Identification of Candidate Genes for Male Sterile in Soybean by RNA-Seq

doi:10.7668/hbnxb.20196210

Abstract

Abstract:

The large-scale production of soybean hybrids and the utilization of hybrid vigor are key to germplasm resource innovation and variety improvement.By mining and utilizing soybean male sterility genes,more advantageous soybean varieties can be cultivated,thereby improving soybean yield and quality.This study selected the male sterile mutant population that appeared in the hybrid offspring of Baoquanling green soybean(female parent)and Heinong 44(male parent)as the experimental material.Flower buds were selected from soybean fertile and sterile plants,the anthers and pollen cytology were observed,and a high-throughput sequencing platform was used to perform transcriptome sequencing on the flower and anther tissues of soybean fertile and sterile plants.Annotated,screened and analyzed the sequencing results.The results indicated that abnormal development of the tapetum layer and high pollen sterility in sterile plants were important causes of male infertility.The pentose and glucuronate interconversion pathway(ko00040:Pentose and glucuronate interconversion)was a key pathway affecting soybean fertility,and the genes encoding pectin methylesterase(PEM)and pectin lyase(PL)were downregulated in expression.The differentially expressed gene protein interaction network indicated that the pectin lyase gene was a key gene affecting the interconversion pathway between pentose and glucuronic acid.The determination of pectin lyase content showed that the pectin lyase content in sterile plant anthers was significantly reduced.The qRT-PCR detection results were consistent with the RNA-Seq sequencing results.It is speculated that the downregulation of these genes may cause a decrease in pectin lyase activity,thereby affecting the development of the tapetum and leading to male infertility.

Key words: Soybean, Male infertility, Transcriptomics, Tapetum, Pectate lyase

CLC Number:

CHEN Tianyu, XU Chao, ZHAO Liang, WANG Ziye, ZHANG Qi, DU Jidao. Identification of Candidate Genes for Male Sterile in Soybean by RNA-Seq[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 1-11. doi: 10.7668/hbnxb.20196210.

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Figures/Tables 10

Tab.1 The corresponding primers of qRT-PCR

基因ID Gene ID	正向引物序列(5'—3') Forward primer(5'—3')	反向引物序列(5'—3') Reverse primer(5'—3')
GmEF1-α	TGCAAAGGAGGCTGCTAACT	CAGCATCACCCTTCTTCAAA
Glyma.01G042700	CACCGGAGCAAGAGTGGA	TGCCTTGAGAACGGCCTG
Glyma.02G020600	GAACCGGACCCTTGTGGA	ATACCAGCCCCACCAGCA
Glyma.04G173900	TGCCAAGGTGCAGATGGG	TAAACCGGTTGCCCTGGC
Glyma.07G150200	ATTCAAGGCGCGCTTTGC	CGTGAGGACCAAGGCCAT
Glyma.08G296900	TGCAATCTGGGCCTGAGC	GCGACCCACTCTGCACTT
Glyma.13G064700	TGCCAAGGTGCAGATGGG	GGCGTTGTCGTTGTTCGG
Glyma.18G255900	TGCCAAGGTGCAGATGGG	GGCAGCGTTGTTGTTGGG
Glyma.19G020200	TTCGGAGCCAGCAACGTT	GAGCCCTGGATGACGTCG
Glyma.20G240800	ATGGCGGGGATGCAGAAG	AGAGAGGACCGTGGAGGC

Tab.2 Statistics on the number of trait segregation in the offspring of sterile lines

单株编号 Number	可育植株 (实际值) Fertile	不育植株 (实际值) Sterile	总株数 Total number of plants	卡方值 (单株) χ²	组别 Group	未分离株行数 (实际值) Unseparated plant line	分离株行数 (实际值) Separated plant line	总株行数 Total number of plants and rows	卡方值 (单组) χ²
1	38	13	51	0.006 5	F2-1	7	13	20	0.021 6
2	33	10	43	0.069 6	F2-2	6	14	20	0.108 4
3	34	14	48	0.444 4	F2-3	7	13	20	0.021 6
4	45	14	59	0.051 0	F2-4	7	13	20	0.021 6
5	43	15	58	0.022 9	F2-5	6	14	20	0.108 4
6	35	13	48	0.111 1	F2-6	5	15	20	0.541 6
7	48	15	63	0.047 7	F2-7	7	13	20	0.021 6
8	38	12	50	0.026 7	F2-8	8	12	20	0.433 4
9	41	13	54	0.026 7	F2-9	7	13	20	0.021 6
10	42	15	57	0.052 6	F2-10	6	14	20	0.108 4
总计Total	397	134	534	0.859 2	总计	66	134	200	1.373 4

