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Inheritance and fine mapping of a restorer-of-fertility (Rf) gene for the cytoplasmic male sterility in soybean
Plant Science 188–189 (2012) 36–40
Contents lists available at SciVerse ScienceDirect
Plant Science journal homepage: www.elsevier.com/locate/plantsci
Inheritance and fine mapping of a restorer-of-fertility (Rf) gene for the cytoplasmic male sterility in soybean D.K. Dong a,1 , Z. Li b,1 , F.J. Yuan a , S.L. Zhu a , P. Chen c , W. Yu b , Q.H. Yang a , X.J. Fu a , X.M. Yu a , B.Q. Li a , D.H. Zhu a,∗ a
Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, PR China Fuyang Institute of Agricultural Sciences, Fuyang, Anhui 236031, PR China c Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA b
a r t i c l e
i n f o
Article history: Received 21 December 2011 Received in revised form 7 February 2012 Accepted 11 February 2012 Available online 17 February 2012 Keywords: Glycine max Cytoplasmic male sterility Restorer-of-fertility Genetic mapping
a b s t r a c t The cytoplasmic male sterility (CMS) line FuCMS5A and its restorer line FuHui9 were crossed to produce a segregating F2 population for pollen fertility assay and the genetic mapping of restorer-of-fertility (Rf) gene. Results showed that the individual F2 plants were fertile or semi-fertile based on their pollen fertility characteristics. The average ratios of viable pollen were 96.90% and 50.00% for each class of individuals. The segregation of F2 plants showed a good fit to a 1:1 ratio, which reflects a typical heredity pattern of gametophytic CMS with fertility restorer being controlled by a single dominant gene. Using bulk segregation analysis (BSA) and genetic mapping, the Rf gene was mapped on molecular linkage group J (chromosome 16), between the simple sequence repeat (SSR) makers BARCSOYSSR-161064 and BARCSOYSSR-16-1082 with the distances of 0.59 and 0.83 cM, respectively. Four SSR markers (BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077) were cosegregating with this Rf gene in the mapping population. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The exploitation of male sterile lines has made great contribution to crop breeding as they provide cost-effective methods for feasibly producing adequate F1 seeds in agricultural industry. Three types of male sterility systems, i.e. genic male sterility (GMS), photoperiod-thermo-sensitive genic male sterility (PTGMS) and cytoplasmic-nuclear male sterility (CMS) were discovered and used in various crops. However, the first two types of male sterile systems were less desirable due to the segregation of large proportion of fertile plants in the GMS system and the susceptible nature of
Abbreviations: Rf, restorer-of-fertility gene; CMS, cytoplasmic male sterility; SSR, simple sequence repeat; GMS, genic male sterility; PTGMS, photoperiod-thermosensitive genic male sterility; MAS, marker assisted selection; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; cM, centi-Morgan; ORF, open reading frames; LG, linkage group. ∗ Corresponding author at: 198#, Shiqiao Rd., Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, PR China. Tel.: +86 571 8640 4248; fax: +86 571 8640 0488. E-mail address: [email protected] (D.H. Zhu). 1 Contributed equally to this work. 0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2012.02.007
PTGMS system to the environments. So far, CMS is still the most practically applicable system for soybean hybrid breeding. CMS in soybean was firstly reported by Davis [1] in 1985 and further characterized by Sun et al. in China. Up to now, multiple sources of sterile cytoplasm were discovered, including Ru Nan Tian E Dan (designated as RN type), XXT (XX type), ZD8319 (ZD type), N8855, and N21566 [2–6]. With the three-line system of CMS, several soybean hybrids have been developed and released in China. These hybrids have showed a more than 20% yield advantage over the control varieties [7–10]. The success of CMS in hybrid breeding relies to a great extent on the capacity of fertility restoration when crossed with male parent lines which carry dominant restorer of fertility (Rf) genes that restore male fertility in hybrid cultivars. Despite its importance in soybean hybrid breeding, the genetic architecture of fertility restoration remains unclear. This makes the developing of new restorer lines a time-consuming and labor intensive process since the only way to identify the restorer lines is to cross lines in question with male sterile lines and assess the fertility of their offsprings. Up to date, several Rf genes have been mapped on various genetic loci using different genetic populations as shown in Table 1 [11–15]. In these previous mapping studies, various sources of male sterile cytoplasm systems were used with complicated mechanisms
D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
37
Table 1 SSR markers linked with Rf genes for soybean CMS in previous reports. Genetic population
Sources of sterile cytoplasm
Linked marker
Linkage group
Distance (cM)
Reference
JLCMS82A × JLR1 F2 JLCMS82A × JIHUI1 F2 NJCMS1A × Zhongdou 5 F2 NJCMS2A × Zhongdou 5 F2 N21566/N21249//N21249 BC1 F1 N21566 × N21249 F2
Ru Nan Tian E Dan
Satt547
J
7.56
Zhao et al. [11]
Ru Nan Tian E Dan
Sctt011 Satt547 Satt626 Satt300 Satt135
J
3.6 5.4 9.75 11.18 11.47
Wang et al. [12]
N8855 N8855 N21566
Satt331 Satt477 Satt331 Satt477
involved in male sterility. Some CMS were sporophytic and others were gametophytic type. Therefore, the relatively loose mapping of Rf gene is still far from being used for marker assisted selection (MAS). In this study we report on the hereditary analysis and fine mapping of an Rf gene for ZD type CMS in soybean. This may lead to isolation and cloning of this Rf specific gene. This work will greatly facilitate the maker assisted selection procedures in soybean CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene.
