Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

Sudthana Khlaimongkhon, Sriprapai Chakhonkaen, Keasinee Pitngam, Khanittha Ditthab, Numphet Sangarwut,Natjaree Panyawut, Thiwawan Wasinanon, Chareerat Mongkolsiriwatana, Julapark Chunwongse, Amorntip Muangprom,



Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

Sudthana Khlaimongkhon1, Sriprapai Chakhonkaen2, Keasinee Pitngam2, Khanittha Ditthab2, Numphet Sangarwut2,Natjaree Panyawut2, Thiwawan Wasinanon2, Chareerat Mongkolsiriwatana3, Julapark Chunwongse1, Amorntip Muangprom1, 2

(Center for Agricultural Biotechnology, Kasetsart University, Nakhon Pathom 73140, Thailand; National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand; Division of Genetics, )

The discovery of thermo-sensitive genic male sterility (TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F2populations derived from crosses between IR68301S, anTGMS rice line, and IR14632 (), Supanburi 91062 () and IR67966-188-2-2-1 (), respectively. Approximately 1:3 ratio between sterile and normal pollen of F2plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat (SSR) and insertion-deletion (InDel) markers were used to identify markers linked to thegene. The linkage analysis based on the three populations indicated that thelocus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F2population, the results showed that thelocus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from thegene to these two flanking markers were 1.10 and 0.82 cM, respectively. InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to thegene using F2populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

hybrid rice; thermo-sensitive genic male sterility; insertion/deletion; simple sequence repeat; marker-assisted selection

Rice (L.) is one of the most important global food crops.The world’s population has doubled since the early 1960s, therefore, existing yields of the major cereal crops will be insufficient to meet the food needs of the future (FAO, 2018). In addition, arable lands have declined worldwide, thus an increase in yield will be necessary to meet this demand (Bruinsma, 2003). Rice production in Thailand signifies a portion of the Thai economy and is a major export product.However,yield of Thai rice is less than that of the neighboring countries such as Indonesia, Vietnam, Malaysia, Laos and the Philippines (USDA, 2016). Yields have increased with the development of hybrid rice currently planted in many countries. The trend of hybrid rice development in both commercialization and research of private companies has been increasing. Hybrid rice technology has increased yields by 20%–30% under unchanged irrigation conditions (Yuan, 1998; Virmani, 2003). Hybrid rice seeds of large scale commercial production have been released to the market in China since 1976. The Ministry of Agriculture of China develops super rice breeding programs to increase rice yield per planting area, resulting thataverage rice yieldsin China rose from 1.89 t/hm2in 1949 to 6.71 t/hm2in 2012 (Cao and Zhan, 2014). In Thailand, average rice yield is only 2.89 t/hm2in 2018 (Thai Rice ExporterAssociation, 2018). Many countries which adopt hybrid rice technology are able to achieve higher yields.

Hybrid rice seed production involves the use of male sterility systems. Two well established male sterility systems in rice are cytoplasmic genetic male sterility (CMS), a three-line system, and environmentally sensitive genic male sterility (EGMS), a two-line system including photoperiod-sensitive genic male sterility (PGMS) and thermo-sensitive genic male sterility (TGMS). The discovery of PGMS and TGMS has led to development of a simple and highly efficient two-line breeding system in hybrid rice seed production. Several EGMS-related genes have been mapped such as(Wang et al, 1995),(Yamaguchi et al, 1997; Pitnjam et al, 2008),(Subudhi et al, 1997;Lang et al, 1999),(Dong et al, 2000),(Wang et al, 2003;Nas et al, 2005; Jiang et al, 2006; Yang et al, 2007),(Lee et al, 2005),(t) (Li et al, 2005),(Hussain et al, 2011),(Sheng et al, 2013),(Qi et al, 2014),(Peng et al, 2010),(Liu et al, 2001; Zhou et al, 2011),(Zhanget al,1994),(Dinget al,2012a, b),(Xu et al, 2011),(Zhou et al, 2012),(Peng et al, 2008) and(Jia et al, 2001). Candidate genes for EGMS have been reported, Myb-like DNA-binding domain containing protein (Zhou et al, 2011), a nuclear ribonuclease Z gene (Xu et al, 2011; Zhou et al, 2014), ORMDL (Pitnjam et al, 2008; Chueasiri et al, 2014) and the putative pollen specific protein encoded by(Nas et al, 2005). In addition, several genes controlling male sterility phenotype have been reported in rice such as undeveloped() (Jung et al, 2005), tapetum degeneration retardation () (Li et al, 2006), CER-like protein causing wax-deficient() (Jung et al, 2006), and LRR receptor kinase affecting(Nonomura et al, 2003).

