Characterization of dwarf mutants and molecular mapping of a dwarf locus in soybean

Journal of Integrative Agriculture 2016, 15(10): 2228–2236 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Characterization of dwarf mutants and molecular mapping of a dwarf locus in soybean CHENG Wen1, GAO Jin-shan2, FENG Xing-xing2, SHAO Qun1, YANG Su-xin2, FENG Xian-zhong1, 2 1

Key Laboratory of Systems Biology in Universities of Shandong/College of Life Science, Shandong Normal University, Jinan 250014, P.R.China

2

Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, P.R.China

Abstract Plant height is one of the most important traits in soybean. The semi-dwarf soybean cultivars could improve the ability of lodging resistance to obtain higher yield. To broaden the dwarfism germplasm resources in soybean, 44 dwarf mutants were identified from a gamma rays mutagenized M2 population. Two of these mutants, Gmdwf1 (Glycine max dwarf 1) and Gmdwf2 (Glycine max dwarf 2), were investigated in this study. Genetic analysis showed that both mutants were inherited in a recessive manner and their mutated regions were delimited to a 2.610-Mb region on chromosome 1 by preliminary mapping. Further fine mapping study proved that the two mutants had a common deletion region of 1.552 Mb in the target region, which was located in a novel locus site without being reported previously. The dwarfism of Gmdwf1 could not be rescued by gibberellin (GA) and brassinolide (BR) treatments, which indicated that the biosynthesis of these hormones was not deficient in Gmdwf1. Keywords: soybean, dwarf mutant, mapping, BR, GA

1. Introduction Induced plant mutation breeding had been widely used for creating genetic variability in yield contributing traits and to improve the yield of crop plants. The use of induced mutants in plant breeding programs has led to the official release of more than 2 700 plant mutant varieties throughout the world till 2009, which cover most of cultivated crops (Shu

Received 8 October, 2015 Accepted 22 February, 2016 Correspondence FENG Xian-zhong, Tel: +86-431-85655051, Fax: +86-431-85542298, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61312-0

2009). Gamma rays irradiation is a major physical technique used to generate plant mutations that cause potentially useful agronomical traits for decades to improve in major seed-propagated crops, such as wheat, rice, barley, cotton, peanuts, and beans. Soybean gamma rays irradiation inducing mutation breeding was begun in the early 1950s, and nearly hundred varieties resulting from this method have been released to date in the world (Shu 2009; Hwang et al. 2014). Plant height is one of the most important agronomic traits for crop architecture and yield. Dwarfism could improve lodging resistance, which is one of a desirable agronomic characteristic for plant breeding. The ‘Green Revolution’ was a breakthrough that increased crop yield by introgression of semi-dwarf traits into cereal crop cultivars in the beginning of the 20th century (Peng et al. 1999; Khush 2001; Hedden 2003). It has been reported that soybean seed yield in-

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creases by 350 kg ha–1 and the lodging score increases by 0.008 for every 10 cm increase in plant height in breeding study (Wilcox and Sediyama 1981). Many determinate semi-dwarf soybean cultivars were released and showed high-yielding and lodging-resistance in environments, however, the inheritance of a mutation for dwarfishness has not been intensively studied and few dwarfing genes have been isolated in soybean (Hwang et al. 2015). Plant height is determined by complex genetic networks, and a variety of plant height-related genes have been identified and cloned in recent years (Yamaguchi 2008; Piao et al. 2014). Most of these genes are involved in phytohormone synthesis and/or signal transduction; gibberellins (GAs) and brassinosteroids (BRs) are two major hormones involved in plant height regulation (Wang and Li 2008). Mutations in the biosynthesis or perception of GAs and BRs always produce dwarf phenotypes (Sun 2011; Yang et al. 2011; Li et al. 2014). Many rice GA-related mutants have been reported to show dwarf phenotypes with rough and deep green leaves (Sakamoto et al. 2004), and several rice BR-deficient mutants also show dwarf phenotypes but with abnormal morphologies including abnormal leaves with twisted and stiff blades (Hong et al. 2004). Other plant hormones are also involved in plant height determination; jasmonic acid inhibits plant growth by repressing GA biosynthetic gene transcription (Heinrich et al. 2013), and strigolactone stimulates cell division to regulate internode elongation rather than acting on cell elongation, which is independent of GAs (de Saint et al. 2013). Other genes also participate in plant height determination, such as genes related to the cell wall (Endler and Persson 2011) and polyamine synthesis (Imai et al. 2006; Takahashi and Kakehi 2009), homeotic genes (Jasinski et al. 2005) and transcription factors (Dubouzet et al. 2003; Curaba et al. 2004; Yamaguchi 2008; Guo et al. 2010). Few molecular studies have been reported in plant height of soybean recently. RNA-seq analysis revealed that not only gibberellin-related genes but also many other genes involved in hormone biosynthetic pathways are affected in the Gmdwarf1 mutant (Zhang et al. 2014). Another soybean dwarf mutant was identified from a fast neutron-irradiated mutant population with reduced plant height, and further genome-wide analysis showed an 803 bp deletion region (including the first partial exon of Glyma15g05831) that may be responsible for the dwarf phenotype (Hwang et al. 2015). In this study, 44 novel dwarf mutants were screened from a M2 gamma rays mutagenesis population of a Chinese local cultivar, Hedou 12. A new genetic locus controlling soybean plant height was mapped to the upper arm of chromosome 1. The dwarfism of Gmdwf1 could not be rescued by GA or BR treatment, which suggested that this mutant was not