M A1 D2 O
10.4 19.2 8.1 13.3
Yang et al. [13] Dong et al. [14] Li et al. [15]
The CMS sterile line FuCMS5A was crossed with its restorer line FuHui9 to obtain the F1 hybrid seeds in 2009. Then the F1 seeds were grown to produce the segregating F2 population in 2010. The sterile line, restorer line and F2 seeds were grown in 2011 for pollen sterility analysis and Rf gene mapping.
for the region of interest. Information on specific SSR primers was from Cregan et al. [17], Song et al. [18] and the soybase database (http://www.soybase.org). The polymerase chain reaction (PCR) mixture (10 L in volume) consisted of 20 ng genomic DNA, 0.3 mol/L each primer, and 5 L GoTaq Green Mix (promega). A touchdown process was employed to increase the product specificity in consideration of various SSR primers with different annealing temperatures. The thermal cycling procedure was as following: pre-denatured at 94 ◦ C for 2 min; followed by 13 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 1 min, the annealing temperature decreased 1 ◦ C by each cycle; then followed by 25 cycles of 94 ◦ C for 30 s, 47 ◦ C for 30 s, and 72 ◦ C for 1 min; PCR products were finally extended at 72 ◦ C for 5 min and then stored at 4 ◦ C for further analysis. The PCR products were separated using 8% polyacrylamide gels electrophoresis (PAGE) in 1× TBE and then visualized by silver staining according to Xu et al. [19]. Briefly, the PAGE gels were stained in 0.1% silver nitrate solution for 10 min, washed for 30 s, and then visualized in 2% NaOH solution with 0.5% formaldehyde until the DNA bands appeared clearly.
2.2. Pollen sterility analysis
2.4. Data analysis
Blooming flowers from parents and the F2 individuals were sampled between 8:00 am and 11:00 am every day. Anthers were dissected, squashed into an aqueous solution of I2 -KI, and observed using microscope. The viable pollen grains were stained in dark color and they were in regular shape and uniform size. While sterile pollen grains were shown in yellow or light brown colors, with varying shapes or grain sizes. Pollen grains from three independent visual fields were counted according to their fertile characteristics. For a single plant, at least three flowers that sampled from different days were examined and scored to avoid sampling bias. On the basis of the ratios of viable pollens, the F2 plants were classified into two groups: fertile group (most pollen grains were viable) and semi-fertile group (about half of the pollen grains were viable).
Chi square test was performed using PROC FREQ procedure of SAS 8.0 software (SAS Institute Inc., Cary, NC). The Joinmap 3.0 program was used in linkage analysis [20]. Recombination frequencies were converted to map distances in centiMorgans (cM) by the Kosambi function [21]. Linkage map was produced using MapChart 2.2 software [22].
2. Materials and methods 2.1. Plant material
2.3. BSA analysis and genetic mapping Genomic DNAs were isolated from young leaves of both parents and the F2 individuals using CTAB method [16]. DNAs of 15 F2 individuals randomly selected from the fertile group were composited to create the fertile DNA pool. DNAs of 15 plants from the semi-fertile group were composited to form the semi-fertile DNA pool. Initially, 240 SSR primers from the 20 soybean linkage groups were screened for polymorphism among parents and the two DNA pools. After the preliminary genetic mapping of Rf gene region, new SSR markers were synthesized to increase the maker density
3. Results 3.1. Pollen fertility and the segregation of the F2 individuals A total of 174 F2 plants were examined for pollen fertility. The fertile individuals and semi-fertile individuals were clearly distinguished according to their pollen characteristics (Fig. 1). Eighty-six F2 plants were classified into fertile group, and the remaining 88 individuals were in semi-fertile group, while no individual with nearly 100% sterile pollen was observed in this study. The average fertile pollens were 96.90% and 50.00% for fertile and semi-fertile groups with ranges from 89.37% to 99.44% and 41.03% to 57.24%, respectively (Table 2). Chi square test showed that the segregation of fertile and semi-fertile individuals in the F2 population perfectly fit a 1:1 ratio (Table 2). 3.2. BSA analysis and molecular mapping of the Rf gene Initially, 240 SSR primers were screened for polymorphism among parents and the two bulked DNA pools. A total of 93 SSR showed polymorphism between parents with the polymorphic
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D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
Fig. 1. Pollen grains stained by I2 -KI (bars = 20 m). (a) Viable pollen grains were stained in dark color and they were in regular shape and uniform size. (b) Sterile pollen grains were shown in yellow or light brown colors, with varying shapes or grain sizes. (c) Pollen grains from semi-fertile F2 plants, about half pollen grains were in light color. (d) Pollen grains from another type of semi-fertile F2 plants, about half pollen grains were gigantic pollens or were in various sizes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 2 The proportion of fertile pollens and the Chi square test of segregation for fertile and semi-fertile plants in F2 population. Group
Fertile Semi-fertile
Fertile pollen ratio (%)
Chi square test
Min
Mean ± S.D.
Max
No. of individuals
Expected segregation ratio
2
P value
89.37 41.03
96.90 ± 2.23 50.00 ± 3.24
99.44 57.24
86 88
1:1
0.012
0.91
ratio of 38.75%. Only two primers, Sat 366 and Sat 224 were simultaneously polymorphic between fertile and semi-fertile DNA pools. Further analysis mapped the Rf gene on soybean linkage group (LG) J (chromosome 16), between Sat 366 and Sat 224. However, these markers were relatively far away from the Rf gene due to the high recombination rates (Figs. 2a and 3). Based on the preliminarily mapping results, new SSR primers for region between Sat 366 and Sat 224 were synthesized to improve the marker density for fine mapping. Thirteen new polymorphic markers were then obtained. For all these 15 markers that showed
polymorphism between DNA pools, heterozygotic amplicons of both parents were observed in the amplifications of semi-fertile DNA pool. Examples of these heterozygotic amplifications were shown in Fig. 2. Linkage analysis showed that all these 15 SSR markers (Sat 366, Sat 224, and 13 new SSR markers in this region) were linked with Rf gene with LOD scores overtopping 10. All markers were arranged in the identical order to those previously published except for some cosegregations, i.e. BARCSOYSSR-16-1056 and BARCSOYSSR-161057; BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and
Fig. 2. PAGE gel results of SSR markers. (a) Amplification of Sat 224, a number of recombinations were observed (lanes indicated by arrows). (b) Amplification of Sctt011. P1, the restorer line FuHui9; P2, the sterile line FuCMS5A; B1, Bulked DNA pool from fertile F2 individuals; B2, Bulked DNA pool from semi-fertile F2 individuals.