IR68301S is a TGMS rice line obtained from International Rice Research Institute (IRRI), the Philippines. Male sterility in IR68301S is stable in a high temperature (≥ 28ºC), and it has been used for the development of two-line hybrid in Thailand. In this study, we mapped a gene controlling TGMS in IR68301S, and identified candidate genes using expression analysis. The knowledge in this study will be useful for rice breeding programs.

Materials and methods

Rice materials and phenotypic screening

An F2population, generated from a cross between IR68301S (B2), anTGMS mutant rice line, and IR14632 (B30), arice line, was used for identification of linked markers to thegene. These plants were obtained from IRRI. In addition, the other F2populations generated from crosses between B2 and Supanburi 91062 (B11), a Thairice line having high yield, and between B2 and IR67966-188-2-2-1(KM9), arice line, were also used. Theandcrosses were used to increase chances for polymorphic marker identification, and then these markers were used for linkage analysis. The F2populations were planted in paddy fields for observation on pollen sterility/fertility in summer of 2011, 2012 and 2013, in which the maximum temperatures were 33 ºC–37 ºC. Pollen sterility and fertility of the F2plants were evaluated on flowering day by observing anthers with the naked eyes and under a microscope after staining with 1% I2-KI solution. The χ2test was applied to determine the F2segregation ratio of sterility and fertility.

Genotyping and linkage analysis

Genomic DNA was extracted from fresh leaves of individual F2sterile plants generated from all the three crosses using the cethy trimethylammonium bromide (CTAB) method (Murray and Thomson, 1980). Polymorphic markers between parents and bulked segregant analysis (BSA) using pooled DNA from 10 individuals each for fertile and sterile plants, were applied to identify linked markers togene. The resulting linked markers were used to determine the genotype of the individual F2plants. Primers for single nucleotide polymorphism (SNP), and insertion-deletion (InDel) markers were designed from sequences obtained from GRAMENE database (http://www.gramene.org) andRiceVarMap (http://ricevarmap.ncpgr.cn/).Amplification of DNA fragments using simple sequence repeat (SSR) and InDels was performed as previously described by McCouch et al (2002) and Pitnjam et al (2008). The resulting PCR products were electrophoretically separated on 3% agarose gel or 6% acrylamide gel, and DNA patterns were observed by ethidium bromide or silver staining.The recombination frequency () was calculated with the formula:

= (1+2)/

whereis the total number of sterile plants surveyed,1is the number of sterile individuals with the homozygous band of the fertile parent, and2is the number of individuals with heterozygous bands (Peng et al, 2010). Recombination frequency was converted into genetic distance (cM) for linkage analysis.

Expression analysis

Segregating wild type male fertile and TGMS male sterile BC3F5plants generated from Pathum Thani 1, a Thai elite line, crossed with B2 were used for expression analysis, These plants were grown under natural condition at National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (14º04′50.6′′N and 100º36′09.0′′E), Pathum Thani Province, Thailand. Pre-germinated grains wereseeded in a tray and then the seedlings were transplanted into plastic pots (without hole at the bottom, diameter of 20.5 cm and height of 16.0 cm) with one seedling per pot. About one month before flowering, each of at least five plants/line were moved to controlled growth rooms at temperatures(24 ± 2) ºC and (32 ± 2) ºC under 80% relative humidity, 12 h light/12 h darkness until flowering. For expression analysis, young panicles about 13−14 cm in length (dyad stage) were harvested from BC3F5wild type and TGMS mutant plants at the same day. Total RNA was extracted using TRIZOLTMreagent (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) experiments were performed with cDNA synthesis kit (Fermentus, Lithuania) using cDNAs transcribed from total RNA. Based on information in GRAMENE database, genes located between two flanking markers tightly linked togene were used for expression analysis by qRT-PCR.The qRT-PCR was performed using Bio-RAD iCycler iQ5 Machine (BioRAD, USA) and all reactions were conducted in 96-well plates (BioRad, USA). The qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems) assay was used in a total volume of 10 µL per reaction. Each reaction mixture contained 1 µL cDNA, 2µL distilled water, 1 µL (10 µmol/L) forward primer, 1 µL (10 µmol/L) reverse primer and 5 µL of 2× qPCRBIO SyGreen Mix. All qRT-PCR plates were carried out with following cycling condition: 95 ºC for 3 min, following 35 cycles of 95 ºC for 30 s, 57 ºC to 63 ºC for 30 s and 72 ºC for 30 s, then 95 ºC for 1 min and a melting curve from 60 ºC to 95 ºC in 0.5 ºC increments.was used as an internal control. Three replications were performed for all reactions. The 2−ΔΔCtmethod was used to calculate relative gene expression(Livak and Schmittgen, 2001). The significantly of qRT-PCR were calculated byprogram (http://cran.r-project.org/)with ANOVA and Duncan’s new multiple range test.

