Research Report

Marker assisted selection for S5 neutral allele in inter-subspecific hybridization of rice  

Revathi P , Dinesh Chandra , Ram Deen , Arun Kumar Singh , Sohanvir singh , Moti Lal , Vijay pal bhadana , Ram Tilathoo
Directorate of Rice Research, Crop Improvement Section, Hyderabad, India
Author    Correspondence author
Molecular Plant Breeding, 2015, Vol. 6, No. 1   doi: 10.5376/mpb.2015.06.0001
Received: 11 Nov., 2014    Accepted: 19 Dec., 2014    Published: 27 Jan., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Revathi et al., Marker assisted selection for S5 neutral allele in inter-subspecific hybridization of rice, Molecular Plant Breeding, 2015, Vol.6, No. 1 1-7 (doi: 10.5376/mpb.2015.06.0001)

Abstract

The present level of heterosis in indica hybrids can be improved by exploiting inter-subspecific gene pool. Hybrid sterility in inter-subspecific hybridization is the major bottleneck, however use ofwide compatible varieties (WCVs) carrying neutral alleles helps in producing fertile hybrids. Conventionally WC varieties are identified by tedious test crossing. To identify WC varieties, 325 tropical japonica and 7 indica lines have been genotyped for the presence of S5n (neutral allele) with the help of S5-MMS (multiplex marker system) which clearly distinguish indica, japonica and S5 neutral allele. Of the 325 lines, 90 tropical japonica and Swarna (indica) showed the presence of S5n. One hundred and fifty F1 hybrids, indica × japonica (I × J), were evaluated for their hybrid sterility and spikelet fertility percentage ranged from 4 to 97%. The I × J hybrids with S5n showed higher spikelet fertility; however some hybrids without S5n showed higher spikelet fertility and among them few tropical japonica lines identified to carry indica allele by S5-MMS marker system, therefore this functional marker is a powerful tool for molecular screening of WC varieties. Hybrids with S5n showed higher sterility indicating existence of other than embryo sac sterility mechanism of I X J crosses, since S5n overcomes only embryo sac hybrid sterility. In wild rice O.rufipogon accessions the S5n allelic status was determined by using S5-MMS marker system. To conclude, S5-MMS is a powerful tool PCR based and with low cost, highly efficient as marker system in identifying WC varieties in different mapping populations including wild rice accessions. Whichsaves one year of breeder’s valuable time in inter-subspecific hybridization in rice.

