本研究以铁低效品种吉育93和铁高效品种吉育99为亲本，杂交后得到的重组自交系群体为材料，通过表性鉴定分析筛选出在低铁胁迫下铁效率表现极端的群体，分别构建铁高效池和铁低效池，利用全基因组重测序技术获得了大量的SNPs和InDels，通过关联分析挖掘与大豆铁吸收有关基因的候选区域。通过一代测序和KASP分型鉴定开发了可高效用于铁吸收效率分子标记辅助选择的KASP标记。本研究是基于高通量测序技术和重组自交系群体，对大豆铁效率相关基因进行定位，主要研究结果如下：

1. 重组自交系群体（F7代），在低铁胁迫条件下，不同品系大豆叶绿素含量在不同时期存在显著差异。极端低铁效率品系中，11个品系已经死亡，占整体品系的6.3%，19个品系在R2期之前停止生长，占整体品系的10.9%。

2. 对亲本及两个极端性状混池，共四组样品进行重测序分析，共获得120.68 Gbp数据量，过滤后得到的Clean Bases为119.63 Gbp，Q30 达到80%以上，样品与参考基因组平均比对效率为99.06 %，平均覆盖深度为27.00 ×。

3. 通过SNP位点和InDel位点的关联分析，铁效率有关的4个目标区域定位在3号、4号、5号染色体上，其中3号染色体上定位到了2个候选区域，,4个候选区间大小总计 10.7 Mbp。对候选区域内的SNPs开发KASP标记，通过一代测序技术筛选得到了可用于分型的3个KASP分子标记，JY10392、JY10551和JY10972。利用筛选出的KASP标记在重组自交系群体中验证结果，发现JY10392和JY10551是共显性标记，基因型和铁效率表型高度一致。

4. 对定位区间内的基因进行分析，通过GO注释分析，被注释到可能与铁离子吸收有关的基因有7个，分别是Glyma.4g15170、Glyma.4g15220、 Glyma.4g15397、 Glyma.5g16400、 Glyma.5g16287、 Glyma.5g16245、 Glyma.5g16288。

In this study, the recombinant inbred line populations obtained after hybridization with iron inefficient variety Jiyu 93 and iron efficient variety Jiyu 99 as parents were used as materials. The populations with extreme iron efficiency under low iron stress were selected through phenotypic identification analysis. Iron efficient pools and iron inefficient pools were constructed respectively. A large number of SNPs and indels were obtained by whole genome re-sequencing technology, The candidate regions of genes related to iron absorption in soybean were mined by association analysis. At the same time, KASP markers that can be used for iron absorption efficiency molecular marker assisted selection were developed by first-generation sequencing and KASP typing. Based on high-throughput sequencing technology and recombinant inbred line population, this study located genes related to soybean iron efficiency. The main results are as follows:

1. The chlorophyll content of recombinant inbred line population (F7 generation) was significantly different in different lines at different stages under low iron stress. There are two groups extremely low iron efficiency strains. 11 strains have died, which accounts for 6.3% of the whole strain. 19 strains stopped growing and did not grow to R2 stage, which accounts for 10.9% of the whole strain.

2. Four groups of samples from parents and two extreme traits were re-sequenced and analyzed. A total of 120.68 Gbp data were obtained. The filtered clean bases were 119.63 Gbp, and the q30 reached more than 80%. The average comparison efficiency between samples and reference genome was 99.06%, and the average coverage depth was 27.00 ×。

3. Through the association analysis of SNP locus and indel locus, the target regions related to iron efficiency were located on chromosomes 3, 4 and 5. There were four candidate regions, of which two candidate regions were located on chromosome 3. The total size of the four candidate intervals is 10.7 Mbp.KASP markers were developed for SNPs in the candidate region. Three KASP molecular markers, jy10392, jy10551 and jy10972, were screened by first-generation sequencing technology.Using the selected KASP markers to verify the results in the recombinant inbred line population, it was found that jy10392 and jy10551 were codominant markers, and the genotype and iron efficiency phenotype were highly consistent.

4. The genes in the candidate interval were analyzed. Through GO annotation analysis, seven genes that may be related to iron ion absorption were annotated, namely glyma.4g15170,glyma.4g15220, glyma.4g15397, glyma.5g16400, glyma.5g16287, glyma.5g16245 and glyma.5g16288.