Results

Pollen staining

Mature anthers of the TGMS mutant and wild type grown in controlled growth rooms under fertile and sterile conditions were observed. There was no pollen produced in anthers of TGMS mutant plants under sterile condition (32 ± 2) ºC (Fig. 1-A), while underthe fertile condition of (24 ± 2) ºC, pollen production was similar to that of wild type plants (Fig. 1-B). At these two conditions, wild type plants produced normal pollen (Fig. 1-C and -D).

Fig. 1. Pollen staining of thermo-sensitive genic male sterility (TGMS) mutant and wild type.

A, Abortive anther of the TGMS mutant grown under high temperature (32 ± 2)ºC. B, Normal anther of the TGMS mutant grown under low temperature (24 ± 2) ºC. C, Normal anther of wild type grown under high temperature (32 ± 2) ºC. D, Normal anther of wild type grown under low temperature (24 ± 2) ºC.

Genetic analysis of tms gene controlling TGMS in IR68301S

By growing the F2populations under sterile condition of(32 ± 2) ºC, the segregation patterns of fertile to sterile plants in the F2populations were near to 3:1 ratio in all the three populations (Table 1). The results indicated that the TGMS of IR68301S was controlled by a single recessive gene.

Mapping of tms gene controlling TGMS in IR68301S

A total of 119 SSR, 10 SNP and 283 InDel markers were used to identify polymorphism between parents from the three crosses. The results showed that 53, 3 and 7 markers were polymorphic between the parents from B2 × B30, B2 × B11 and B2 × KM9, respectively.

The resulting polymorphic markers were applied to identify linked markers to thegene using BSA. And 24 out of the 53 markers showed linkage to thegene in F2male sterile plants from B2 × B30. In addition, three and four markers showed linkage to thegene in F2male sterile plants from B2 × B11 and B2 × KM9, respectively (Supplemental Table 1). According to GRAMENE and RiceVarMap databases, these markers are on chromosome 2 at the position of 4.19 to 7.44 Mb.

Table 1. Segregation patterns of F2 populations for fertility and sterility.

B2, IR68301S; B30, IR14632; B11, Supanburi 91062; KM9, IR67966-188-2-2-1.

The value of χ20.05is 3.84.

The linked markers identified from each cross were used to genotype the F2male sterile. These F2male sterile plants selected from flowering plants in summers (33 ºC–37 ºC) were completely sterile. A total of 217, 259 and 450 F2male sterile plants from B2 × B30, B2 × B11 and B2 × KM9 were used for genotyping, respectively. Segregation patterns were shown in Fig. 2. A linkage analysis based on the three F2populations showed that thegene in IR68301S was located on chromosome 2 (Fig. 3). Using F2sterile individuals of B2 × B30, the results showed that thegene was located between SSR marker RM12676 at 5.74 Mb and InDel marker 2gAP0050058 at 5.81 Mb. The genetic distance of thegene from the two markers was 1.10 and 0.82 cM, respectively. In addition, InDel marker 2gAP004045 showed complete co-segregation with the TGMS phenotype (Fig. 3-A). Using F2sterile individuals of B2 × B11, the results showed thatgene was located between RM126016 at 4.69 Mb and InDel marker vf0206114052 at 6.11 Mb. The genetic distance of thegene from the two markers was 14.97 and 2.94 cM, respectively (Fig. 3-B). Using F2population of B2 × KM9, the results showed that four SSR markers, RM126016, RM12649, RM6378 and RM12674 were linked to thegene. The genetic distance of thegene from these markers were 12.50, 8.75, 8.25 and 6.25 cM, respectively (Fig. 3-C).