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deficient in BR or GA biosynthesis.

2. Materials and methods 2.1. Plant materials 10 000 dry seeds of Hedou 12, a local Chinese cultivar, were mutated with gamma rays at a dose of 200 Gy at the Atomic Energy Agency of Shandong Academy of Agricultural Sciences in China in April, 2009. The mutagenized seeds were grown in June, 2009 and 6 865 M2 lines were harvested in October, 2009. Twenty progeny seeds of each M2 line were sowed in the field. The phenotypes of the M2 plants were observed from July to October, 2010. Forty-four dwarf mutants were identified from the M2 population during the growing season. Twelve progeny seeds of each dwarf mutant line were sowed to confirm the plant height phenotype in a greenhouse during the winter of 2010 and 20 seeds from each M3 dwarf mutant line were sowed at Shandong Normal University field in June, 2011 for further backcrossing and genetic analysis. The Gmdwf1 and Gmdwf2 progenies from the M3 generation were backcrossed to the wild type, Hedou 12, for four generations to purify the genetic background from July 2011 to October 2012. The Gmdwf1 and Gmdwf2 mutants were crossed with the cultivar Williams 82 in the summer of 2013 and the F2 segregating populations were used for mapping in 2014. Reciprocal crossings between Gmdwf1 and Gmdwf2 were performed for allelic detection and the progeny phenotypes were analyzed in 2014.

2.2. Phenotype analysis The plant heights of Gmdwf1 and Gmdwf2 were measured from the cotyledonary to the apex at R8 stage grown in the field. The internodes were counted from the cotyledonary node to the apex node at R8 stage. Pods sizes were captured at R6 stage. Leaves of the 7th node were collected during V9 stage to produce a digital image by flattening and scanning with a HP scanner (LaserJet Pro 200 color MP, Co., USA), and their middle leaflet of compound leaves’ areas were calculated using the Image J software (http:// rsb.info.nih.gov/ij/). Pod numbers were counted manually at R8 stage. All above data were collected from 12 individual plants.

2.3. Genetic mapping and deletion detection InDel markers for mapping were developed based on the whole genome re-sequencing of Hedou 12 (Song et al. 2015) (Appendix A). Mutants from the F2 segregating populations between Gmdwf1 and Williams 82 and between

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Gmdwf2 and Williams 82 were used for genetic mapping. DNA was extracted from young leaves using the cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980). The polymerase chain reaction (PCR) protocol used to amplify the genetic markers was 3 min at 95°C for one cycle, 30 s at 95°C, 30 s at 54°C, and 30 s at 72°C for 36 cycles, and 10 min at 72°C for one cycle. Each PCR reaction consisted of a final concentration of 1× PCR buffer, including 1.5 mmol L–1 MgCl2, 0.5 mmol L–1 dNTP mix, 0.25 μmol L–1 forward/reverse primers, about 100 ng genomic DNA and 1 U of Taq polymerase (TaKaRa, Japan) in a 20-μL final volume. The PCR conditions of detection of the deletion in target region were the same as genetic mapping. The marker informing for fine mapping and deletion detection was listed in Appendix A.

