seedling of ‘I-69’
28	‘I-107’杨 P. × euramericana ‘Neva’	欧美杨 P. × euramericana
29	‘I-108’杨 P. × euramericana ‘I-108’	欧美杨 P. × euramericana
30	‘银中’杨 P. alba × P. berolinensis ‘Yinzhong’	银白杨×中东杨 P. alba × P. berolinensis
31	‘湘林90’杨 P. deltoides ‘XL-90’	50号杨×W07杨 P.deltoides cl. ‘55/65’)×P. deltoides ‘W07’
32	‘丹红’杨 P. deltoides ‘Danhong’	50号杨×36号杨 P. deltoides cl. ‘55 /65’ × P. deltoides ‘2KEN8’
33	‘巨霸’杨 P. deltoides ‘Juba’	50号杨×36号杨 P. deltoides cl. ‘55 /65’ × P. deltoides ‘2KEN8’

表1

表2

表3

图1

表4

图2

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编号 code	位点 locus	登录号 accession number	引物序列 primer sequences	基序 motif	期望片段/ bp expected fragment
1	SALeSSR0069	Pr032826640	F:TGACCCACAAAGTGAAACAGA R:CTCAGCCAACTCCGAAGAA	(AGG)₆	151
2	SALeSSR0074	Pr032826645	F:TTTCCGTCCAACTCATCTTCT R:AGCAGTCACGAGGCGAACT	(TTTC)₄	458
3	SALeSSR0108	Pr032826679	F:AAGCTGGACCGCATGAAC R:ACGGGCTATGATGGAGGA	(TGG)₉	378
4	SALeSSR0169	Pr032826740	F:CTTCGCATCTCCGTTTCAC R:CGTCGCTTTGGTTCTGGTT	(CCT)₆	300
5	SALeSSR0259	Pr032826830	F:CGCTTTGGGATGACTGATT R:GTTGCTGGAGTGTTTCTTGG	(CAG)₆	217
6	SALeSSR0264	Pr032826835	F:ACAGAAACGCAAGCCTCAA R:AAAGAGCAACTGCCGAGAA	(ACC)₉	251
7	SALeSSR0279	Pr032826850	F:AACTTCATAGCCCATACACCTT R:CCATCAACAACAACCCAAAC	(CTC)₅	326
8	SALeSSR0296	Pr032826867	F:GTGATACTGGCACCTCTTTCC R:TGGGTGGAACTGTTTCTATTGC	(CAG)₅	275
9	SALeSSR0340	Pr032826911	F:TTCATGCCTTTGGCTGTG R:TGTCCTCTTTGCCCGTCT	(ATG)₆	315
10	SALeSSR0346	Pr032826917	F:CACCCGCAAGTTTCCCTA R:CTGCCCTAAAGCCGTTGT	(AGC)₇	190
11	SALeSSR0696	Pr032827267	F:CTTGCTCGTCCCGTAGATT R:ACCAGGAGTGGCAGAAACC	(GCT)₅	186
12	SALeSSR0939	Pr032827510	F:TCACTTCCACCCGCATCA R:AGACCCGAGGTTTCAGACAG	(TCT)₇	381

位点 locus	等位基因数 number of allele	等位基因长度/bp length of allele	多态信息含量 PIC
SALeSSR0069	11	132~165	0.826 2
SALeSSR0074	7	452~488	0.766 4
SALeSSR0108	9	338~377	0.834 8
SALeSSR0169	9	292~316	0.829 8
SALeSSR0259	8	193~229	0.819 9
SALeSSR0264	6	233~260	0.751 0
SALeSSR0279	5	327~339	0.762 8
SALeSSR0296	6	263~284	0.637 2
SALeSSR0340	10	290~347	0.827 2
SALeSSR0346	13	352~394	0.808 5
SALeSSR0696	6	174~189	0.755 0
SALeSSR0939	7	351~384	0.761 1
合计 total	97
平均 average	8.1		0.781 7

品种 varieties	核心引物 core primers
品种 varieties	SALeSSR0259	SALeSSR0340	SALeSSR0346
‘苏柳172’	211/217	314/317	352/361/373
‘苏柳795’	211/214/217	317	361/382
‘苏柳799’	211/217	317	361/382
‘苏柳932’	193/211	314	358/373
‘苏柳1010’	211/217/220	314	361/373/388
‘苏柳1011’	211/217/220	314	361/376/394
‘苏柳52-2’	217	290/311	361/382
‘苏柳8-26’	211/217	290/311	361/382
‘苏柳9-6’	211/214	290	361/382
‘苏柳887’	214/217	290/311	358/382
‘苏柳1050’	217/229	347	367/394
‘花叶柳’	214/217	293/314	358/385
‘竹柳’	205/217	314/317	352/361/373
‘SV1’	205/211	290	379/388
‘SX61’	208/220	290	361/382
‘S25’	211/214	290	382/391
‘SX64’	217/220	290/311	388
‘SX67’	208/220	290/311	364/388
‘Fish Creek’	208/220	311	361/376
‘Onondaga’	214/217	290/311	361/382
‘Otisco’	214/217	290/311	388
‘南林95’杨	193/208	296/311	379
‘南林895’杨	193/208	296/314	361/379
‘I-69’杨	208	314/317	361/382
‘I-72’杨	208	296	361/382
‘35’杨	208	296/305	361/382
‘苏杨7号’	193/208	314	361/382
‘I-107’杨	208	296/311	361/382
‘I-108’杨	208	296/314	361/382
‘银中’杨	208	329	376/385
‘湘林90’杨	208	314/317	382
‘丹红’杨	208	299/317	361/382
‘巨霸’杨	208	299/317	382

33个杨柳品种指纹图谱构建

Construction of fingerprints for 33 varieties in Salicaceae

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