不同品种南瓜内生细菌多样性及PICRUSt基因功能预测分析

doi:10.3864/j.issn.0578-1752.2021.18.019

摘要/Abstract

摘要：

【目的】比较5个不同品种南瓜内生细菌多样性特征并开展基因功能预测分析,为挖掘和利用南瓜内生细菌功能菌株及开拓南瓜辅助育种新方向提供理论依据与参考。【方法】采集5个不同品种南瓜茎部,基于MiSeq高通量测序技术比较分析南瓜茎内生细菌多样性与丰富度,内生细菌门、属分类水平的群落结构组成,以及不同品种南瓜茎内生细菌优势菌群占比。【结果】不同品种南瓜茎部内生细菌多样性与丰富度指数均无显著差异。5个品种南瓜茎内生细菌群落结构由18个门,30个纲,101个目,199个科,362个属,567个种和863个OTU组成,但不同品种南瓜茎中的内生细菌分类阶层总数存在差异。门分类水平,变形菌门（Proteobacteria）细菌是不同品种南瓜茎部的主要优势细菌门类,不同南瓜品种内生细菌群落结构组成相似,但丰度占比不同;属分类水平,假单胞菌属（Pseudomonas）、分枝杆菌属（Mycobacterium）、剑菌属（Ensifer）、德沃斯氏菌属（Devosia）细菌是不同品种南瓜茎部的主要优势细菌属,优势内生细菌属组成及其丰度随南瓜品种的不同呈现明显差异。杂交品种与父母本品种的门、属分类水平优势内生菌组成及占比均基本相同。PICRUSt分析显示,不同品种南瓜茎部内生细菌主要涉及6个生物代谢通路及46个子功能。不同品种南瓜茎部内生细菌基因二级功能层预测基因种类基本无差异,但基因拷贝数却随着品种不同而异。环境条件是影响南瓜植株内生细菌多样性和丰富度的重要因素;不同品质南瓜植株具有相异的内生细菌群落结构,品质优良南瓜品种具有富集内生细菌的趋势;假单胞菌属（Pseudomonas）、分枝杆菌属（Mycobacterium）、剑菌属（Ensifer）、德沃斯氏菌属（Devosia）等13个菌属是5个不同品种南瓜茎部的共有优势内生细菌属;而糖霉菌属（Glycomyces）、Candidatus_Rubidus属和Actinokineospora属细菌是品质最优品种——‘桂丰7号’香芋南瓜茎部特有的优势内生细菌属;同时,‘桂丰7号’香芋南瓜茎部内生细菌基因功能中,涉及碳水化合物代谢、氨基酸代谢、辅助因子和维生素代谢等37个二级功能层预测基因拷贝数在5个南瓜品种中数量最多。【结论】特有的优势内生细菌属和更为丰富的二级功能预测基因拷贝数均是‘桂丰7号’香芋南瓜品质优于其余4个品种南瓜的重要原因,糖霉菌属（Glycomyces）、Candidatus_Rubidus属和Actinokineospora属细菌有望作为提高南瓜品质的备选功能内生细菌属。

关键词: 南瓜, 内生细菌, 多样性, MiSeq高通量测序, 功能预测

Abstract:

【Objective】The aim of this study was to provide a theoretical basis for utilizing functional strains of endophytic bacteria in different varieties of pumpkins, as well as to explore new directions for pumpkin breeding. 【Method】The endophytic bacterial community composition and PICRUSt gene functions of 5 pumpkins varieties were analyzed based on MiSeq high-throughput sequencing results. The diversity and richness of endophytic bacterial community compositions in terms of 5 different pumpkin varieties were compared at phylum and genus levels.【Results】In total, 18 phyla, 30 classes, 101 orders, 199 families, 362 genera, 567 species and 863 OTUs could be obtained as the endophytic bacterial composition in 5 pumpkin varieties. There was no significant difference in endophytic bacteria diversity among pumpkin varieties. Significant differences in total numbers of endophytic bacteria were observed at different classification levels among pumpkin varieties. The proteobacteria was the dominant bacteria in terms of different 5 pumpkin varieties at the phylum level, but the abundance and proportion in every pumpkin variety were quite different. Moreover, Pseudomonas, Mycobacterium, Ensifer and Devosia were the dominant endophytic bacteria at genus level of different 5 pumpkin varieties, but their composition and abundance varied with pumpkin varieties. The composition and proportion of dominant endophytes at the phylum and genus level were similar between the hybrid and parent species. PICRUSt analysis also showed that endophytic bacteria inhabited in terms of different pumpkin varieties, mainly involved in 6 biological metabolic pathways and 46 sub-functions. However, there were no significant differences of endophytic bacterial genes in terms of different pumpkin varieties at the secondary functional layer. However, the numbers of gene functional copies varied with different varieties. The diversity and abundance of endophytic bacteria in terms of pumpkin were significantly affected by environmental factors. Moreover, the different endophytic bacterial community structures could be found in different pumpkin varieties. Meanwhile, a higher abundance of endophytic bacteria tended to enrich in terms of higher quality hybrids. Bacteria in 13 genera of bacteria, including Pseudomonas, Mycobacterium, Ensifer, and Devosia, etc, were the dominant endophytic bacterial genera in terms of 5 different pumpkin varieties. By contrast, Glycomyces, Candidatus_Rubidus, and Actinokineospora were the specific dominant endophytic bacterial genera in terms of Guifeng NO.7. In addition, 37 secondary functional layer functions were all the highest endophytic bacterial genes in terms of Guifeng NO.7 among the five pumpkin varieties, which involved the metabolisms of carbohydrate, amino acid, cofactors, and vitamins.【Conclusion】The higher dominant endophytic bacterial genus and the richer secondary functional genes were essential reasons for higher quality of Guifeng NO.7 than that of other four pumpkin varieties. Glycomyces, Candidatus_Rubidus, and Actinokineospora could be considered as the alternative benefit endophytic bacterial genera for improving pumpkin quality.