D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
Wang et al, (2010)
Lg J (Chromosome 16)
39
Reference Linkage Map
Satt215 Sat_366
Sat_366 Satt620
0.87 2.02
8.36
13.70
Sctt011
BARCSOYSSR-16-1053 BARCSOYSSR-16-1055 BARCSOYSSR-16-1056 BARCSOYSSR-16-1057 BARCSOYSSR-16-1064 BARCSOYSSR-16-1070 Sctt011 Rf (Restorer-of-Fertility) BARCSOYSSR-16-1076 BARCSOYSSR-16-1077 BARCSOYSSR-16-1082 BARCSOYSSR-16-1085 Satt244 Satt547
0.31 0.29 1.48 0.59 0.83
3.60 Rf
4.77 0.92
5.40 Satt547
Sat_350
7.16
Sctt011 2.15
Satt244
2.75 Satt547
1.51
Sat_396
5.82
3.67 Sat_224
a
b
c
Sat_224
Fig. 3. SSR markers closely linked with Rf gene of soybean CMS. Genetic distances (cM) between markers were on the left side of each linkage group. (a) The mapped Rf gene for CMS with RN type sterile cytoplasm by Wang et al. [12]. (b) The mapped Rf gene for CMS with ZD type sterile cytoplasm in our research. (c) Reference map according to Cregan et al. [17].
BARCSOYSSR-16-1077; BARCSOYSSR-16-1082 and BARCSOYSSR16-1085 (Fig. 3). With the additional SSR marker data, the Rf gene was mapped between BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082 on LG J (chromosome 16) with the distance of 0.59 cM and 0.83 cM, respectively. In addition, four SSR markers BARCSOYSSR-16-1070, Satt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077 were cosegregating with the Rf gene in our mapping population (Fig. 3). 4. Discussion Two types of CMS, i.e. sporophytic and gametophytic sterility have been reported in soybean. For example, the RN type, N21566 and Zhongyou89B male sterility system were classified as gametophytic male sterility, and their fertility restorations were controlled by one dominant gene [5,6,11]. However, the N8855-derived sterile lines were classified as sporophytic sterility with two Rf genes involved in the fertility restoration [3,13,14,23]. The sterile cytoplasm in this study was derived from the variety ZD8139 (also named Zhongdou19) [24], and designated as ZD type cytoplasm. Our results showed that only two classes of individuals with distinct fertility levels (fertile vs. semi-fertile) were observed in the F2 population according to their pollen sterilities, and the segregation of fertile vs. semi-fertile individuals fit in a ratio of 1:1, which was typical for gametophytic male sterility with fertility restoration controlled by one dominant gene. Therefore, we conclude that ZD type male sterility is a gametophytic male sterility with fertility restoration controlled by a single dominant gene. This confirms the results of Zhang et al. using a CMS line derived from Zhongyou89B which inherited the same source of sterile cytoplasm from ZD8319 (Zhongdou19) [5]. All SSR markers linked with the Rf gene in this study were arranged identically in order as compared to the reference map
or corresponded to their physical positions except for some cosegregations. This result testified the accuracy of this linkage group. The cosegregating events were possibly caused by the relatively close physical positions between markers and the relatively small genetic populations used in this study. Linkage analysis showed that the Rf gene for ZD type CMS was mapped on soybean LG J (chromosome 16), between SSR markers BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082. It is worth pointing out that another Rf gene for RN type CMS was also mapped in this region [11,12]. Since RN type CMS and ZD type CMS both exhibit as gametophytic male sterility and their Rf genes were mapped in the same region. There are two genetic possibilities: 1. RN and ZD type CMS lines possess materially identical male sterile cytoplasm as well as the Rf gene or 2. RN and ZD type CMS may contain different cytoplasms, but similar mechanisms (maybe the same Rf gene) are involved in the male sterility and sterility restoration. Further allelism test and comparative analysis of these two types of CMS will shed new light on the mechanisms involved in soybean CMS. Fine mapping in the F2 population delimited the Rf locus to an approximately 228 kb region on LG J according to the physical position of the linked SSR markers [18]. A total of 120 kb sequence around the Rf locus and the cosegregated markers were downloaded from Phytozome database and subsequently analyzed (data not shown). Eleven transcripts were annotated in Phytozome database, in which 3 were peroxidase related, 1 was triose-phosphate transporter related, 1 was DHHC-type zinc finger protein related and six others encode unknown proteins (data not shown). A number of open reading frames (ORF) with the length over 300 bp were also predicted. BlastX results showed that they were highly similar to Peroxidase, Zinc Finger Protein, Retrotransposon hopscotch polyprotein/Putative reverse transcriptase, Cytochrome bd biosynthesis ABC-type transporter (ATPase and
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D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
permease component), ABC superfamily ATP binding cassette transporter (ABC/membrane protein), Retrovirus-related like protein, or belong to unknown protein families. Further comparative sequencing and expression analysis are under way in our lab to identify the potential Rf gene for ZD type soybean CMS. Our results showed that soybean ZD type CMS is gametophytic male sterility; and the restoration of sterility is controlled by a single dominant gene. The Rf gene was mapped on LG J (chromosome 16) between SSR markers BARCSOYSSR-16-1064 and BARCSOYSSR16-1082, with the distance of 0.59 and 0.83 cM, respectively. Four markers were cosegregated with the Rf gene. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene. Acknowledgments This work was supported by “Zhejiang Provincial Nature Science Foundation of P.R. China (Y3080103)”, “Genetically Modified Organisms Breeding Major Projects of P.R. China (2012ZX08009004)” and “Special Fund for Agro-scientific Research in the Public Interest (201103007)”. References [1] W.H. Davis, Route to hybrid soybean production, US Patent, 1985. [2] H. Sun, L.M. Zhao, S.M. Wang, Y.Q. Wang, J.P. Li, A review on soybean heterosis and its utilization, Chin. J. Oil Crop Sci. 25 (2003) 92–96. [3] Y. Bai, J. Gai, Inheritance of male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A of soybean [Glycine max (L) Merr.], Euphytica 145 (2005) 25–32. [4] J.Y. Zhang, H. Sun, L.M. Zhao, B. Peng, W.L. Zhang, S.G. Li, X.M. Zhao, Classification of male-sterile lines with rn sterile cytoplasm and their restorers, Soybean Sci. 29 (2010) 559–564. [5] L. Zhang, Z.P. Huang, J.K. Li, Y.L. Zhang, C. Hu, O.H. Dai, Genetic study on soybean nucleo-cytoplasmic male sterile restore gene, J. Anhui Agr. Sci. 35 (2007) 2513–2515. [6] T. Zhao, J. Gai, Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic-nuclear male-sterile line NJCMS3A in soybean, Euphytica 152 (2006) 387–396.
