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The two line hybrid rice system, pollen sterility is regulated by recessive gene that responds to temperature thermo-sensitive genetic male sterility, TGMS. The recessive gene controlling male sterility of TGMS lines is expressed when they are grown in condition with higher or lower critical temperature. The objective of this study was to transfer tgms gene controlling thermo-sensitive genetic male sterility to Thai rice cultivars by backcross breeding method. The male sterile line was used as a donor parent while Thai rice cultivars ChaiNat1, PathumThani1, and SuphanBuri1 were used as recurrent parent. The BC2F4 lines were developed from backcrossing and selfing. Moreover, the simple sequence repeat (SSR) markers were developed for identifying tgms gene and the linked marker was used for assisting selection in backcrossing. The identification lines were confirmed by pollen observations. The results showed the success of introgression of thermo-sensitive genetic male sterility to Thai rice cultivars. These lines will be tested for combining ability and used as female parent in hybrid rice production in Thailand.
T. Sreewongchai · W. Matthayatthaworn (ï€ª) · C. Phumichai · P. Sripichitt
Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand
e-mail: email@example.com Tel: +66-2-5793130. Ext: 125. Fax: +66-2-5798580.
Keywords Hybrid rice · Thermo-sensitive genic male-sterile · Non-pollen type · Molecular marker · Marker assisted backcrossing
Rice (Oryza sativa L.) is the most important staple food crop for half the world's population. The Green Revolution technology developed at the International Rice Research Institute (IRRI) in the 1960s increased world rice production. However, during the past decade, production potential of modern cultivars has remained stagnant. It is imperative to increase rice production to feed the increasing world population (Khush 2005). Hybrid rice technology has tremendously improved rice productivity as effectively demonstrated in China and other Asian countries. Hybrid rice includes three line and two line hybrid rice that is developed via cytoplasmic male sterility and photo/thermo-sensitive genic male sterility, respectively. The application of thermo-sensitive genic male sterile (TGMS) lines has made a great contribution to hybrid rice production by providing the two-line system instead of the commonly used three-line system. Developing TGMS lines is one of the basic steps in obtaining superior two-line rice hybrids.
Thermo-sensitive genic male sterility, developed in china (Yuan 1987), Japan (Maruyama et al. 1990), and IRRI (Virmani and Voc 1991) is controlled by a single recessive nuclear gene which in interacts with temperature to express pollen fertility or sterility (Borkakati and Virmani 1996; Maruyama et al. 1990). The non-pollen abortion is more difficult to revere from sterile to fertile phase than typical abortion. Under the same critical sterility point temperature (CSPT), the male sterility of non-pollen abortion is more stable than of typical abortion, thus TGMS line of non-pollen abortion could be safer than that of typical abortion in two line hybrid seed production (Peng et al. 2010).
Marker-assisted selection (MAS) can be used for monitoring the presence or absence of these genes in breeding populations and can be combined with conventional breeding approaches (Jena and Mackill 2008). In hybrid rice breeding, the transfer of TGMS gene to different background will be highly facilitated by using the MAS technique in a simple and accurate way with those identified molecular markers linked to TGMS genes.
This study took advantage of the know information regarding chromosomal location of the tmsX gene and the availability of many mapped microsatellite markers in rice (Matthayatthaworn et al. 2011). The objective of this study was to develop the new TGMS line by transferring the non-pollen type from TGMS line to Thai rice by using marker assisted backcrossing.
Materials and Methods
TGMS line, a non-pollen type thermo-sensitive genic male sterile, has been identified to carry single recessive gene (Matthayatthaworn et al. 2011). The TGMS line origin from China is poor adaptability for growing in Thailand, while CNT1, PTT1, and SPR1 are popular Thai rice cultivar to well adaptability for Thailand. In the backcross breeding program, a TGMS line and 3 Thai rice cultivar, CNT1, PTT1 and SPR1 were used as recurrent parents. The backcross breeding procedures were showed in Fig.1.
Markers for genotyping
T2 marker were developed by (Matthayatthaworn et al. 2011) locating on chromosome 2. It tightly linked to tmsX locus and showed polymorphism between the two parents of each crosses. This marker was used in genotyping the offspring of each generation. The amplified products were analyzed on the 6 % PAGE gel and detected by silver staining (Benbouza et al. 2006).
Phenotyping for male sterile
In order to determine the non-pollen critical sterility point temperature (CSPT) in new Thai TGMS line, rice plants at stem elongation stage were grown in growth chamber in which daily mean temperatures were set for 11.5 hours day-length at 20°C, 25°C, and 30°C. Separated three tillers from each genotype were sampled for each treatment to examine the type of pollen abortion. Pollen and spikelet fertility were determined at the heading stage using a 1 % I2-KI solution straining. Plant with no stained pollen were considered to be completely male sterile, whereas plants having more than 95 % darkly stained pollen were classified as male fertile. At the same time, self-pollination was tested to confirm the sterility and fertility (Wang et al. 2003).