2.4. GA and BR treatments Gmdwf1 and Hedou 12 seeds were sowed in pots in a greenhouse (12 h light and 12 h dark at 23°C). Seven days after sowing, four seedlings of each group were smeared with GA solutions (0, 0.5, 1, and 5 μmol L–1) every 2 days in the morning for four times, and the phenotype was observed on the 9th day after the first treatment, each treatment repeated three times. For BR treatment, Gmdwf1 and Hedou 12 seeds were germinated in 12-cm glass plates covered with wet Whatman 3 mm papers in dark at 23°C for 72 h. Four germinated seeds transferred into square growth boxes covered with nylon mesh containing 1/2 Hoagland solution with different BR

concentrations (0, 0.02 and 0.1 μmol L–1) in greenhouse (12 h light and 12 h dark at 23°C) for 19 d , three replicates for each treatment. The plant height was observed in 22 days after sowing.

3. Results 3.1. Identification of dwarf mutants In this study, 10 000 Hedou 12 seeds were treated with gamma rays at a dose of 200 Gy, and 1 547 visible mutants were obtained from M2 population. Among these mutants, 44 dwarf mutants were observed (Fig. 1). Of the 44 dwarf mutants, 17 of them exhibited stunted growth and low seed yield (plant height less than 40 cm, while the wild type is over 100 cm at R8 stage), 27 of them showed semi-dwarf (plant height higher than 40 cm but less than wild type) and lodging resistance in field environment. Besides of dwarf phenotype, 9 mutants had pale green leaves, 4 with curled leaves and 4 were semi-fertile. In the M3 generation, 41 dwarf mutants derived from the M2 mutants were heritable; only 3 mutant lines did not exhibit dwarf phenotype (Appendix B). Of the M3 heritable dwarf mutants, two of them with severe dwarf phenotype were further investigated in this study (Fig. 2-A and B).

3.2. Characterization of the Gmdwf1 and Gmdwf2 mutants Both Gmdwf1 and Gmdwf2 mutants had shorter stems and

Fig. 1 Photos of dwarf mutants identified in the field from M2 generation. Red arrows indicate the mutant plants, the wild type Hedou 12 plants are shown in the right side.

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fewer internodes. The plant height of the wild type (Hedou 12) was (116.33±3.30) cm (n=12) at R8 stage. However, the heights of Gmdwf1 and Gmdwf2 were reduced to (18.67±3.73) cm (n=12) (16.05% of the wild type) and (16.67±1.80) (n=12) cm (14.33% of the wild type), respectively, when they were grown in the same field (Fig. 2-E). The internode number was reduced from 26.00±1.29 (n=12) in the wild type to 17.50±0.96 (n=12) and 17.30±2.98 (n=12) in the Gmdwf1 and Gmdwf2 mutants, respectively (Fig. 2-F). The average internode length decreased from 4.67 to 1.07 cm in Gmdwf1 and 0.96 cm in Gmdwf2 in field, respectively. Apart from the reduced plant height phenotype, both mutants also showed small, twisted leaves with short petioles (Fig. 2-C and G). The leaf size of Gmdwf1 was 25% of the wild type and Gmdwf2 was 20% of the wild type. The pod number was only 17% of the wild type in Gmdwf1 and 14% in Gmdwf2 (Fig. 2-H). However, there was no significant difference in pod size and seed size among the

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wild type and the two mutants (Fig. 2-D, Appendix C). The 100-seed weight of Hedou 12 is (25.09±1.17) g, while the Gmdwf1 is (24.57±1.31) g and Gmdwf2 is (24.16±1.32) g (Appendix C). This indicted that the dwarf mutation affected the number of pod and thereby yield. Although the architecture of both mutants had no significant difference with the wild type, however, they could produce more branches than Hedou 12 (no branches produced) while it was sowed in greenhouse (Appendix C). The difference of branching phenotype between field and greenhouse might be due to the variations of light, humidity and nutrition conditions in two growth conditions.

3.3. Gmdwf1 and Gmdwf2 dwarfism was controlled by a single recessive mutation To analyze the genetic properties of Gmdwf1 and Gmdwf2, the two mutants were backcrossed with the wild type plant,

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Fig. 2 Phenotypes of the Gmdwf1 and Gmdwf2 mutants. A, the seedling of Gmdwf1 (left) and Hedou 12 (right) at the V9 stage, bar=10.0 cm. B, the seedling of Gmdwf2 (left) and Hedou 12 (right) at the V9 stage, bar=10.0 cm. C, the middle leaflet of the seventh compound leaf of Gmdwf1, Gmdwf2 and Hedou 12 at the V9 stage, bar=5.0 cm. D, pods of Hedou 12, Gmdwf1 and Gmdwf2 at the R6 stage, bar=1.0 cm. E, comparisons of plant height among Hedou 12, Gmdwf1 and Gmdwf2 at R8 stage (n=12). F, comparisons of internode number among Hedou 12, Gmdwf1 and Gmdwf2 at R8 stage (n=12). G, comparisons of middle leaflet area of the 7th compound leaf among Hedou 12, Gmdwf1 and Gmdwf2 at V9 stage (n=12). H, comparisons of pod number of Hedou 12, Gmdwf1 and Gmdwf2 at R8 stage (n=12). ***, Student’s t-test, P<0.001.