[1] Liu KS. Soybeans: Chemistry, Technology and Utilization[J]. Soybeans Chemistry Technology & Utilization, 1997.
[2] 紫林.加大科研力度，强化国产食用大豆的市场地位——访国家大豆产业技术体系首席科学家、中国农业科学院作物科学研究所研究员韩天富[J].中国食品,2018(20):22-23.
[3] 中华人民共和国海关总署.2020年我国大豆进口量首次突破1亿吨（进出口监测预警专题）[EB/OL].
http://www.customs.gov.cn//customs/resource/cms/article/333551/3605482/2021040213270759945.doc, 2021-04-02
[4] Weiss M. Inheritance and Physiology of Efficiency in Iron Utilization in Soybeans [J]. Genetics, 1943, 28(3):253-268.
[5] Spiller S, Terry N. Limiting Factors in Photosynthesis: II. IRON STRESS DIMINISHES PHOTOCHEMICAL CAPACITY BY REDUCING THE NUMBER OF PHOTOSYNTHETIC UNITS[J]. Plant Physiology, 1980, 65(1):121-125.
[6] J. C. BROWN,R. S. HOLMES,L. O. TIFFIN. Iron chlorosis in soybeans as related to the genotype of rootstalk. [J]. Soil Science,1968, 96(2):387-394.
[7]D. Kramer,V. Römheld,E. Landsberg,H. Marschner. Induction of transfer-cell formation by iron deficiency in the root epidermis of Helianthus annuus L.[J]. Planta,1980,147(4):335-339
[8] Youxi Yuan,Huilan Wu,Ning Wang,Jie Li,Weina Zhao,Juan Du,Daowen Wang,Hong-Qing Ling. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis[J]. Cell Research,2008,18(3): 385-397.
[9]Liu Xing Xing,He Xiao Lin,Jin Chong Wei. Roles of chemical signals in regulation of the adaptive responses to iron deficiency.[J]. Plant signaling & behavior,2016,11(5),e1179418.
[10] Gayomba Sheena R.,Zhai Zhiyang,Jung Ha il, et al. Local and systemic signaling of iron status and its interactions with homeostasis of other essential elements[J]. Frontiers in Plant Science,2015:716-721
[11]Hirayama Takashi,Lei Gui Jie,Yamaji Naoki, et al.The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis.[J]. Plant & cell physiology,2018, 59(9):1739-1752.
[12] Marschner H, V Römheld, Kissel M . Different strategies in higher plants in mobilization and uptake of iron[J].Journal of Plant Nutrition, 1986,9:3-7.
[13]Fox, Tama, Christine, et al. MOLECULAR BIOLOGY OF CATION TRANSPORT IN PLANTS.[J]. Annual Review of Plant Physiology & Plant Molecular Biology, 1998, 49(1):669-669.
[14] Marschner V R & H . Plant‐induced pH changes in the rhizosphere of “Fe‐efficient” and “Fe‐inefficient” soybean and corn cultivars[J]. Journal of Plant Nutrition, 1984:1-7.
[15] Walker EL, Connolly EL. Time to pump iron: iron-deficiency-signaling mechanisms of higher plants[J]. Current Opinion in Plant Biology, 2008, 11(5):530-535.
[16] Korshunova YO, Eide D, Clark WG, et al. The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range[J]. Plant Molecular Biology, 1999, 40(1):37-44.
[17] Xiong H, Kakei Y, Kobayashi T, et al. Molecular evidence for phytosiderophore-induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil.[J]. Plant, Cell & Environment, 2013, 36(10):1888-1902.
[18] Astolfi S, Pii Y, Mimmo T, et al. Single and Combined Fe and S Deficiency Differentially Modulate Root Exudate Composition in Tomato: A Double Strategy for Fe Acquisition?[J]. International Journal of Molecular Sciences, 2020, 21(11):4038.
[19] Atencio Leorrie,Salazar Justin,Lauter Adrienne N. Moran,Gonzales Michael D.,O'Rourke Jamie A.,Graham Michelle A.. Characterizing short and long term iron stress responses in iron deficiency tolerant and susceptible soybean (Glycine max L. Merr.)[J]. Plant Stress,2021(prepublish)
[20]Römheld V. Different strategies for iron acquisition in higher plants[J]. Physiol. Plant, 1987, 70(2):231-234.