[7] B. Peng, L.M. Zhao, S.M. Wang, Y.X. Cheng, H. Sun, Y.Q. Wang, J.P. Li, W.L. Zhang, Studies on breeding of ‘HybSoy 2’ soybean and high yield seed production technique, J. Jilin Agr. Sci. 33 (2008) 3–4. [8] B. Peng, L.M. Zhao, W.L. Zhang, J.Y. Zhang, H. Sun, S.M. Wang, J.P. Li, M.H. Li, A breeding report of hybrid soybean ‘HybSoy 3’, J. Jilin Agr. Sci. 35 (2010) 4–5. [9] L. Zhang, O.H. Dai, Z.P. Huang, J.K. Li, L.Y. Zhang, C. Hu, Breeding of hybrid soybean Zayoudou No. 1, Soybean Bull. 2 (2007) 14–16. [10] L.M. Zhao, H. Sun, S.M. Wang, Y.Q. Wang, M. Huang, J.P. Li, Breeding of hybrid soybean ‘HybSoy 1’, Chin. J. Oil Crop Sci. 26 (2004) 15–17. [11] L.M. Zhao, Y.M. Wang, H. Sun, H.K. Zhao, Y.X. Cheng, B. Peng, S.M. Wang, W.L. Zhang, Y.S. Dong, Identification of SSR markers linked to the fertility restorer gene for the CMS in soybean, Soybean Sci. 26 (2007) 835–839. [12] Y. Wang, L. Zhao, X. Wang, H. Sun, Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean, Plant Breed. 129 (2010) 9–12. [13] S.P. Yang, M.P. Duan, Q.C. Meng, J. Qiu, J.M. Fan, T.J. Zhao, D.Y. Yu, J.Y. Gai, Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean, Plant Breed. 126 (2007) 302–305. [14] J.S. Dong, S.P. Yang, D.Y. Yu, J.Y. Gai, Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male sterile line NJCMS2A in soybean, Soybean Sci. 27 (2008) 181–185. [15] S.G. Li, T.J. Zhao, J.Y. Gai, Mapping male fertility gene with SSR markers in parents of cytoplasmic-nuclear male-sterile line NJCMS3A in soybean, Acta Agron. Sin. 36 (2010) 1061–1066. [16] M.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res. 8 (1980) 4321–4325. [17] P.B. Cregan, T. Jarvik, A.L. Bush, R.C. Shoemaker, K.G. Lark, A.L. Kahler, N. Kaya, T.T. VanToai, D.G. Lohnes, J. Chung, J.E. Specht, An integrated genetic linkage map of the soybean genome, Crop Sci. 39 (1999) 1464–1490. [18] Q. Song, G. Jia, Y. Zhu, D. Grant, R.T. Nelson, E.-Y. Hwang, D.L. Hyten, P.B. Cregan, Abundance of SSR motifs and development of candidate polymorphic SSR markers (BARCSOYSSR 1.0) in soybean, Crop Sci. 50 (2010) 1950–1960. [19] S.B. Xu, Y.F. Tao, Z.Q. Yang, J.Y. Chu, A simple and rapid methods used for silver staining and gel preservation, Hereditas (Beijing) 24 (2002) 335–336. [20] J.W. Van Ooijen, R.E. Voorrips, JoinMap 3.0, Software for the Calculation of Genetic Linkage Maps, Plant Research International, Wageningen, the Netherlands, 2001. [21] D.D. Kosambi, The estimation of map distances from recombination values, Ann. Hum. Genet. 12 (1943) 172–175. [22] R.E. Voorrips, MapChart: software for the graphical presentation of linkage maps and QTLs, J. Hered. 93 (2002) 77–78. [23] Y.N. Bai, J.Y. Gai, Development of a new cytoplasmic-nuclear male-sterility line of soybean and inheritance of its male-fertility restorability, Plant Breed. 125 (2006) 85–88. [24] Z.Y. Xu, L. Li, L.J. Qiu, R.Z. Chang, M.B. Wang, Z. Li, P. Guo, Selection of three lines and localization of the restorer genes in soybean using SSR markers, Sci. Agric. Sin. 32 (1999) 32–38.