Expression analysis of candidate genes located inside the flanking markers

Based on information in GRAMENE database, there are ten genes (Table 2) located between the two flanking markers, RM12676 and 2gAP0050058. Nine out of the ten genes were annotated as expressed proteins, and one was annotated as a conserved hypothetical protein. These genes were tested for expression in panicles of wild type and TGMS mutantplants under fertile and sterile conditions. The results showed that the expressions of seven genes were detected in the tested tissues, but those of,andcould not be detected (data not shown). All the seven expressed genes showed different expression levels under fertile and sterile conditions or between the TGMS mutant and the wild type. Genes,andshowed significantly different expression levelsbetween the two conditions in the TGMS rice plants but no significantly different expression levels were detected between the two conditions in the wild-type rice plants (Fig. 4).andshowed higher expression levelsunder fertile conditionthan sterile condition, whileshowed higher expression levelsunder sterile conditionthanthe fertile conditionsThese three genes showed higher expression levels in the TGMS mutants than in the wild type under both conditions. These two genesandshowed a similar pattern of expression both in the mutant and wild-type rice plants, by showing higher expression under lower temperature condition. These genesandshowed a similar pattern of expression by showing higher expression under lower temperature condition in the TGMS mutants, but higher expression under higher temperature condition in wild-type plants (Fig. 4).

Fig. 2. Samples of genotyping F2male sterile plants.

P1, Female parent (IR68301S, B2); P2, Male parent (IR14632, B30); 1–10, F2male sterile individuals of B2 × B30 (×) using Os02g12370 marker; P3, Male parent (Supanburi 91062, B11); 11–20, F2male sterile individuals of B2 × B11 (×) using vf0206114052 marker.

Fig. 3. Genetic linkage map of thermo-sensitive genic male sterility gene on chromosome 2.

The linkage maps were analyzed based on male sterile F2populations of IR68301S × IR14632 (A), IR68301S × Supanburi 91062 (B) and IR68301S × IR67966-188-2-2-1 (C). Distances of each marker in centiMorgans (cM) fromgene were given on the left side of the genetic map.

Table 2. Genes located between the two flanking markers RM12676 and 2gAP0050058.

Fig. 4. Relative expression levels of genes in panicles by quantitative real-time PCR.

HT, High temperature condition (32 ºC); LT, Low temperature condition (24 ºC); TGMS, Thermo-sensitive genic male sterility.

Data represent Mean ± SE, and the same lowercase letter(s) indicate no significant difference at< 0.05 by Duncan’s new multiple range test.

Discussion

The segregation patterns of fertile to sterile plants in the three F2populations followed the 3:1 ratio, which is typical of Mendelian low, indicating that the TGMS of anIR68301S is controlled by a single recessive gene, which is in agreement with other studies (Borkakati and Virmani, 1996; Lopez et al, 2003; Wang et al, 2003; Hussain et al, 2011; Qi et al, 2014).

Using the three mapping populations, thegene in IR68301S was mapped on chromosome 2. Using B2 × B30 F2population, thegene was located between SSR marker RM12676 and InDel marker 2gAP0050058. In addition, InDel marker 2gAP004045, located between these two markers, showed complete co-segregation with the TGMS phenotype. These markers are useful tool for developing new TGMS lines by marker-assisted selection (MAS) and identifying the TGMS individuals at earlier stages of line development in rice breeding program. Using F2population fromparents (B2 and B11),only two markers were linked to thegene. The genetic distance of thegene from the two markers was 14.97 and 7.50 cM. Since major subspecies of rice in Thailand is, the polymorphic and tightly linked markers generated fromparents will be more practical than the linked markers generated from×crosses. Therefore, several markers nearby the flanking region were designed and test for polymorphism between the twoparents (B2 and B11). The 30 InDel markers covering flanking region from 5.50 to 6.28 Mb were designed to test polymorphism between these two parents (Supplemental Table 2). The results showed that only vf0206114052 at 6.11 Mb showed polymorphism and linked (2.94 cM) to thegene. Therefore, this marker will be useful inbreeding programs. The two tightly linked InDel markers, vf0206114052 and 2gAP004045, will be useful for×and×rice breeding programs, respectively.

Candidate genes for EGMS have been reported. The locus of(photo period-sensitive male sterility) and(photo- or thermo-sensitive genic male sterility) represent the same locus on chromosome 12 conferring PGMS and TGMS traits (Zhou et al, 2012). This report revealed that a SNP C-to-G between the fertile lines and sterile lines led to increasing of methylation in putative promoter region of this non-coding gene, resulting in premature programmed cell death (PCD) in developing anthers, thus causing PGMS inand TGMS insubspecies.

Theis a candidate gene ofwhich locates on chromosome 2 at the position of 6.39 Mb (htpp://www.gramene.org) and this gene encodes RNase ZS1. Experimental results revealed that at high temperature RNase ZS1loses its function and causes defective pollen production (Zhou et al, 2014). This enzyme maintained mRNAs ofUbat normal level and led to male fertility.LOC_Os02g12290, a nuclear ribonuclease Z gene was identified as the candidate for thegene, located on chromosome 2 in Guangzhan 63S, aPTGMS line (Xu et al, 2011).