[21] Brown J. Iron-stress response in tomato (Lycopersicon esculentum) 1. Sites of Fe reduction, adsorption andtransport.[J]. Physiol Plant, 1974, 31.
[22] Jolley VD, Brown JC, Blaylock MJ, et al. A role for potassium inthe use of iron by plants. [J]. Plant Nutr, 1988.11(6-11): 1159–1175.
[23] Gibson AH. Methods for legumes in glasshouses and controlled environment cabinets[J]. Methods for Evaluating Biological Nitrogen Fixation, 1980.
[24] Soerensen KU, Terry RE, Jolley VD, et al. The interaction of iron﹕tress response and root nodules in iron efficient and inefficient soybeans[J]. Journal of Plant Nutrition, 1988, 11(6-11):853-862.
[25] Terry RE, Jolley VD. Nitrogenase activity is required for the activation of iron-stress response in iron-inefficient T203 soybean[J]. Journal of Plant Nutrition, 1994, 17(8):1417-1428.
[26] Pii Y, Marastoni L, Springeth C, et al. Modulation of Fe acquisition process by Azospirillum brasilense in cucumber plants[J]. Environmental & Experimental Botany, 2016, 130:216-225.
[27] Zanin L, Tomasi N, Cesco S, et al. Humic Substances Contribute to Plant Iron Nutrition Acting as Chelators and Biostimulants[J]. Frontiers in Plant Science, 2019,1-10.
[28] Cianzio SR, Fehr WR. Genetic control of iron deficiency chlorosis in soybeans.[J]. Iowa State J Res, 1980, 54.
[29] Prohaska KR, Fehr WR. Recurrent Selection for Resistance to Iron Deficiency Chlorosis in Soybeans[J]. Cropence, 1981, 21(4):524-526.
[30] Walter R. Fehr. Control of iron-deficiency chlorosis in soybeans by plant breeding[J]. Journal of Plant Nutrition,1982,5(4-7):611-621.
[31] Lin S, Cianzio S, Shoemaker R. Mapping genetic loci for iron deficiency chlorosis in soybean[J]. Mol Breed, 1997, 3(3):219-229.
[32] Lin SF, Grant D, Cianzio S, et al. Molecular characterization of iron deficiency chlorosis in soybean[J]. Journal of Plant Nutrition, 2000, 23(11-12):1929-1939.
[33] Charlson DV, Bailey TB, Cianzio SR, et al. Molecular Marker Satt481 is Associated with Iron-Deficiency Chlorosis Resistance in a Soybean Breeding Population[J]. Crop ence, 2005, 45(6):2394-2399.
[34]Dirk V. Charlson,Silvia R. Cianzio,Randy C. Shoemaker. Associating SSR Markers with Soybean Resistance to Iron Deficiency Chlorosis[J]. Journal of Plant Nutrition,2003,26(10-11):2267–2276.
[35]Ju Wang,Phillip E. McClean,Rian Lee,R. Jay Goos,Ted Helms. Association mapping of iron deficiency chlorosis loci in soybean ( Glycine max L. Merr.) advanced breeding lines[J]. Theoretical and Applied Genetics,2008,116(6):777-787.
[36]Jamie A O'Rourke,Rex T Nelson,David Grant, et al. Integrating microarray analysis and the soybean genome to understand the soybeans iron deficiency response[J]. BioMed Central,2009,10(1):1-17.
[37]Peiffer Gregory A.,King Keith E.,Severin Andrew J., et al. Identification of Candidate Genes Underlying an Iron Efficiency Quantitative Trait Locus in Soybean[J]. Plant Physiology,2012,158(4):1745-1754.
[38] Myles S, Peiffer J, Brown PJ, et al. Association Mapping: Critical Considerations Shift from Genotyping to Experimental Design[J]. Plant Cell, 2009, 21(8):2194-2202.
[39] Wang J, Mcclean PE, LeeR, et al. Association mapping of iron deficiency chlorosis loci in soybean (Glycine max L. Merr.) advanced breeding lines[J]. Theoretical & Applied Genetics, 2008, 116(6):777-787.
[40] Mamidi S, Chikara S, Goos RJ, et al. Genome-Wide Association Analysis Identifies Candidate Genes Associated with Iron Deficiency Chlorosis in Soybean[J]. Plant Genome, 2011, 4(3):154.
[41] Bailey-Serres J, Fukao T, Ronald P, et al. Submergence Tolerant Rice: SUB1's Journey from Landrace to Modern Cultivar[J]. Rice, 2010, 3(2-3):138-147.