Keywords
Rice; Inter sub-specifiec hybridization; WC genotypes; S5 neutral allele; Molecular markers
Rice is one of the most important food crops in the world. Of the total of 24 species of the genus Oryza, only Oryza sativa and O. glaberrima are cultivated in Asia and Africa, respectively. During the course of evolution, the Asian cultivated rice (O. sativa) was differentiated into two distinct eco-geographic races or subspecies, indica and japonica (Kato et al., 1928). In the same way, the Chinese have recognized two rice varietal groups, Hsien (indica) and Keng (japonica) (Khush, 1997). Based on morphological characters and geographic distribution, Matsuo (1952) classified cultivated rice into three types: japonica, javanica, and indica. Indica rice is generally adapted to the humid regions of the tropics and subtropical Asia (India, China, Vietnam, Thailand, Myanmar, the Philippines, Bangladesh, and Sri Lanka) whereas japonica rice is adapted to the temperate regions (Korea, Japan, and northern China, Taiwan, parts of Australia, California, Europe, and Egypt). Also, a third small group called tropical japonica (or javanica) is mainly found in the equatorial region of Southeast Asia, particularly Java and Indonesia). Rice breeders in tropical Asia, impressed by the relatively higher yielding ability of japonica varieties, attributable to semi-tall stature and hence lodging resistance and higher responsiveness to higher doses of fertilizer, believed that recombining of such yield-promoting features of japonicawith wide adaptability and good grain quality of indicas could raise the yield of tropical rice, which had been stagnating for a long time at low yield (Yuan, 1994).
However, the major problemin exploiting intersubspecific heterosis is hybrid semi sterility. The discovery of wide compatibility (WC) genes by Ikehashi and Araki (1986) paved the way for exploiting indica × japonica heterosis in rice (Yuan, 1994). Varieties possessing WC genes are called wide compatible varieties (WCVs) that produce a normal fertile hybrid when crossed with either indica or japonica. Based on the results of WC variety inheritance studies, Ikehashi and Araki (1986) proposed a genetic model to account for wide compatibility. According to this model, there are three alleles at the S5 locus: neutral S5n; indica S5i, and japonica S5j. A zygote formed from the S5n allele with either of the other two alleles, S5n/S5i or S5n/S5j, would be normal fertile, while a zygote with genotype S5i/S5j would be partly sterile. Using morphological markers, they located the S5 locus near the C+ locus on chromosome 6. This chromosomal location has been further confirmed by several studies using isozyme and molecular markers (Li et al., 1991; Liu et al., 1992; Zheng et al., 1992; Yanagihara et al., 1995).
Conventionally, WC varieties are identified by test crossing and evaluating the spikelet fertility of F1 hybrids . This is a tedious and time-consuming process and it is also often inconclusive. The use of molecular markers can overcome these limitations. With the availability of molecular markers, genetic linkage maps, and genome sequences in rice, the major gene S5 was recently cloned by Chen et al., (2008). This finding using a map-based cloning approach showed that S5 encodes an aspartic protease for embryo-sac fertility, and that indica and japonica has alleles different by two nucleotides. A discontinuous 136-bp deletion that was separated by a TAAT motif in the first exon of the gene encoding aspartic protease was reported in WC varieties carrying a neutral allele compared with the indica and japonica allele. This large deletion has led to subcellular mislocalization of the encoded protein that resulted in its non-functionality in WC varieties carrying S5n. Sundaram et al.,(2010) developed a PCR-based co-dominant S5 functional multiplex marker system (S5-MMS) to detect a neutral allele carrying deletion and an indica and japonica allele carrying SNPs in a single PCR tube reaction by multiplexing. Revathi et al., (2010) utilized S5-indel primer for screening 103 rice genotypes of indica and tropical japonica and identified 48 genotypes as wide compatible varieties for inter subspecific hybridization to increase the level of heterosis. In our investigation, we used the S5 multiplex marker system to identify potential genetic resources for wide compatibility for use in hybrid rice breeding.
1 Materials and methods
A total of 325 tropical japonica accessions provided by the International Rice Research Institute (IRRI) and seven indica genotypes (Swarna, CSR36, APMS6A, APMS6B, CST 7-1, Improved Sambha Mahsuri, and Sampada) were subjected to molecular screening for the presence of a WC S5 neutral allele. One hundred and fifty F1 hybrids were generated using seven indica genotypes as female parents and tropical japonica genotypes as male parents. These F1 hybrids were grown at Ramachandra Puram farm, Directorate of Rice Research, Hyderabad during 2012, Rabi/ season (November-March). The F1s were evaluated for their spikelet fertility percentage. An F2 population of Swarna × IRGC48960 were raised and phenotyping and genotyping observation was recorded. A total 20 Oryza rufipogon accessions were genotyped with the help of S5 multiplex marker system and results were recorded. To test the restoration of tropical japonica lines, APMS 6A (WA-CMS line) was crossed with 20 tropical japonica lines and F1 hybrids were raised and studied for their spikelet fertility percentage.
1.1 Spikelet fertility
The panicles that emerged from the primary tiller were bagged before anthesis to avoid outcrossing and the number of filled grains and chaffs in the panicle were counted at the time of maturity. The ratio of filled grains to the total number of spikelets, expressed as spikelet fertility percentage, is as follows:
Number of filled grains per panicletotal number of grains per panicle' type="#_x0000_t75">SPF % =     × 100
Plants were classified into four classes based on spikelet fertility percentage (SPF%): fertile (more than 75% spikelet fertility), partially fertile (51-75%), partially sterile (1-50%), and completely sterile (0%).
1.2 Genotyping: DNA isolation and PCR analysis
Total genomic DNA was isolated from 20-day-old young leaves of all the genotypes by a mini-preparation method (Dellaporta et al., 1983). The PCR was carried out using 25-30 ng/μL of template DNA containing 2.5 mM of each dNTP, 0.25 μM of each forward and reverse primer, 1 U of Taq DNA polymerase, and 1X PCR buffer in a total volume of 10 μL in a thermal cycler (Eppendorf, USA). The cycling conditions were an initial denaturation at 94˚C for 5 min followed by 30 cycles of PCR amplification under the next parameters: 30 s at 94˚C, 30 s at 55˚C, and 1 min at 72˚C, followed by a final extension at 72˚C for 5 min. The amplified PCR products along with a 100-bp molecular marker (Bangalore Genie, India) were separated on a 3.0% Seakem® LE agarose gel (Lonza, USA), stained with ethidium bromide and documented using the Gel documentation system (Alpha Innotech, USA). Based on the banding pattern, gels were scored for the presence and absence of bands as restorers and non-restorers. Table 1 presents the sequence of primers and their amplification product sizes.