Screening marker for MAS
The T2 marker could use as MAS in breeding programs only on TGMS/PTT1 cross because this marker showed polymorphism between TGMS line and PTT1. However, in this study there are more two crosses, TGMS/CNT1 and TGMS/SPR1 therefore needs to be developed more markers specific to this region. For further molecular marker to select plant carrying tgms gene in breeding programs, we designed9 SSR marker from the region surrounding tmsX gene that reported by (Peng et al. 2010). Two markers showed polymorphism between TGMS line and CNT1. Unfortunately, no marker showed polymorphism between TGMS line and SPR1. From screening markers it could use as MAS in the crosses of TGMS/PTT1 and TGMS/CNT1 but in the cross of TGMS/SPR1 used conventional breeding program.
Phenotyping and MAS in BC2F1 and BC2F2 generations
SSR marker, T2 was used to identify BC2F1 plant which carrying the tmsX allele. The banding patterns of the BC2F1 individuals could be classified in to those homozygous for PTT1 and heterozygous displaying both bands of TGMS line and PTT1. The selected plant was heterozygous at this locus. The seeds of selected plants were harvested. As the same in the cross of TGMS/CNT1, the favorite genotype plants were selected and harvested on heterozygous plants. On the other hand, the cross of TGMS/SPR1 could not classified homozygous and heterozygous. Thus, the seeds on all plant were harvested.
The BC2F2 seeds were planted under the temperature higher 30°C, and all lines from the crosses of TGMS/PTT1 and TGMS/CNT1 were genotyped using mentioned SSR markers. Of these lines had 3 segregation classes of homozygous dominant, heterozygous, and homozygous recessive bands (Fig. 2.).These types of bands were confirmed by using phenotype observation. As a result, homozygous dominant and heterozygous bands were fertile plants and homozygous recessive band was sterile plants. On the other hand, the cross of TGMS/SPR1 was observed by phenotype and it could identify of fertile and sterile plants in BC2F2 generation.
Performance of new TGMS line in different temperature regimes
The TGMS line (donor parent) showed male sterility when exposed to high temperature. However, when exposed to low temperature at the critical stage, it showed male fertility. The new TGMS lines with Thai rice genetic background from three crosses showed no pollen when grown in dairy temperature higher than 30 °C conditions. On the other hand, at low temperature (20°C) these plants produced male fertile. Intermediary, at 25°C these plants produced unstained pollen which it considered to be male sterile (Fig. 3.). These results indicated that the successful of introgression of TGMS trait to Thai rice cultivars.
The application of the TGMS line has greatly facilitated the application of two-line system in hybrid rice production (Wang et al. 2003). In order to facilitate the transfer of TGMS allele to desirable genetic background via marker-assisted selection (MAS) (Chen et al. 2011) and more accurately identify the TGMS individuals at earlier stages, closely linked markers of the tmsX gene were developed and used as marker assisted selection (Peng et al. 2010)
The possibility of marker-assisted selection for tmsX has been demonstrated in this study. Development of specific simple sequence repeat (SSR) marker for non-pollen type thermo-sensitive genic male sterile gene in rice (Oryza sativa L.) have facilitated the development of tropical TGMS lines with Thai cultivar background (Matthayatthaworn et al. 2011). In generating advanced line, the use of these markers can select heterozygous-fertile (Tt) and homozygous-sterile (tt) plants at seedlings before transplanting. Thus, these markers able reduce the experimental area and effort expended to select line for phenotype fertile and sterile plants (Collard et al. 2005; Sreewongchai et al. 2010).
In this study we used backcross breeding program incorporated with pedigree method of selection. The BC2F2 generation carried 87.5% of recurrent genetic background. Thus, some offspring line will display the agronomic character different from the recurrent parent. The shorter plant than recurrent parent was one of favorite phenotypes when using pedigree method of selection after backcrossing (Jin et al. 2010; Wongsaprom et al. 2010). Moreover, these result showed that marker assisted backcrossing (MAB) and conventional backcrossing (CB) for recessive gene using same time of selection. The MAB more advantage via worked with small amount of plants in breeding program. On the other hand, the CB could be select favorite phenotype in large population but cost saving.
The performances of new TGMS line results indicate that stable male sterility in TGMS system there is a possibility in developing tropical rice hybrids. However, study about critical temperature in different genetic background must necessary (Peng et al. 2010). Seed setting on TGMS line should be at least 30 % to economize their seed multiplication (Lopez and Virmani 2000). The stable male sterility of new TGMS lines is critical for the application of two-line hybrid rice system in Thailand.
Acknowledgments This work was financially supported by Kasetsart University, Thailand.