Fig.1 Phenotypic identification of soybean fertile and sterile plants A,B.Feasible and sterile plants during soybean pod setting period;C,D.Soybean fertile plants and sterile plants anthers;E,F.Soybean pollen from fertile and sterile plants;G,H.Soybean fertile plants and sterile plants anthers.

Tab.3 Transcriptome sequencing data information

样品 Sample	Reads总数 Reads sum	总碱基数 Base sum	GC/%	N/%	Q20/%	Q30/%
FA1	22 843 955	6 820 359 173	44.16	0.04	98.15	94.58
FA2	20 990 838	6 267 855 523	44.09	0.03	97.96	94.14
FA3	21 102 342	6 293 914 777	43.96	0.03	98.04	94.28
FF1	20 283 651	6 052 402 966	43.89	0.03	97.95	94.15
FF2	20 693 432	6 171 776 753	43.89	0.03	97.97	94.20
FF3	22 807 796	6 817 058 932	43.86	0.02	97.75	93.56
SA1	20 907 079	6 242 338 350	43.79	0.03	97.86	93.90
SA2	21 282 893	6 357 207 698	43.79	0.03	97.93	94.01
SA3	21 481 838	6 419 512 460	43.71	0.02	97.95	94.11
SF1	21 556 089	6 444 548 048	43.72	0.02	97.84	93.84
SF2	22 528 378	6 731 511 923	43.58	0.02	97.82	93.70
SF3	20 992 211	6 266 325 309	43.72	0.03	97.85	93.78

Fig.2 DEGs sets and gene co-expression trend

Fig.3 KEGG enrichment analysis of differentially expressed genes A.The KEGG enrichment bubble plot for FA vs SA treatment;B.The KEGG enrichment bubble plot for FF vs SF treatment;C,D.The network diagram of differentially expressed genes and KEGG pathway in FA vs SA treatment and FF vs SF treatment,respectively.A:1.Pentose and glucuronate interconversions,2.Plant-pathogen interaction,3.Endocytosis,4.Circadian rhythm-plant,5.Phosphatidylinositol signaling system,6.Glycerophospholipid metabolism,7.Glycolysis/Gluconeogenesis,8.Sphingolipid metabolism,9.Linoleic acid metabolism,10.Carbon fixation in photosynthetic organisms,11.Glycosphingolipid biosynthesis-ganglio series,12.Amino sugar and nucleotide sugar metabolism,13.Inositol phosphate metabolism,14.Glycosaminoglycan degradation,15.Anthocyanin biosynthesis,16.Fructose and mannose metabolism,17.Cysteine and methionine metabolism,18.Fatty acid elongation,19.Phagosome,20.Galactose metabolism;B:1.Pentose and glucuronate interconversions,2.Arachidonic acid metabolism,3.Glycerophospholipid metabolism,4.Plant-pathogen interaction,5.Alpha-linolenic acid metabolism,6.Valine,leucine and isoleucine degradation,7.Endocytosis,8.Glycerolipid metabolism,9.Fructose and mannose metabolism,10.Ether lipid metabolism,11.Linoleic acid metabolism,12.Carbon fixation in photosynthetic organisms,13.Amino sugar and nucleotide sugar metabolism,14.Glycolysis/Gluconeogenesis,15.Oxidative phosphorylation,16.Phagosome,17.Inositol phosphate metabolism,18.Isoquinoline alkaloid biosynthesis,19.Sphingolipid metabolism,20.Phosphatidylinositol signaling system.