Table 1 List of S5 multiplex marker system primer sequences

2 Results and discussion
A total 332 genotypes, including tropical japonica genotypes and indica genotypes, underwent molecular screening to assess the allelic status of the S5 locus. Out of 325 genotypes tested, 90 tropical japonica genotypes were identified to carry an S5 neutral allele and out of seven indica genotypes, Swarna, was identified to carry an S5 neutral allele. The list of WC genotypes possessing an S5 neutral allele identified with the help of an S5-InDel SSR marker presented in Table 2. A total of 150 inter-subspecific hybrids were generated by crossing indica genotypes as female parents and tropical japonica genotypes as male parents. F1 hybrids were raised during the next Kharif season to evaluate their spikelet fertility percentage (SPF%). Phenotyping for spikelet fertility ranged from 4% to 97% as partial sterility to complete fertility. Genotyping of F1 hybrids for their S5 allele status was done with the help of S5-MMS (Figure 1). Heterozygosity of the F1 hybrids were confirmed with the help of a multiplex marker system and results appear in Figure 2.
Three categories of inter-subspecific hybrids have been observed, hybrids with high spikelet fertility with the presence of an S5 neutral allele, hybrids with low SPF% without S5n, and hybrids with low SPF% with S5n. A few hybrid combinations with low to high spikelet fertility with or without an S5 neutral allele are presented in Table 3. A few inter-subspecific hybrids without the presence of an S5 neutral allele showed higher fertility, indicating that some other neutral alleles are involved in overcoming partial sterility of inter-subspecific hybrids. A few hybrid combinations showed high hybrid sterility with the presence of an S5 neutral allele, which explains the existence of other than an embryo sac sterility mechanism for indica by japonica crosses, since S5novercomes only embryo sac hybrid sterility. Liu et al., (1992) reported that the S5 neutral allele overcomes only embryo sac sterility and it is not sufficient to overcome indica ×japonica hybrid sterility completely.


Figure 1 Molecular screening using S5-indel marker


Figure 2 Screening of indica × japonica F1 hybrids using S5 multiplex marker system


Table 2 List of wide compatibility genotypes possessing S5n identified by S5-indel SSR marker


Table 3 Spikelet fertility percentage and S5n allele status of inter subspecific hybrids a

In rice, around 50 loci controlling indica × japonica hybrid sterility and wide compatibility have been identified, including loci causing female gamete-embryo sac abortion and pollen sterility (Ouyang et al., 2009). Our results are in agreement with earlier reports that many loci are involved in controlling wide compatibility and that the S5 neutral allele alone is not sufficient to overcome indica×japonica hybrid sterility.
To test the restoration ability of tropical japonica lines, APMS6A was crossed with 20 tropical japonica with S5n and these indica × japonica hybrids showed sterility to partial sterility, indicating that tropical japonica lines unable to restore the fertility of WA-CMS may be due to poor restoration ability or the presence of other sterility mechanisms. Virmani (2005) reported that restorer frequency in temperate and tropical japonica rice is negligible and, for the development of rice hybrids in a japonica genetic background, restorer lines have to be bred by transferring Rf genes from indica rice. Wei et al., (2010) and Yang et al., (2012) showed that the S5 neutral allele exists in wild rice and that Oryza sativa and O. nivara had S5n in the homozygous state (S5n S5n), whereas O. rufipogon had it in the heterozygous state (S5n S5i or S5nS5j). To validate these results, with the help of a PCR-based S5-multiplex marker system, a total 20 O. rufipogon accessions were screened and, surprisingly, all 20 O. rufipogon accessions were identified to carry S5n in the heterozygous state (Figure 3). These results confirm that an S5 neutral allele might have evolved from O. rufipogon.


Figure 3 S5 multiplex marker system of Oryza rufipogon accessions

An F2 population of Swarna × IRGC48960 were subjected to genotyping with the help of the S5 multiplex marker system. Of 155 plants, 38 showed a Swarna parent-type amplification of the S5 neutral allele presence, 41 plants showed an IRGC48960 genotype amplification pattern of the japonica allele, and 76 plants showed a heterozygous pattern of amplification explaining the 1:2:1 segregation ratio of a perfect expected F2 ratio. The amplification pattern of S5-MMS in an F2 population appears in Figure 4.


Figure 4 Genotyping of F2 population of Swarna × IRGC48960

To conclude, the S5 multiplex marker system is a powerful PCR-based low-cost, highly efficient marker system for identifying WC genotypes carrying the S5 neutral allele from germplasm lines, different mapping populations, and wild genomes. It saves breeders one year of valuable time in evaluating testcrosses for producing intersubspecific hybrids. This S5-MMS is a very useful tool for incorporating the S5 neutral allele into elite indica or japonica lines for their use in inter subspecific hybridization.
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