棉属人工异源四倍体后代性状鉴定及花器转录组学分析

doi:10.3724/SP.J.1006.2023.34061

摘要/Abstract

摘要：

实验室前期通过远缘杂交及加倍人工合成草棉与雷蒙德氏棉异源四倍体, 旨在拓宽棉花遗传背景, 为棉花种质创新提供新材料。本试验以人工异源四倍体3个世代为材料, 将其与亲本及陆地棉标准系TM-1进行纤维性状、生理生化比较、SSR标记鉴定以及花器转录组学分析。结果表明, 人工异源四倍体纤维颜色介于亲本之间, 呈浅黄色, 纤维长度与TM-1接近。人工异源四倍体的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性以及丙二醛(MDA)含量均显著高于亲本和TM-1, 表明人工异源四倍体经过远缘杂交及加倍后, 抗逆性得到加强。选取11对SSR引物在材料中共检测到55个多态性位点, 每对引物变异位点为2~10个, 平均为5个; 多态信息含量(PIC)变幅为0.498~0.892, 平均为0.742; 表明SSR标记在试验材料中表现出丰富的遗传多样性。S₂、S₃、S₄同时扩增出亲本互补条带和特异性条带, 表明该异源四倍体在分子水平上出现了染色体片段的重组。S₂、S₃、S₄与TM-1相同的条带分别是37条、19条、20条, 其中17条是各世代与TM-1共有, 表明人工异源四倍体与TM-1有相同的遗传背景。花器转录组学结果表明, 人工异源四倍体与TM-1中有5653个DEGs, 其中3062个上调, 2591个下调。GO功能分析表明, DEGs在光合作用、光系统II、RNA指导的DNA聚合酶活性等途径差异显著。KEGG富集分析表明, DEGs在光合作用-天线蛋白、光合作用和异黄酮的生物合成等相关代谢通路较为活跃。通过富集分析筛选人工异源四倍体与TM-1中与育性相关的DEGs发现, 人工异源四倍体在花粉发育与识别方面的差异基因大都是下调表达, 这可能是导致人工异源四倍体结实率低的关键因子。通过聚类从Nr注释信息获得3个G型凝集素类受体丝氨酸/苏氨酸蛋白激酶(基因Gh_D01G067500、Gh_D05G168100、Gh_A01G062500)。研究结果表明人工异源四倍体综合了父母本的优良性状, 在纤维颜色、抗逆、光合方面表现出优势。期望通过和陆地棉杂交恢复其育性, 进一步育成生产上有价值的新材料。

关键词: 人工异源四倍体, 生理生化测定, SSR分子标记, 转录组测序, 差异表达基因

Abstract:

The artificial allotetraploid of Gossypium herbaceun and G. raimondii was created in the laboratory to broaden the genetic background of cotton and provide novel materials for the germplasm innovation of cotton. In this study, fiber characters, physiological and biochemical comparison, SSR markers and floral transcriptomes analysis were conducted among three generations of the artificial allotetraploid and their parents, including G. hirsutum L. acc. TM-1. The results showed that the fiber color of artificial allotetraploid, light yellow, mid-parent of G. herbaceun and G. raimondii, and the fiber length was close to TM-1. The activities of SOD, POD, CAT, and MDA content in artificial allotetraploid plants were significantly higher than parents and TM-1, indicating that the stress resistance of artificial allotetraploid was enhanced after distant hybridization and chromosome doubling. 55 polymorphic loci were detected from 11 pairs of SSR primers, with 2-10 mutation loci per primer and an average of 5. PIC ranged from 0.498 to 0.892, with an average of 0.742. The results indicated that SSR markers could reflect rich genetic diversity information among the test materials. The complementary of parents and specific bands were amplified in S₂, S₃, and S₄, simultaneously, indicating that chromosome segment recombination occurred in the artificial allotetraploid at the molecular level. There were 37, 19, and 20 bands of S₂, S₃, and S₄ identical with TM-1 respectively, among which 17 bands were shared with TM-1 by all generations, indicating that the artificial allotetraploid had homology in genetic background with TM-1. The floral organ transcriptome analysis showed that there were 5653 differentially expressed genes (DEGs) between artificial allotetraploid and TM-1, in which 3062 were up-regulated and 2591 were down-regulated. GO functional analysis showed that DEGs had significant differences in photosynthesis, photosystem II, RNA directed DNA polymerase activity and other pathways. KEGG enrichment analysis showed that DEGs were more active in photosynthetic-antenna proteins, photosynthesis and isoflavone biosynthesis. The enrichment analysis of fertility related DEGs between artificial allotetraploids and TM-1 revealed that most of the DEGs of artificial allotetraploids in pollen development and recognition were down-regulated, which may be the key factor leading to low seed setting percentage of artificial allotetraploid. Three G-type lectin receptor serine/threonine protein kinases (Gh_D01G067500, Gh_D05G168100, Gh_A01G062500) were obtained from Nr annotation information by clustering. The results showed that the artificial allotetraploid performed advantage in fiber color, resistance to stress and photosynthesis, combining the excellent quality of its parents. It is expected to restore its fertility by crossbreeding with G. hirsutum, and to further cultivate a valuable novel material for production.