Key words: pumpkin (Cucurbita moschata Duch.), endophytic Bacteria, diversity, Miseq High-Throughput Sequencing, function rediction

黄子粤,刘文君,覃仁柳,庞师婵,肖健,杨尚东. 不同品种南瓜内生细菌多样性及PICRUSt基因功能预测分析[J]. 中国农业科学, 2021, 54(18): 4018-4032.

HUANG ZiYue,LIU WenJun,QIN RenLiu,PANG ShiChan,XIAO Jian,YANG ShangDong. Endophytic Bacterial Community Composition and PICRUSt Gene Functions in Different Pumpkin Varieties[J]. Scientia Agricultura Sinica, 2021, 54(18): 4018-4032.

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样品 Sample	南瓜品种名称 Pumpkin variety	可溶性固形物 Soluble solid (%)	V_C含量 Vitamin C (mg/100 g)	可溶性蛋白 Soluble protein (g/100 g)	淀粉含量 Starch content (g/100 g)
1	桂丰7号香芋南瓜 Guifeng NO.7	7.97±0.9a	2.96±0.55a	3.09±0.22a	7.13±0.70a
2	桂丰8号南瓜Guifeng NO.8	6.87±0.15b	0.53±0.39d	2.79±0.33a	4.76±1.11b
3	杂交F₁代 Hybrid（G1519×G1511）	5.27±0.06c	1.7±0.04b	1.72±0.69b	3.52±0.05c
4	G1519	5.33±0.06c	0.95±0.27cd	1.59±0.16b	1.84±0.05d
5	G1511	4.97±0.12c	1.27±0.46bc	2.6±0.25a	1.58±0.10d

样品 Sample	香浓指数 Shannon index	辛普森指数 Simpson index	Ace指数 Ace index	Chao1指数 Chao1 index	覆盖率 Coverage rate
桂丰7号香芋南瓜 Guifeng NO.7	3.65±0.47a	0.11±0.06a	524.14±29.83a	508.14±43.73a	0.99
桂丰8号南瓜 Guifeng NO.8	3.45±0.73a	0.16±0.16a	535.75±94.62a	499.81±53.18a	0.99
杂交F₁代 Hybrid (G1519×G1511)	4.05±0.60a	0.08±0.06a	565.45±83.11a	554.50±78.32a	0.99
G1519	3.88±0.33a	0.07±0.05a	533.63±11.88a	531.93±16.20a	0.99
G1511	3.77±0.69a	0.11±0.08a	559.50±40.47a	571.41±34.24a	0.99

品种 Varity	门 Phylum	纲 Class	目 Order	科 Family	属 Genus	种 Species	OTU
桂丰7号香芋南瓜 Guifeng NO.7	19	28	88	158	294	434	604
桂丰8号南瓜 Guifeng NO.8	17	29	87	161	289	432	606
杂交F₁代 Hybrid (G1519×G1511)	17	27	87	166	312	473	686
G1519	18	29	89	171	306	449	628
G1511	17	29	89	165	299	444	646
总计 Total	18	30	101	199	362	567	863

门分类水平 Phylum level	桂丰7号 Guifeng NO.7	桂丰8号 Guifeng NO.8	杂交F₁代 Hybrid (G1519×G1511)	G1519	G1511
变形菌门 Proteobacteria	63.74%	76.26%	63.49%	76.55%	63.66%
放线菌门 Actinobacteria	30.55%	14.55%	28.55%	18.98%	32.28%
厚壁菌门 Firmicutes	3.31%	2.76%	4.51%	2.81%	2.10%
拟杆菌门 Bacteroidetes	1.46%	2.47%	1.64%	-	1.06%
衣原体门 Chlamydiae	-	2.27%	-	-	-
其他 Others	-	1.70%	1.77%	-	-

属分类水平 Genus level	桂丰7号 Guifeng NO.7	桂丰8号 Guifeng NO. 8	杂交F₁代 Hybrid (G1519×G1511)	G1519	G1511
假单胞菌属 Pseudomonas	26.86%	33.02%	19.45%	12.93%	25.88%
分支杆菌属 Mycobacterium	10.08%	1.75%	4.22%	3.06%	4.33%
Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium	2.41%	2.95%	5.98%	5.27%	1.28%
剑菌属 Ensifer	2.66%	1.56%	1.36%	8.28%	1.18%
unclassified_f__Rhizobiaceae	1.54%	3.79%	1.83%	6.77%	1.04%
德沃斯氏菌属 Devosia	1.64%	4.24%	3.14%	4.16%	1.74%
苯基杆菌属 Phenylobacterium	1.63%	1.03%	2.82%	3.26%	4.25%
鞘氨醇单胞菌属 Sphingomonas	3.07%	1.72%	3.31%	2.86%	1.93%
链霉菌属 Streptomyces	2.42%	1.50%	3.10%	1.32%	3.91%
细杆菌属 Microbacterium	1.29%	1.25%	1.46%	1.11%	4.93%
生根瘤菌属 Mesorhizobium	3.00%	1.49%	1.05%	1.91%	1.47%
假黄色单胞菌属 Pseudoxanthomonas	2.11%	1.41%	2.48%	1.03%	1.72%
芽孢杆菌属 Bacillus	1.77%	2.05%	1.96%	1.06%	1.09%
糖霉菌属 Glycomyces	3.48%	-	-	-	-
Candidatus_Rubidus	2.22%	-	-	-	-
Actinokineospora	1.55%	-	-	-	-
纤发菌属 Methylibium	-	-	-	1.84%	-