[42] Salvi S, Tuberosa R. The crop QTLome comes of age.[J]. Curr Opin Biotechnol, 2015, 32:179-185.
[43] Gitishree D, Kumar PJ, Kwang-Hyun B. Insight into MAS: A Molecular Tool for Development of Stress Resistant and Quality of Rice through Gene Stacking[J]. Frontiers in Plant Science, 2017, 8: 985-994.
[44] Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors[J]. Proceedings of the National Academy of Sciences, 1977:5463–5467.
[45]Ashley N. Egan,Jessica Schlueter,David M. Spooner. Applications of next‐generation sequencing in plant biology[J]. American Journal of Botany,2012,99(2): 175-185.
[46] Poland JA, Rife TW. Genotyping-by-Sequencing for Plant Breeding and Genetics[J]. Plant Genome, 2012, 5(3):92-102.
[47] He J, Zhao X, Laroche A, et al. Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding[J]. Frontiers in Plant Science, 2014, 5(484):1-8.
[48] Spindel J, Wright M, Chen C, et al. Bridging the genotyping gap: using genotyping by sequencing (GBS) to add high-density SNP markers and new value to traditional bi-parental mapping and breeding populations[J]. Theoretical and Applied Genetics, 2013, 126(11):2699-2716.
[49] Huang X, Feng Q, Qian Q, et al. High-throughput genotyping by whole-genome resequencing[J]. Genome Research, 2009, 19(6):1068-1076.
[50]Robert J Elshire,Jeffrey C Glaubitz,Qi Sun, et al. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species.[J]. PLoS ONE,2017,6(5):e19379.
[51]Jarrod A Chapman,Martin Mascher,Aydın Buluç, et al. A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome[J]. Genome Biology,2015,16(1):26
[52] Phung N, Mai CD, Hoang GT, et al. Genome-wide association mapping for root traits in a panel of rice accessions from Vietnam[J]. BMC Plant Biology, 2016, 16(1):64.
[53] B. Emma Huang,Klara L. Verbyla,Arunas P., et al. MAGIC populations in crops: current status and future prospects[J]. Theoretical and Applied Genetics,2015,128(6):999–1017.
[54] Lu F, Romay MC, Glaubitz JC, et al. High-resolution genetic mapping of maize pan-genome sequence anchors[J]. Nature Communications, 2015, 6:6914.
[55] Michelmore RW, Kesseli I. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.[J]. Proceedings of the National Academy of Sciences of the United States of America, 1991:9828–9832.
[56] Zou C, Wang P, Xu Y. Bulked sample analysis in genetics, genomics and crop improvement[J]. Plant Biotechnology Journal, 2016:1941-1955.
[57] Win KT, Vegas J, Zhang C, et al. QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods[J]. Theoretical & Applied Genetics, 2017, 130(1):199-211.
[58] Wambugu P, Ndjiondjop MN, Furtado A, et al. Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice[J]. Plant Biotechnology Journal, 2017, 16(1):100-110.
[59] Hayward AC, Tollenaere R, Daltonmorgan J, et al. Molecular marker applications in plants.[J]. Methods in Molecular Biology, 2015, 1245:13.
[60] Paul M. Magwene,John H. Willis,John K. Kelly. The Statistics of Bulk Segregant Analysis Using Next Generation Sequencing[J]. PLOS Computational Biology,2011,7(11):1-9
[61] Thomson, Michael J. High-Throughput SNP Genotyping to Accelerate Crop Improvement[J]. Plant Breeding & Biotechnology, 2014, 2(3):195-212.
[62]Li, Zhong-feng, Guo, et al. Identification of the dwarf gene GmDW1 in soybean (Glycine max L.) by combining mapping-by-sequencing and linkage analysis[J]. Theoretical & Applied Genetics International Journal of Breeding Research & Cell Genetics, 2018.:1001-1016.