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between Gmdwf1 and Gmdwf2 showed the same dwarf phenotype as Gmdwf1, which indicated that Gmdwf2 was an allelic mutation of Gmdwf1. This result suggested that Gmdwf1 and Gmdwf2 were controlled by a single recessive locus.

3.4. Genetic mapping of Gmdwf1 and Gmdwf2

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Twenty-one dwarf individuals from the F2 mapping population of Gmdwf1 and Willams 82 were used for preliminary mapping. After 106 InDel markers were used for genotyping, two InDel markers on chromosome 1, InDel_001 (2 recombination events/42 tested chromosomes) and InDel_010 (0 recombination events/44 tested chromosomes) were found to be closely linked to the mutant phenotype in this population (Fig. 3-A). The rest 195 dwarf plants from the same F2 population were used for further mapping. Finally, Gmdwf1 mutation locus was delimited to a 2.610-Mb region between InDel_125 marker (10.036 Mb on chromosome 1) and InDel_012 marker (12.646 Mb on chromosome 1) (Fig. 3-A). For fine mapping, seven pairs of primers between 10.036 and 12.646 Mb on chromosome 1 were developed to nar-

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Hedou 12. The BC1F1 plants showed the same plant height as wild type. In the BC1F2 populations, 95 dwarf mutants and 301 wild type plants were segregated in Gmdwf1 population (fitted segregation ratio of WT:mutant=3:1, χ2=0.284, P=0.594>0.05), and 32 dwarf mutants and 104 wild type in Gmdwf2 population (fitted segregation ratio of WT:mutant=3:1, χ2=0.080, P=0.777>0.05). These results indicated that both Gmdwf1 and Gmdwf2 were caused by a single recessive mutation. To identify the genetic locus responsible for the Gmdwf1 and Gmdwf2, two independent mapping populations were established between these two mutants and Williams 82 respectively. We observed that 195 dwarf plants from 823 plants of F2 population originated from the crossing between Gmdwf1 and Williams 82 (fitted segregation ratio of WT:mutant=3:1, χ2=0.3812, P=0.5370>0.05) and 36 dwarf plants from 149 plants of F2 population derived from the crossing between Gmdwf2 and Willams82 (fitted segregation ratio of WT:mutant=3:1, χ2=0.0283, P=0.866>0.05). This result also confirmed that both mutants behaved as single recessive mutations. The progeny from eight seeds of the reciprocal crossing

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Fig. 3 Map-based cloning of Gmdwf1 and Gmdwfa loci. A, genetic mapping of Gmdwf1 and Gmdwf2. The horizontal line represents soybean chromosome 1; InDel markers, deletion checking primers and the number of recombinants between the marker and Gmdwf1 locus are shown above and below vertical lines. The red broken horizontal line represents the deletion region. B, the deletion region of Gmdwf1 checking by polymerase chain reaction (PCR). The DNA templates of lanes 1, 3, 5, and 7 are Hedou 12; lanes 2, 4, 6, and 8 are Gmdwf1. The primers of lanes 1 and 2 are OL_003; lanes 3 and 4 are OL_005; lanes 5 and 6 are OL_002; lanes 7 and 8 are OL_004. M, DL2000 DNA marker (TaKaRa, Japan). C, the deletion region of Gmdwf2 checking by PCR. The DNA templates of lanes 1, 3, 5, and 7 are Hedou 12; lanes 2, 4, 6, and 8 are Gmdwf2. The primers of lanes 1 and 2 are InDel_014; lanes 3 and 4 are OL_006; lanes 5 and 6 are InDel_012; lanes 7 and 8 are InDel_004. M, DL2000 DNA marker (TaKaRa).