The gene controlling TGMS in IR68301S was located between the two flanking markers at the position of 5.74 to 5.81 Mb. There were no previously reported EGMS genes at this position. There were 10 genes located between the flanking markers, however, we detected the expressions level of only 7 genes in the TGMS mutant and the wild type under both conditions. All these genes responded to temperatures by showing different expression levels under fertile and sterile conditions in the TGMS rice plants.Threegenes,andshowed similar expression levelsunder fertile and sterile conditions in wild type rice plants but they showed significantly different levels of expression in the mutant plants under sterile condition. Interestingly,showed significantly higher expression level under sterile condition than under fertile condition in the TGMS rice plants, while it showed similar expression levelsunder both conditions in the wildtype rice plants. Theencodesa tetratricopeptide-like helical domain protein (TPR). Shin et al (2014) reported that the N-terminal tetratricopeptide repeat1 (TPR1) domain is essential for its interaction with pectate lyase-like proteins (PLLs) in petunia, maize and. Lacking the TPR1 domain, no interaction resulting in pollen that failed to germinate.pollen calmodulin-binding protein (OsPCBP) is a Ca2+-dependent calmodulin-binding (CaMBP) protein and it contains six TPR motifs that can interact with protein in pollen to regulate germination and also involve in starch accumulation. The absence or reduction of starch can cause abortive pollen (Zhang et al, 2012). This information indicated that the TPR domain is associated with pollen development.encodes a zinc ion binding protein andencodes a zinc finger protein. The zinc finger family proteins are transcription factors (TF) involved in abiotic stress. Bai et al (2015) reported that three zinc finger TF genes (LOC_Os06g14180, LOC_Os12g37800, LOC_Os11g14000) are up-regulated and two zinc finger TF genes (LOC_Os01g50750 and LOC_Os05g44550) are down-regulated following cold stress treatment in rice male sterile lines. In, the result of RNA sequencing showed that, a zinc finger TF protein gene associated with flowering time and tolerance to abiotic stress (Yang et al, 2014). Yang et al (2015) reported that zinc finger CCCH domain-containing proteins are two (Unigene43080_Zhong-531and Unigene43085_Zhong-531) of differential expression genes (DEGs) which respond to temperature interacting with nitrogen at meiosis stage of rice spikelet. In our study,andshowed higher expression levels under lower temperature in the TGMS plants, while in wild type rice plants,showed lower expression levelsunder lower temperature andshowed similar expression levels under both conditions.encodes an F-box domain and kelch repeat containing protein.F-box proteins are critical for degradation of cellular proteins. The F-box protein-encoding genes have specific and/or overlapping expression during floral transition as well as panicle and seed development. The F-box protein encoding genes are involved in different abiotic stress conditions (Jain et al, 2007; Chunthong et al, 2017). The TPR domains were predicted in rice F-box proteins responsible for processing and/or translation of mRNAs. DNA-binding domains such as zinc finger, ring finger and helix loop helix are also found in rice F-box proteins, which may be directly or indirectly involved in transcriptional regulation (Small and Peeters, 2000).

encodes a conserved hypothetical protein. It showed higher level of expression under the higher temperature condition than under the lower temperature condition in the wildtype rice plants, but in the TGMS rice plants the expression of this gene was similar under both conditions.encodes a putative uncharacterized protein. It showed similar expression patterns in the TGMS and wild type rice plants but the expression levels were higher in the TGMS than in wild type rice plants under both conditions. Although the functions of,andare not clear but their expressions in our study suggested that they could be involved in TGMS. Therefore, further study is needed to identify the candidate gene controlling TGMS in IR68301S rice line.

Acknowledgements

This study was supported by Center for Agricultural Biotechnology, Kasetsart University, Center of Excellence on Agricultural Biotechnology (AG-BIO/PERDO- CHE), Agricultural Research Development Agency (ARDA), and National Science and Technology Development Agency, Thailand. The authors thank Dr. Peera Jaruampornpan for her critical reading and comments on the manuscript.

supplemental datA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/ journal/16726308; http://www.ricescience.org.

Supplemental Table 1. Markers on chromosome 2 linked to thegene using F2male sterile plants from IR68301S × IR14632, IR68301S × Supanburi 91062 and IR68301S × IR67966-188-2-2-1.

Supplemental Table 2. InDel markers on chromosome 2 covering flanking region from 5.50 to 6.28 Mb.

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http://dx.doi.org/10.1016/j.rsci.2018.08.006

9 July 2018;

24 August 2018

Amorntip Muangprom (Amorntip.mua@biotec.or.th)

(Managing Editor: Wang Caihong)