Contents lists available at SciVerse ScienceDirect
Plant Science journal homepage: www.elsevier.com/locate/plantsci
Inheritance and fine mapping of a restorer-of-fertility (Rf) gene for the cytoplasmic male sterility in soybean D.K. Dong a,1 , Z. Li b,1 , F.J. Yuan a , S.L. Zhu a , P. Chen c , W. Yu b , Q.H. Yang a , X.J. Fu a , X.M. Yu a , B.Q. Li a , D.H. Zhu a,∗ a
Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, PR China Fuyang Institute of Agricultural Sciences, Fuyang, Anhui 236031, PR China c Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701, USA b
a r t i c l e
i n f o
Article history: Received 21 December 2011 Received in revised form 7 February 2012 Accepted 11 February 2012 Available online 17 February 2012 Keywords: Glycine max Cytoplasmic male sterility Restorer-of-fertility Genetic mapping
a b s t r a c t The cytoplasmic male sterility (CMS) line FuCMS5A and its restorer line FuHui9 were crossed to produce a segregating F2 population for pollen fertility assay and the genetic mapping of restorer-of-fertility (Rf) gene. Results showed that the individual F2 plants were fertile or semi-fertile based on their pollen fertility characteristics. The average ratios of viable pollen were 96.90% and 50.00% for each class of individuals. The segregation of F2 plants showed a good fit to a 1:1 ratio, which reflects a typical heredity pattern of gametophytic CMS with fertility restorer being controlled by a single dominant gene. Using bulk segregation analysis (BSA) and genetic mapping, the Rf gene was mapped on molecular linkage group J (chromosome 16), between the simple sequence repeat (SSR) makers BARCSOYSSR-161064 and BARCSOYSSR-16-1082 with the distances of 0.59 and 0.83 cM, respectively. Four SSR markers (BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077) were cosegregating with this Rf gene in the mapping population. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The exploitation of male sterile lines has made great contribution to crop breeding as they provide cost-effective methods for feasibly producing adequate F1 seeds in agricultural industry. Three types of male sterility systems, i.e. genic male sterility (GMS), photoperiod-thermo-sensitive genic male sterility (PTGMS) and cytoplasmic-nuclear male sterility (CMS) were discovered and used in various crops. However, the first two types of male sterile systems were less desirable due to the segregation of large proportion of fertile plants in the GMS system and the susceptible nature of
Abbreviations: Rf, restorer-of-fertility gene; CMS, cytoplasmic male sterility; SSR, simple sequence repeat; GMS, genic male sterility; PTGMS, photoperiod-thermosensitive genic male sterility; MAS, marker assisted selection; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; cM, centi-Morgan; ORF, open reading frames; LG, linkage group. ∗ Corresponding author at: 198#, Shiqiao Rd., Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, PR China. Tel.: +86 571 8640 4248; fax: +86 571 8640 0488. E-mail address: [email protected] (D.H. Zhu). 1 Contributed equally to this work. 0168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2012.02.007
PTGMS system to the environments. So far, CMS is still the most practically applicable system for soybean hybrid breeding. CMS in soybean was firstly reported by Davis [1] in 1985 and further characterized by Sun et al. in China. Up to now, multiple sources of sterile cytoplasm were discovered, including Ru Nan Tian E Dan (designated as RN type), XXT (XX type), ZD8319 (ZD type), N8855, and N21566 [2–6]. With the three-line system of CMS, several soybean hybrids have been developed and released in China. These hybrids have showed a more than 20% yield advantage over the control varieties [7–10]. The success of CMS in hybrid breeding relies to a great extent on the capacity of fertility restoration when crossed with male parent lines which carry dominant restorer of fertility (Rf) genes that restore male fertility in hybrid cultivars. Despite its importance in soybean hybrid breeding, the genetic architecture of fertility restoration remains unclear. This makes the developing of new restorer lines a time-consuming and labor intensive process since the only way to identify the restorer lines is to cross lines in question with male sterile lines and assess the fertility of their offsprings. Up to date, several Rf genes have been mapped on various genetic loci using different genetic populations as shown in Table 1 [11–15]. In these previous mapping studies, various sources of male sterile cytoplasm systems were used with complicated mechanisms
D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
37
Table 1 SSR markers linked with Rf genes for soybean CMS in previous reports. Genetic population
Sources of sterile cytoplasm
Linked marker
Linkage group
Distance (cM)
Reference
JLCMS82A × JLR1 F2 JLCMS82A × JIHUI1 F2 NJCMS1A × Zhongdou 5 F2 NJCMS2A × Zhongdou 5 F2 N21566/N21249//N21249 BC1 F1 N21566 × N21249 F2
Ru Nan Tian E Dan
Satt547
J
7.56
Zhao et al. [11]
Ru Nan Tian E Dan
Sctt011 Satt547 Satt626 Satt300 Satt135
J
3.6 5.4 9.75 11.18 11.47
Wang et al. [12]
N8855 N8855 N21566
Satt331 Satt477 Satt331 Satt477
involved in male sterility. Some CMS were sporophytic and others were gametophytic type. Therefore, the relatively loose mapping of Rf gene is still far from being used for marker assisted selection (MAS). In this study we report on the hereditary analysis and fine mapping of an Rf gene for ZD type CMS in soybean. This may lead to isolation and cloning of this Rf specific gene. This work will greatly facilitate the maker assisted selection procedures in soybean CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene.