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row down the target region (Appendix A). Four of them, OL_005(10.507 Mb), OL_001(11.392 Mb), OL_007(12.024 Mb), and OL_002(12.059Mb), produced clear PCR bands from the wild type Hedou 12 and Williams 82, but did not amplify PCR products from both dwarf plants of the F2 population and Gmdwf1 mutant (Fig. 3-B). These results indicate that there is a deletion in the genome of Gmdwf1. To find the border region of this deletion, another two pairs of primers, OL_003 (10.503 Mb) and OL_004 (12.070 Mb) were employed to check this deletion. With these two pairs of primers, we obtained the same PCR products in both the mutants and wild type plants (Fig. 3-B). These results indicated that the deletion region located between 10.507 and 12.059 Mb of chromosome 1, which covered 1.552 Mb genome region (Fig. 3-A). Genetic analysis showed that Gmdwf2 was an allelic mutation of Gmdwf1, and independent preliminary mapping confirmed that Gmdwf2 was also located in the same region of chromosome 1. Two pairs of primers, OL_006 (10.049 Mb) and InDel_012 (12.646 Mb), which were outside of two pairs of primers, OL_005 and OL_002, did not produce any PCR products in Gmdwf2. These results indicated Gmdwf2 has a bigger deletion at least between OL_006 (10.049 Mb) and InDel_012 (12.646 Mb) (Fig. 3-C). Above results displayed that both of Gmdwf1 and Gmdwf2 shared the 1.552 Mb deletion region of chromosome 1, which was responsible for the dwarf phenotype.

3.5. Candidate genes for the dwarf phenotype Fifty-eight genes have been predicted in above mapped 2.610 Mb region from the Glycine max Wm82.a2.v1 soybean gene annotation database (accessible at Phytozome ver. 10.0, www.phytozome.net) (Appendix D). Of the 58 candidate genes, 42 genes were in the 1.552 Mb deletion region while the other 16 were not. When we conducted de novo gene annotation with AUGUSTUS (Stanke et al. 2006) for this sequence and queried the next-Gen sequence databases for small RNAs (http://mpss.udel.edu/soy_sbs), no additional genes or small RNAs were predicted in this region. Among 42 genes located in the deletion region, Glyma.01G066600 is the most possible candidate gene, which is predicted to be orthologous to Arabidopsis thaliana BIM2 (BES1-interacting Myc-like protein 2). BIM2 is reported to be involved in BR responses (Yin et al. 2005). Glyma.01G069400 is another possible candidate gene, which encodes a phytosulfokine-alpha (PSK) precursor which is a unique plant peptide growth factor. Phytosulfokine (PSK) is a secreted disulfated pentapeptide that controls root and shoot growth depending on BR (Stuhrwohldt et al. 2011; Hartmann et al. 2013; Sauter 2015). Glyma.01G070200 is a peroxidase superfamily protein, which is a possible can-

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didate gene as well. Hwang et al. (2015) recently reported deletion of Glyma15g05831 (peroxidase superfamily protein) caused stunted soybean, as peroxidases involved in the metabolism of indole-3-acetic acid (IAA) through a conventional hydrogen-peroxide-dependent pathway. This region also contains three transcription factors, Glyma.01G067800 encodes a putative transcription factor (MYB92), Glyma.01G068600 encodes basic helix-loop-helix (bHLH) DNA-binding superfamily protein, Glyma.01G069300 is basic leucine-zipper 42 (bZIP42) and involved in regulation of transcription. Apart from above genes in this region, there are 23 predicted genes related to metabolism, kinase, DNA helicase, RNA polymerase and pre-mRNA splicing, cell structure, immunity, four genes with no homologues, and nine genes with no functional annotation.

3.6. Dwarfism of Gmdwf1 could not be rescued by GA and BR We tested whether exogenous GA could rescue the Gmdwf1 dwarf phenotype. Hedou 12 and Gmdwf1 seedlings were treated with 0.5, 1 and 5 μmol L–1 GA after the VC stage (unfolding of the unifoliate leaves, 7 days after sowing). The plant height of Hedou 12 increased rapidly after treated with GA, compared with the treatment of water (Fig. 4-A, Appendix E). Furthermore, 5 μmol L–1 GA treatment of Hedou 12 produced much longer internodes compared with the 0.5 and 1 μmol L–1. However, there was no significant difference among Gmdwf1 seedlings treated with different concentrations of GA and water (Fig. 4-A, Appendix E). These results showed that the dwarfism of Gmdwf1 could not be rescued by GA, and the GA signal transduction might be inhibited in Gmdwf1. Gmdwf1 and Hedou 12 seedlings were also treated with BR to examine their effects on their growth. When treated with 0.02 μmol L–1 BR, both Hedou 12 and Gmdwf1 produced more roots; but with 0.1 μmol L–1 BR, the root numbers were extremely reduced in both Hedou 12 and Gmdwf1. The plant height of Hedou 12 became shorter when treated with 0.1 μmol L–1 BR, however, we have not detected significant difference of the plant height of Gmdwf1 when treated with 0.1 μmol L–1 BR (Fig. 4-B, Appendix E). These results indicated that the dwarf phenotype of Gmdwf1 could not be rescued by exogenous BR, but both Hedou 12 and Gmdwf1 could respond BR to show root hair phenotypes.