Fig.4 GO enrichment analysis of differentially expressed genes A—C.BP,CC,MF of the FA vs SA treatment;D—F.BP,CC,MF of the FF vs SF treatment.A:1.Pectin catabolic process,2.Cell wall modification,3.Regulation of pH,4.Monovalent inorganic cation transport,5.Clathrin coat assembly,6.Pollen tube growth,7.Vesicle budding from membrane,8.Endocytosis,9.Clathrin-dependent endocytosis,10.Intracellular signal transduction,11.Xyloglucan metabolic process,12.Cell wall organization,13.Cell wall biogenesis,14.Carbohydrate transport,15.Rab protein signal transduction,16.Cell tip growth,17.Carbohydrate metabolic process,18.Developmental cell growth,19.Multi-multicellular organism process,20.Pollination;B:1.Plasma membrane,2.Extracellular region,3.Cell wall,4.Clathrin-coated vesicle,5.Clathrin-coated pit,6.Endomembrane system,7.Actin cytoskeleton,8.Appoplast,9.Uniplex complex,10.Apical part of cell,11.Apical plasma membrane,12.Pollen tube,13.Golgi apparatus,14.Plasma membrane region,15.Integral component of mitochondrial inner membrane,16.Cytoskeleton,17.Vacuolar proton-transporting V-type ATPase complex,18.Integral component of plasma membrane,19.Vacuole,20.Amyloplast;C:1.Pectinesterase activity,2.Pectinesterase inhibitor activity,3.Calcium ion binding,4.Aspartyl esterase activity,5.Solute proton antiporter activity,6.Clathrin heavy chain binding,7.1-phosphatidylinositol binding,8.Calmodulin binding,9.GTPase activator activity,10.Phosphatidylinositol-4,5-bisphosphate binding,11.Xyloglucan:xyloglucosyl transferase activity,12.Calcium transmembrane transporter activity,phosphorylative mechanism,13.SNARE binding,14.Rho GDP-dissociation inhibitor activity,15.Beta-galactosidase activity,16.Carbohydrate:proton symporter activity,17.Actin filament binding,18.Proton-exporting ATPase activity,phosphorylative mechanism,19.Uniporter activity,20.Amino acid transmembrane transporter activity;D:1.Pectin catabolic process,2.Cell wall modification,3.Regulation of pH,4.Monovalent inorganic cation transport,5.Callose coat assembly,6.Regulation of jasmonic acid mediated signaling pathway,7.Pollen tube growth,8.Vesicle budding from membrane,9.Clathrin-dependent endocytosis,10.Intracellular signal transduction,11.Cell tip growth,12.Endocytosis,13.Peptidyl-serine phosphorylation,14.Developmental cell growth,15.Multi-multicellular organism process,16.Pollination,17.Response to wounding,18.Regulation of intracellular pH,19.Regulation of defense response,20.Developmental growth involved in morphogenesis;E:1.Plasma membrane,2.Endomembrane system,3.Cell wall,4.Extracellular region,5.Clathrin-coated pit,6.Clathrin-coated vesicle,7.Actin cytoskeleton,8.Integral component of plasma membrane,9.Apical part of cell,10.Apical plasma membrane,11.Olgi apparatus,12.Cell periphery,13.Pollen tube,14.Plasma membrane region,15.Apoplast,16.Membrane,17.External encapsulating structure,18.Plasma membrane bounded cell projection,19.Cell projection,20.Cell junction;F:1.Pectinesterase activity,2.Pectinesterase inhibitor activity,3.Aspartyl esterase activity,4.Solute:proton antiporter activity,5.Calcium ion binding,6.Clathrin heavy chain binding,7.1-phosphatidylinositol binding,8.Phosphatidylinositol-4,5-bisphosphate binding,9.Calmodulin binding,10.GTPase activator activity,11.Calcium-dependent protein serine/threonine kinase activity,12.Rho GDP-dissociation inhibitor activity,13.SNARE binding,14.Pectate lyase activity,15.Carbohydrate:proton symporter activity,16.Actin filament binding,17.Proton-exporting ATPase activity,phosphorelay intermediate,18.Transmembrane receptor protein serine/threonine kinase activity,19.Receptor ligand activity,20.Receptor regulator activity.

Fig.5 Differential expression gene protein interaction network diagram

Fig.6 Heat map of expression levels of differentially expressed genes(A)and qRT-PCR validation(B) Different lowercase letters indicate significant difference(P<0.05).The same as Fig.7.

Fig.7 Determination of pectin lyase activity

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