M A1 D2 O
10.4 19.2 8.1 13.3
Yang et al. [13] Dong et al. [14] Li et al. [15]
The CMS sterile line FuCMS5A was crossed with its restorer line FuHui9 to obtain the F1 hybrid seeds in 2009. Then the F1 seeds were grown to produce the segregating F2 population in 2010. The sterile line, restorer line and F2 seeds were grown in 2011 for pollen sterility analysis and Rf gene mapping.
for the region of interest. Information on specific SSR primers was from Cregan et al. [17], Song et al. [18] and the soybase database (http://www.soybase.org). The polymerase chain reaction (PCR) mixture (10 L in volume) consisted of 20 ng genomic DNA, 0.3 mol/L each primer, and 5 L GoTaq Green Mix (promega). A touchdown process was employed to increase the product specificity in consideration of various SSR primers with different annealing temperatures. The thermal cycling procedure was as following: pre-denatured at 94 ◦ C for 2 min; followed by 13 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s, and 72 ◦ C for 1 min, the annealing temperature decreased 1 ◦ C by each cycle; then followed by 25 cycles of 94 ◦ C for 30 s, 47 ◦ C for 30 s, and 72 ◦ C for 1 min; PCR products were finally extended at 72 ◦ C for 5 min and then stored at 4 ◦ C for further analysis. The PCR products were separated using 8% polyacrylamide gels electrophoresis (PAGE) in 1× TBE and then visualized by silver staining according to Xu et al. [19]. Briefly, the PAGE gels were stained in 0.1% silver nitrate solution for 10 min, washed for 30 s, and then visualized in 2% NaOH solution with 0.5% formaldehyde until the DNA bands appeared clearly.
2.2. Pollen sterility analysis
2.4. Data analysis
Blooming flowers from parents and the F2 individuals were sampled between 8:00 am and 11:00 am every day. Anthers were dissected, squashed into an aqueous solution of I2 -KI, and observed using microscope. The viable pollen grains were stained in dark color and they were in regular shape and uniform size. While sterile pollen grains were shown in yellow or light brown colors, with varying shapes or grain sizes. Pollen grains from three independent visual fields were counted according to their fertile characteristics. For a single plant, at least three flowers that sampled from different days were examined and scored to avoid sampling bias. On the basis of the ratios of viable pollens, the F2 plants were classified into two groups: fertile group (most pollen grains were viable) and semi-fertile group (about half of the pollen grains were viable).
Chi square test was performed using PROC FREQ procedure of SAS 8.0 software (SAS Institute Inc., Cary, NC). The Joinmap 3.0 program was used in linkage analysis [20]. Recombination frequencies were converted to map distances in centiMorgans (cM) by the Kosambi function [21]. Linkage map was produced using MapChart 2.2 software [22].
2. Materials and methods 2.1. Plant material
2.3. BSA analysis and genetic mapping Genomic DNAs were isolated from young leaves of both parents and the F2 individuals using CTAB method [16]. DNAs of 15 F2 individuals randomly selected from the fertile group were composited to create the fertile DNA pool. DNAs of 15 plants from the semi-fertile group were composited to form the semi-fertile DNA pool. Initially, 240 SSR primers from the 20 soybean linkage groups were screened for polymorphism among parents and the two DNA pools. After the preliminary genetic mapping of Rf gene region, new SSR markers were synthesized to increase the maker density
3. Results 3.1. Pollen fertility and the segregation of the F2 individuals A total of 174 F2 plants were examined for pollen fertility. The fertile individuals and semi-fertile individuals were clearly distinguished according to their pollen characteristics (Fig. 1). Eighty-six F2 plants were classified into fertile group, and the remaining 88 individuals were in semi-fertile group, while no individual with nearly 100% sterile pollen was observed in this study. The average fertile pollens were 96.90% and 50.00% for fertile and semi-fertile groups with ranges from 89.37% to 99.44% and 41.03% to 57.24%, respectively (Table 2). Chi square test showed that the segregation of fertile and semi-fertile individuals in the F2 population perfectly fit a 1:1 ratio (Table 2). 3.2. BSA analysis and molecular mapping of the Rf gene Initially, 240 SSR primers were screened for polymorphism among parents and the two bulked DNA pools. A total of 93 SSR showed polymorphism between parents with the polymorphic
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Fig. 1. Pollen grains stained by I2 -KI (bars = 20 m). (a) Viable pollen grains were stained in dark color and they were in regular shape and uniform size. (b) Sterile pollen grains were shown in yellow or light brown colors, with varying shapes or grain sizes. (c) Pollen grains from semi-fertile F2 plants, about half pollen grains were in light color. (d) Pollen grains from another type of semi-fertile F2 plants, about half pollen grains were gigantic pollens or were in various sizes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 2 The proportion of fertile pollens and the Chi square test of segregation for fertile and semi-fertile plants in F2 population. Group
Fertile Semi-fertile
Fertile pollen ratio (%)
Chi square test
Min
Mean ± S.D.
Max
No. of individuals
Expected segregation ratio
2
P value
89.37 41.03
96.90 ± 2.23 50.00 ± 3.24
99.44 57.24
86 88
1:1
0.012
0.91
ratio of 38.75%. Only two primers, Sat 366 and Sat 224 were simultaneously polymorphic between fertile and semi-fertile DNA pools. Further analysis mapped the Rf gene on soybean linkage group (LG) J (chromosome 16), between Sat 366 and Sat 224. However, these markers were relatively far away from the Rf gene due to the high recombination rates (Figs. 2a and 3). Based on the preliminarily mapping results, new SSR primers for region between Sat 366 and Sat 224 were synthesized to improve the marker density for fine mapping. Thirteen new polymorphic markers were then obtained. For all these 15 markers that showed
polymorphism between DNA pools, heterozygotic amplicons of both parents were observed in the amplifications of semi-fertile DNA pool. Examples of these heterozygotic amplifications were shown in Fig. 2. Linkage analysis showed that all these 15 SSR markers (Sat 366, Sat 224, and 13 new SSR markers in this region) were linked with Rf gene with LOD scores overtopping 10. All markers were arranged in the identical order to those previously published except for some cosegregations, i.e. BARCSOYSSR-16-1056 and BARCSOYSSR-161057; BARCSOYSSR-16-1070, Sctt011, BARCSOYSSR-16-1076 and
Fig. 2. PAGE gel results of SSR markers. (a) Amplification of Sat 224, a number of recombinations were observed (lanes indicated by arrows). (b) Amplification of Sctt011. P1, the restorer line FuHui9; P2, the sterile line FuCMS5A; B1, Bulked DNA pool from fertile F2 individuals; B2, Bulked DNA pool from semi-fertile F2 individuals.