Key words: artificial allotetraploid, physiological and biochemical detection, SSR molecular marker, transcriptome sequencing, differentially expressed gene

陈天, 李昱樱, 荣二花, 吴玉香. 棉属人工异源四倍体后代性状鉴定及花器转录组学分析[J]. 作物学报, 2024, 50(2): 325-339.

CHEN Tian, LI Yu-Ying, RONG Er-Hua, WU Yu-Xiang. Character identification and floral organ transcriptome analysis on artificial allotetraploids of Gossypium hirsutum L.[J]. Acta Agronomica Sinica, 2024, 50(2): 325-339.

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链接本文: https://zwxb.chinacrops.org/CN/10.3724/SP.J.1006.2023.34061

https://zwxb.chinacrops.org/CN/Y2024/V50/I2/325

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CK vs CL育性相关差异表达基因"

基因ID Gene ID	GO条目 GO term	Nr 注释 Nr annotation	FDR
Gh_A07G232100↑	花粉萌发 Pollen germination (GO:0009846)	含非特征性WD重复序列alr3466样蛋白 Uncharacterized WD repeat-containing protein alr3466-like	1.14E-11
Gh_A03G188700↑	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白ES332_A03G193800v1 Hypothetical protein ES332_A03G193800v1	3.37E-06
Gh_D04G029900↑	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白 Hypothetical protein ES332_D04G035100v1	1.28E-05
Gh_D12G209800↓	花粉萌发 Pollen germination (GO:0009846) 花粉管发育 Pollen tube growth (GO:0009860)	未表征蛋白 LOC105764824 亚型 X1 Uncharacterized protein LOC105764824 isoform X1	5.42E-05
Gh_A01G062500↓	花粉识别 Recognition of pollen (GO:0048544)	G型凝集素受体丝氨酸/苏氨酸蛋白激酶At2g19130 G-type lectin S-receptor-like serine/threonine-protein kinase At2g19130	0.000114428
Gh_D02G100100↓	花粉识别 Recognition of pollen (GO:0048544)	推定受体蛋白激酶ZmPK1 Putative receptor protein kinase ZmPK1	0.000126516
Gh_D01G067500↓	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白E1A91_D01G078200v1 Hypothetical protein E1A91_D01G078200v1	0.000257193
Gh_D05G168100↓	花粉识别 Recognition of pollen (GO:0048544)	G型凝集素受体丝氨酸/苏氨酸蛋白激酶At4g27290 G-type lectin S-receptor-like serine/threonine-protein kinase At4g27290	0.000261106
Gh_D02G205900↓	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白ES332_D02G232300v1 Hypothetical protein ES332_D02G232300v1	0.000614955
Gh_A01G217800↑	花的发育 Flower development (GO:0009908)	调节NPR5-样蛋白 Regulatory protein NPR5-like	0.001068761