4. Discussion Plant height is one of the most important agronomic traits of crops. Gamma rays irradiation mutagenesis has been used to produce important mutants, which represent promising materials for molecular biological and genetic

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A Hedou 12 Hedou 12 Hedou 12 Gmdwf1

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Fig. 4 A, seedlings of Hedou 12 and Gmdwf1 treated with different concentrations of GA, bar=10.0 cm. B, seedlings of Hedou 12 and Gmdwf1 treated with different concentrations of BR, bar=10.0 cm. Control indicated as a solution with ethanol at the same concentration of that in gibberellin (GA) or brassinolide (BR) solution.

studies. In this study, 44 dwarf mutants were screened from a gamma-ray-treated mutant population, which will facilitate the identification of plant height-related genes in the future. Twenty-seven of them showed semi-dwarf and lodging resistance in field environment, and this provides a good genetic resource for improving lodging-resistance in soybean breeding. In soybean, there are many loci involved in plant height determination and 195 plant height-related QTLs distributed on 20 linkage groups have been reported

(data from www.soybase.org) (Grant et al. 2009). Previous studies reported that there were two QTLs located on linkage group D1a (chromosome 1), named plant height 6–8 (Lark et al. 1995) and plant height 24–7 (Chen et al. 2007), the former QTL links to marker Sat_305, at a physical position of 51.50 Mb of chromosome 1, and the latter QTL links to the marker Satt502, at a physical position of 26.40 Mb. Neither of them lies in our mapped target region (10.507 and 12.059 Mb), this indicates that Gmdwf1 locus represents a novel locus involved in soybean plant height determination. Plant height depends on both cell division and cell expansion of the stem. Except GA and BR, IAA is also involved in regulation of stem elongation (Achard et al. 2009; Lee et al. 2012; de Saint et al. 2013). GA promotes cell elongation by accelerating the degradation of nuclear DELLAs (Swain and Olszewski 1996; Silverstone et al. 2001; Olszewski et al. 2002; Achard et al. 2009). BR promotes GA accumulation by regulating the expression of GA metabolism-related genes to stimulate cell elongation (Tong et al. 2014). IAA enhances internode elongation by increasing bioactive GA levels (Ross et al. 2000, 2003). GA can also stimulate cell division by modulating cell cycle activity in the root meristem via a DELLA-dependent mechanism (Achard et al. 2009). The BR signal pathway is also involved in cell cycling that affects cell division (Cheon et al. 2010; Jiang et al. 2012). Although we proved that both GA and BR could not rescue the dwarf phenotype of Gmdwf1, we could not rule out that other phytohormones such as IAA and strigolactone might be also play roles in this mutant. Among these 42 candidate genes in the targeting region of Gmdwf1 locus, soybean BIM2 homologue (Glyma.01G066600), PSK precursor (Glyma.01G069400) and peroxidase superfamily protein (Glyma.01G070200) are three possible candidates for controlling plant height. However, all the candidate genes are mainly from bioinformatics analysis, their functions still need to be proved by experiments in the future. Plant height is determined by both length and number of stem internode. Besides the internode length, the internode numbers are also decreased in both Gmdwf1 and Gmdwf2. This implied that the shoot initiation may be affected by the mutations as well. This hypothesis is supported by the lost apical dominance phenotype of mutants in greenhouse (Appendix C). As the current determined 1.552 Mb of Gmdwf1 locus is still a wide region, we suspect that either the target gene regulated both length and number of stem internode, or the Gmdwf1 mutant phenotype exhibited more than one gene’s functions.

5. Conclusion In this study, 44 soybean dwarf mutants were screened from a gamma ray induced M2 population, which provided a useful

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resource for soybean breeding. A novel locus regulating soybean plant height was identified between 10.507 and 12.059 Mb of chromosome 1 by genetic mapping of Gmdwf1 and Gmdwf2 mutants.

Acknowledgements This study was supported by the National Natural Science Foundation of China (31171571 and 31571692) and the One Hundred Person Project of the Chinese Academy of Sciences. Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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