D.K. Dong et al. / Plant Science 188–189 (2012) 36–40
Wang et al, (2010)
Lg J (Chromosome 16)
39
Reference Linkage Map
Satt215 Sat_366
Sat_366 Satt620
0.87 2.02
8.36
13.70
Sctt011
BARCSOYSSR-16-1053 BARCSOYSSR-16-1055 BARCSOYSSR-16-1056 BARCSOYSSR-16-1057 BARCSOYSSR-16-1064 BARCSOYSSR-16-1070 Sctt011 Rf (Restorer-of-Fertility) BARCSOYSSR-16-1076 BARCSOYSSR-16-1077 BARCSOYSSR-16-1082 BARCSOYSSR-16-1085 Satt244 Satt547
0.31 0.29 1.48 0.59 0.83
3.60 Rf
4.77 0.92
5.40 Satt547
Sat_350
7.16
Sctt011 2.15
Satt244
2.75 Satt547
1.51
Sat_396
5.82
3.67 Sat_224
a
b
c
Sat_224
Fig. 3. SSR markers closely linked with Rf gene of soybean CMS. Genetic distances (cM) between markers were on the left side of each linkage group. (a) The mapped Rf gene for CMS with RN type sterile cytoplasm by Wang et al. [12]. (b) The mapped Rf gene for CMS with ZD type sterile cytoplasm in our research. (c) Reference map according to Cregan et al. [17].
BARCSOYSSR-16-1077; BARCSOYSSR-16-1082 and BARCSOYSSR16-1085 (Fig. 3). With the additional SSR marker data, the Rf gene was mapped between BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082 on LG J (chromosome 16) with the distance of 0.59 cM and 0.83 cM, respectively. In addition, four SSR markers BARCSOYSSR-16-1070, Satt011, BARCSOYSSR-16-1076 and BARCSOYSSR-16-1077 were cosegregating with the Rf gene in our mapping population (Fig. 3). 4. Discussion Two types of CMS, i.e. sporophytic and gametophytic sterility have been reported in soybean. For example, the RN type, N21566 and Zhongyou89B male sterility system were classified as gametophytic male sterility, and their fertility restorations were controlled by one dominant gene [5,6,11]. However, the N8855-derived sterile lines were classified as sporophytic sterility with two Rf genes involved in the fertility restoration [3,13,14,23]. The sterile cytoplasm in this study was derived from the variety ZD8139 (also named Zhongdou19) [24], and designated as ZD type cytoplasm. Our results showed that only two classes of individuals with distinct fertility levels (fertile vs. semi-fertile) were observed in the F2 population according to their pollen sterilities, and the segregation of fertile vs. semi-fertile individuals fit in a ratio of 1:1, which was typical for gametophytic male sterility with fertility restoration controlled by one dominant gene. Therefore, we conclude that ZD type male sterility is a gametophytic male sterility with fertility restoration controlled by a single dominant gene. This confirms the results of Zhang et al. using a CMS line derived from Zhongyou89B which inherited the same source of sterile cytoplasm from ZD8319 (Zhongdou19) [5]. All SSR markers linked with the Rf gene in this study were arranged identically in order as compared to the reference map
or corresponded to their physical positions except for some cosegregations. This result testified the accuracy of this linkage group. The cosegregating events were possibly caused by the relatively close physical positions between markers and the relatively small genetic populations used in this study. Linkage analysis showed that the Rf gene for ZD type CMS was mapped on soybean LG J (chromosome 16), between SSR markers BARCSOYSSR-16-1064 and BARCSOYSSR-16-1082. It is worth pointing out that another Rf gene for RN type CMS was also mapped in this region [11,12]. Since RN type CMS and ZD type CMS both exhibit as gametophytic male sterility and their Rf genes were mapped in the same region. There are two genetic possibilities: 1. RN and ZD type CMS lines possess materially identical male sterile cytoplasm as well as the Rf gene or 2. RN and ZD type CMS may contain different cytoplasms, but similar mechanisms (maybe the same Rf gene) are involved in the male sterility and sterility restoration. Further allelism test and comparative analysis of these two types of CMS will shed new light on the mechanisms involved in soybean CMS. Fine mapping in the F2 population delimited the Rf locus to an approximately 228 kb region on LG J according to the physical position of the linked SSR markers [18]. A total of 120 kb sequence around the Rf locus and the cosegregated markers were downloaded from Phytozome database and subsequently analyzed (data not shown). Eleven transcripts were annotated in Phytozome database, in which 3 were peroxidase related, 1 was triose-phosphate transporter related, 1 was DHHC-type zinc finger protein related and six others encode unknown proteins (data not shown). A number of open reading frames (ORF) with the length over 300 bp were also predicted. BlastX results showed that they were highly similar to Peroxidase, Zinc Finger Protein, Retrotransposon hopscotch polyprotein/Putative reverse transcriptase, Cytochrome bd biosynthesis ABC-type transporter (ATPase and
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permease component), ABC superfamily ATP binding cassette transporter (ABC/membrane protein), Retrovirus-related like protein, or belong to unknown protein families. Further comparative sequencing and expression analysis are under way in our lab to identify the potential Rf gene for ZD type soybean CMS. Our results showed that soybean ZD type CMS is gametophytic male sterility; and the restoration of sterility is controlled by a single dominant gene. The Rf gene was mapped on LG J (chromosome 16) between SSR markers BARCSOYSSR-16-1064 and BARCSOYSSR16-1082, with the distance of 0.59 and 0.83 cM, respectively. Four markers were cosegregated with the Rf gene. These makers will greatly facilitate the maker assisted selection procedures in CMS breeding programs and it lays a foundation for further map-base cloning of the Rf gene. Acknowledgments This work was supported by “Zhejiang Provincial Nature Science Foundation of P.R. China (Y3080103)”, “Genetically Modified Organisms Breeding Major Projects of P.R. China (2012ZX08009004)” and “Special Fund for Agro-scientific Research in the Public Interest (201103007)”. References [1] W.H. Davis, Route to hybrid soybean production, US Patent, 1985. [2] H. Sun, L.M. Zhao, S.M. Wang, Y.Q. Wang, J.P. Li, A review on soybean heterosis and its utilization, Chin. J. Oil Crop Sci. 25 (2003) 92–96. [3] Y. Bai, J. Gai, Inheritance of male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A of soybean [Glycine max (L) Merr.], Euphytica 145 (2005) 25–32. [4] J.Y. Zhang, H. Sun, L.M. Zhao, B. Peng, W.L. Zhang, S.G. Li, X.M. Zhao, Classification of male-sterile lines with rn sterile cytoplasm and their restorers, Soybean Sci. 29 (2010) 559–564. [5] L. Zhang, Z.P. Huang, J.K. Li, Y.L. Zhang, C. Hu, O.H. Dai, Genetic study on soybean nucleo-cytoplasmic male sterile restore gene, J. Anhui Agr. Sci. 35 (2007) 2513–2515. [6] T. Zhao, J. Gai, Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic-nuclear male-sterile line NJCMS3A in soybean, Euphytica 152 (2006) 387–396.