Gh_D05G374100↓	花粉识别 Recognition of pollen (GO:0048544)	G型凝集素受体样丝氨酸/苏氨酸蛋白激酶B120 G-type lectin S-receptor-like serine/threonine-protein kinase B120	0.001490638
Gh_A07G211000↓	花粉发育 Pollen development (GO:0009555)	假设蛋白ES332_A07G220300v1 Hypothetical protein ES332_A07G220300v1	0.001735562
Gh_D11G192200↓	花粉发育 Pollen development (GO:0009555) 花的发育 Flower development (GO:0009908) 花药发育 Anther development (GO:0048653)	假设蛋白ES332_D11G205000v1 Hypothetical protein ES332_D11G205000v1	0.002122634
Gh_D12G026000↓	花粉管导向 Pollen tube guidance (GO:0010183)	CCG结合1样蛋白 CCG-binding protein 1-like	0.003353379
Gh_D05G207900↓	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白B456_009G216000 Hypothetical protein B456_009G216000	0.005513164
Gh_D05G373900↓	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白ES319_D05G403800v1 Hypothetical protein ES319_D05G403800v1	0.005618249
Gh_A09G137700↑	花的发育 Flower development (GO:0009908)	调节蛋白NPR5 Regulatory protein NPR5	0.005712565
Gh_D12G008900↓	花粉管发育 Pollen tube growth (GO:0009860)	BTB/POZ结构域At1g03010-样蛋白 BTB/POZ domain-containing protein At1g03010-like	0.005938747
Gh_A01G071700↓	花粉识别 Recognition of pollen (GO:0048544)	假设蛋白ES332_A01G085300v1 Hypothetical protein ES332_A01G085300v1	0.005966954
Gh_D04G207700↓	花粉发育 Pollen development (GO:0009555)	REF/SRPP蛋白At1g67360 REF/SRPP-like protein At1g67360	0.00620811
Gh_D02G075200↓	花粉识别 Recognition of pollen (GO:0048544)	G型凝集素受体丝氨酸/苏氨酸蛋白激酶At1g34300 G-type lectin S-receptor-like serine/threonine-protein kinase At1g34300	0.006487744
Gh_A05G248700↑	花粉识别 Recognition of pollen (GO:0048544)	推定受体蛋白激酶ZmPK1 Putative receptor protein kinase ZmPK1	0.007975671
Gh_D05G374300↓	花粉识别 Recognition of pollen (GO:0048544)	受体样丝氨酸/苏氨酸蛋白激酶 SD1-7 Receptor-like serine/threonine-protein kinase SD1-7	0.009414103

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材料编号 Material ID	材料信息 Information	染色体组 Genome	分布 Distribution	种植年份 Year
P₁	草棉 G. herbaceun	A₁	亚洲, 非洲 Asia, Africa	2022
P₂	雷蒙德氏棉 G. raimondii	D₅	墨西哥 Mexico	2022
1	S₂	A₁D₅	新合成 New	2020
2	S₂	A₁D₅	新合成 New	2021
3	S₂	A₁D₅	新合成 New	2021
4	S₂	A₁D₅	新合成 New	2021
5	S₃	A₁D₅	新合成 New	2021
6	S₃	A₁D₅	新合成 New	2022
7	S₄	A₁D₅	新合成 New	2022
TM-1	陆地棉 G. hirsutum	(AD)₁	墨西哥, 美国亚利桑那州 Mexico; Arizona, USA	2022