[63]Houqing Zeng,Guoping Wang,Yuqi Zhang, et al . Genome-wide identification of phosphate-deficiency-responsive genes in soybean roots by high-throughput sequencing[J]. Plant and Soil,2016,398(1): 207-227.
[64] 朱晓岚. 小黑豆抗胞囊线虫SHMT基因序列多样性及功能分析[D]. 沈阳农业大学, 2017.
[65]Ling Zhou,Shi-Bo Wang,Jianbo Jian, et al. Dunwell,Jin Zhang,Jian-Ying Feng,Yuan Niu,Li Zhang,Wen-Long Ren,Yuan-Ming Zhang. Identification of domestication-related loci associated with flowering time and seed size in soybean with the RAD-seq genotyping method[J]. Scientific Reports,2015,5(1):1-8.
[66] Wu XY, Zhou GC, Chen YX, et al. Soybean Cyst Nematode Resistance Emerged via Artificial Selection of Duplicated Serine Hydroxymethyltransferase Genes[J]. Frontiers in Plant Science, 2016, 7(17):998.
[67] 周航. 利用GmARR10基因创制大豆新种质[D].贵州大学,2017.
[68] 李俊成,于慧,杨素欣,冯献忠.植物对铁元素吸收的分子调控机制研究进展[J].植物生理学报,2016,52(06):835-842.
[69] 李晓勉. 高粱SbbHLH1转录因子与植物的铁营养平衡[D].山东大学,2020.
[70] 张伟,赵丽梅,韩喜国,徐长宏,赵婧,邱强,闫晓艳,张鸣浩.石灰性土壤大豆缺铁矫正[J].大豆科学,2011,30(03):463-467.
[71] 张伟,赵丽梅,韩喜国,徐长宏,赵婧,闫晓艳,张鸣浩.吉林省白城地区大豆黄叶原因分析[J].河南农业科学,2011,40(11):57-59.
[72]中华人民共和国农业农村部. 2007年9月大豆市场监测信息[EB/OL].http://www.moa.gov.cn/ztzl/jsshzyxnc/tgnyzhscnl/200710/t20071025_909636.htm,2007-10-25
[73] FroelichD, Fehr W. Agronomic Performance of Soybeans with Differing Levels of Iron Deficiency Chlorosis on Calcareous Soil[J]. Crop Science, 1981, 21:438-441.
[74] Le K, Nguyen, Alexandre, et al. Next-Generation Sequencing Accelerates Crop Gene Discovery.[J]. Trends in plant science, 2018.11.008:263-274.
[75] Diers BW, Cianzio SR, Shoemaker RC. Possible identification of quantitative trait loci affecting iron efficiency in soybean[J]. Journal of Plant Nutrition,1992,15(10):2127-2136
[76]Lin S, Cianzio S, Shoemaker R. Mapping genetic loci for iron deficiency chlorosis in soybean[J]. Mol Breed, 1997, 3(3):219-229.
[77] Lin SF, Baumer J, Ivers D, et al. Nutrient solution screening of Fe chlorosis resistance in soybean evaluated by molecular characterization[J]. Journal of Plant Nutrition, 2000, 23(11-12):1915-1928.
[78] Lin SF, Grant D, Cianzio S, et al. Molecular characterization of iron deficiency chlorosis in soybean[J]. Journal of Plant Nutrition, 2000, 23(11-12):1929-1939.
[79] Charlson DV, Bailey TB, Cianzio SR, et al. Molecular Marker Satt481 is Associated with Iron-Deficiency Chlorosis Resistance in a Soybean Breeding Population[J]. Crop ence, 2005, 45(6):2394-2399.
[80] Peiffer GA, King KE, Severin AJ, et al. Identification of Candidate Genes Underlying an Iron Efficiency Quantitative Trait Locus in Soybean[J]. Plant Physiology, 2012, 158(4):1745-1754.
[81] 张伟,邱强,赵婧,韩喜国,张伟龙,徐长洪,张鸣浩,闫晓艳.基于大豆叶绿素含量和生物产量积累的不同铁效率品种鉴定[J].东北农业科学, 2016,41(04):8-13.DOI:10.16423/ j.cnki.1003-8701.2016.04.003.
[82] 包悦琳. 低铁胁迫对大豆幼苗生理生化指标的影响研究[D].延边大学,2020.
[83]FEHR, WR. Control of iron-defIciency chlorosis in soybeans by plant-breeding [J]. J Plant Nutr, 1982,5(4-7), 611–621.
[84] Reumers J, Rijk PD, Hui Z, et al. Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing[J]. Nature Biotechnology, 2012, 30(1):61-68.
[85]Hill J T , Demarest B L , Bisgrove B W , et al. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq[J]. Genome Research, 2013, 23(4),687-697.
[86]F Rym, Hiroki T , Muluneh T , et al. MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice[J]. Plos One, 2013, 8(7):e68529.
[87]王婷婷,陈鸽,包悦琳,金东淳.大豆根系响应早期缺铁胁迫的转录组分析[J/OL].南
京农业大学学报:1-17[2021-11-29].http://kns.cnki.net/kcms/detail/32.1148.S.20211021.1441.002.html.
[88] 张雪萌. 大豆GmABCG5L基因的克隆及功能的初步鉴定[D].哈尔滨师范大学,2021.