[7] B. Peng, L.M. Zhao, S.M. Wang, Y.X. Cheng, H. Sun, Y.Q. Wang, J.P. Li, W.L. Zhang, Studies on breeding of ‘HybSoy 2’ soybean and high yield seed production technique, J. Jilin Agr. Sci. 33 (2008) 3–4. [8] B. Peng, L.M. Zhao, W.L. Zhang, J.Y. Zhang, H. Sun, S.M. Wang, J.P. Li, M.H. Li, A breeding report of hybrid soybean ‘HybSoy 3’, J. Jilin Agr. Sci. 35 (2010) 4–5. [9] L. Zhang, O.H. Dai, Z.P. Huang, J.K. Li, L.Y. Zhang, C. Hu, Breeding of hybrid soybean Zayoudou No. 1, Soybean Bull. 2 (2007) 14–16. [10] L.M. Zhao, H. Sun, S.M. Wang, Y.Q. Wang, M. Huang, J.P. Li, Breeding of hybrid soybean ‘HybSoy 1’, Chin. J. Oil Crop Sci. 26 (2004) 15–17. [11] L.M. Zhao, Y.M. Wang, H. Sun, H.K. Zhao, Y.X. Cheng, B. Peng, S.M. Wang, W.L. Zhang, Y.S. Dong, Identification of SSR markers linked to the fertility restorer gene for the CMS in soybean, Soybean Sci. 26 (2007) 835–839. [12] Y. Wang, L. Zhao, X. Wang, H. Sun, Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean, Plant Breed. 129 (2010) 9–12. [13] S.P. Yang, M.P. Duan, Q.C. Meng, J. Qiu, J.M. Fan, T.J. Zhao, D.Y. Yu, J.Y. Gai, Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean, Plant Breed. 126 (2007) 302–305. [14] J.S. Dong, S.P. Yang, D.Y. Yu, J.Y. Gai, Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male sterile line NJCMS2A in soybean, Soybean Sci. 27 (2008) 181–185. [15] S.G. Li, T.J. Zhao, J.Y. Gai, Mapping male fertility gene with SSR markers in parents of cytoplasmic-nuclear male-sterile line NJCMS3A in soybean, Acta Agron. Sin. 36 (2010) 1061–1066. [16] M.G. Murray, W.F. Thompson, Rapid isolation of high molecular weight plant DNA, Nucleic Acids Res. 8 (1980) 4321–4325. [17] P.B. Cregan, T. Jarvik, A.L. Bush, R.C. Shoemaker, K.G. Lark, A.L. Kahler, N. Kaya, T.T. VanToai, D.G. Lohnes, J. Chung, J.E. Specht, An integrated genetic linkage map of the soybean genome, Crop Sci. 39 (1999) 1464–1490. [18] Q. Song, G. Jia, Y. Zhu, D. Grant, R.T. Nelson, E.-Y. Hwang, D.L. Hyten, P.B. Cregan, Abundance of SSR motifs and development of candidate polymorphic SSR markers (BARCSOYSSR 1.0) in soybean, Crop Sci. 50 (2010) 1950–1960. [19] S.B. Xu, Y.F. Tao, Z.Q. Yang, J.Y. Chu, A simple and rapid methods used for silver staining and gel preservation, Hereditas (Beijing) 24 (2002) 335–336. [20] J.W. Van Ooijen, R.E. Voorrips, JoinMap 3.0, Software for the Calculation of Genetic Linkage Maps, Plant Research International, Wageningen, the Netherlands, 2001. [21] D.D. Kosambi, The estimation of map distances from recombination values, Ann. Hum. Genet. 12 (1943) 172–175. [22] R.E. Voorrips, MapChart: software for the graphical presentation of linkage maps and QTLs, J. Hered. 93 (2002) 77–78. [23] Y.N. Bai, J.Y. Gai, Development of a new cytoplasmic-nuclear male-sterility line of soybean and inheritance of its male-fertility restorability, Plant Breed. 125 (2006) 85–88. [24] Z.Y. Xu, L. Li, L.J. Qiu, R.Z. Chang, M.B. Wang, Z. Li, P. Guo, Selection of three lines and localization of the restorer genes in soybean using SSR markers, Sci. Agric. Sin. 32 (1999) 32–38.