引物编号 Primer number	正向引物序列 Forword sequence (5'-3')	反向引物序列 Reverse sequence (5'-3')	PCR产物 PCR products	退火温度 Annealing temperature (℃)
BNL4108	TCCACCATTCCCGTAAATGT	TGGCCAAGTCATTAGGCTTT	(GA)31	55
BNL4053	TGAAGGCTTTGAAGCAAACA	AAGCAAGCACCAAGTTAGCC	(CA)10	55
NAU2026	GAATCTCGAAAACCCCATCT	ATTTGGAAGCGAAGTACCAG	(CAG)4	57
NAU1355	ATCTGTTTACGCCACTCTCC	CCAGCCTTTGACATTTTTCT	(GA)21	57
NAU1169	GGGTAGTAGCTTTTATGATAGGG	CCATTCCTTCCCCTAATTCT	(TA)12	57
NAU1157	GAGTTTGGTTCTGGGTTGAG	GATCCTTTTCATCTCCTCCA	(AC)9	57
NAU1052	CGCAGATAAAGGATGGATTT	AGAGCTGGAGGACATAACAAA	(AGT)6	57
NAU1042	CATGCAAATCCATGCTAGAG	GGTTTCTTTGGTGGTGAAAC	(TCAGGC)4	57
NAU1164	CCAACGCTAATTCTACCTCCT	GCGGGTAATTGTAGTACATGC	(TTC)10	57
NBRI_gL015	GAGTTTGGCTCCGAGTTGAG	GCGAGTGAACTCACCGATAA	(GAA)18	53
NBRI_gM7	GTGATACCTGGAAAGATGTGA	TCCTGATGCTGTTTTAGTCTT	(GAAG)6	52

材料 Material	SOD活性 SOD activity (U g^-1)	POD活性 POD activity (U g^-1)	CAT活性 CAT activity (U g^-1)	MDA含量 MDA content (μmol g^-1)
P₁	152.30±10.23 bcC	3029.39±167.15 cC	55.13±4.04 cB	9.3895±0.55 cC
P₂	138.78±10.99 cC	3341.44±135.99 cBC	76.17±5.52 bB	13.2766±1.08 bBC
S₂	221.92±5.29 aA	4214.01±91.94 abA	121.43±4.42 aA	17.7334±0.66 aA
S₃	204.66±7.98 aAB	4336.56±109.44 aA	111.78±5.95 aA	18.7116±0.69 aA
S₄	215.67±9.59 aAB	4155.41±78.71 abA	115.04±7.20 aA	18.7235±1.21 aA
TM-1	176.49±11.28 bBC	3822.59±82.71 bAB	80.90±3.51 bB	15.7484±0.28 abAB

引物 Primer number	多态位点数 Polymorphic loci	总位点数 Total loci	多态信息含量 Polymorphism information content
BNL4108	3	3	0.649
BNL4053	7	9	0.883
NAU2026	10	10	0.892
NAU1355	4	4	0.705
NAU1169	2	2	0.498
NAU1157	3	3	0.656
NAU1052	4	5	0.773
NAU1042	5	5	0.737
NAU1164	4	4	0.742
NBRI_GL015	10	10	0.892
NBRI_gM7	3	4	0.735
总计Total	55	59	—
平均Mean	5	5.36	0.742

引物 Primer name	S₂				S₃				S₄
引物 Primer name	P₁	P₂	新增 New	缺失 Absent	P₁	P₂	新增 New	缺失 Absent	P₁	P₂	新增 New	缺失 Absent
BNL4108	1	1	1	0	1	1	0	0	1	1	0	0
BNL4053	3	1	3	0	2	1	2	1	3	1	0	0
NAU2026	2	2	6	0	0	2	0	2	0	2	0	2
NAU1355	1	1	1	1	1	0	1	2	1	0	1	2
NAU1169	0	0	1	0	0	0	1	1	0	0	1	1
NAU1157	1	2	0	0	1	1	0	1	1	1	0	1
NAU1052	1	0	3	0	1	0	1	0	1	0	1	0
NAU1042	1	0	3	1	1	0	0	1	1	0	0	1
NAU1164	1	1	1	0	1	0	0	2	1	0	1	2
NBRI_GL015	2	2	6	0	1	1	0	2	1	1	2	2
NBRI_gM7	0	0	3	0	0	0	2	0	0	0	2	0
总计Total	13	10	28	2	9	6	7	12	10	6	8	11
平均Mean	1.18	0.91	2.55	0.18	0.82	0.55	0.64	1.09	0.91	0.55	0.73	1