Development and utilization of the functional co-dominant KASP marker for thermo-sensitive genic male sterility in rice

Thermos-sensitive genic male sterility (TGMS) is the important genetic resource in two-line hybrid rice breeding. The pollen fertility of TGMS lines is regulated by a single point mutation on the TGMS genes. Based on the single nucleotide polymorphism (SNP) and indel, KASP markers were developed and utilized in rice molecular breeding due to high-through detection with low cost and save time. In this study, we convert the SNPs on TGMS genes including p/tms12-1 and tms9-1 gene to the functional co-dominant KASP marker and used in two-line hybrid rice breeding. We can differentiate the TGMS lines conferring p/tms12-1 or tms9-1 from another TGMS lines by KASP assay. Of them, the genotype of Pei’ai64S and Hua201S containing p/tms12-1 is homozygous GG genotype with blue signal, and the genotype of HengnongS-1 containing tms9-1 is homozygous CC genotype with red signal. KASP assay for tms9-1 gene was proved that the genetic mode is fit to one recessive Mendalian trait. The test of seed purity was performed by KASP marker for the two-line hybrid varieties of Liangyoupeijiu and Hualiangyou1206, which is consistent with the previous dCAPS marker. Moreover, the tms12-1 and tms9-1 gene were pyramided in the same genetic background and the new TGMS lines were generated. Therefore, the KASP marker for TGMS genes developed in this study can be widely used in two-line hybrid rice breeding. It will provide a visual convenient toolkit for breeders to select the target individual plant by the high-throughput detection in the different two-line rice breeding program.


sterility · SNP · MAS
Rice is the staple crops as the major food resource that supplied for half world's population (Khush 2001. Hybrid rice is planted in many counties due to the potential of the higher production than traditional inbred lines. Presently, hybrid rice was constituted as two systems including three-line and two-line (Cheng et al. 2007). Two-line hybrid rice has many advantages in comparison with the conventional three-line system such as the self-propagation of P/TGMS lines under low-temperature and short-photoperiod conditions, male parents with no need of restorer gene in hybrid rice system, eliminating the adverse cytoplasmic effect on agronomic traits for the hybrid variety and so on (Yuan 1990;Virmani et al. 2003;Si et al. 2011). The photoperiod-and thermo-sensitive genic male sterile gene as the key factors that regulated the fertility conversion of P/TGMS lines under different environmental conditions plan a crucial role in the two-line hybrid rice. Since the past few years, many two-line hybrid rice varieties were bred and widely applied in grain production in China with the finding and development of P/TGMS lines (Si et al. 2011;Mou 2016).
To date, a lot of P/TGMS genes as major locus conferring P/TGMS trait were mapped and indentified into different Chromosome: these include TGMS genes tms1, tms2, tms3, tms4, tms5, tms6, tms6(t), tms9, tms9-1, tms in SA2, tmsX, ptgms2-1 and PGMS genes pms1, pms2, pms3 and p/tms12-1 (Fan and Zhang 2016). Further study showed that TMS9, TMSX, ptgms2-1 were mapped in the region of TMS5 or the vicinity of TMS5 suggesting that tms5, tms9, tmsX and ptgms2-1 are allelic mutants (Fan and Zhang 2016). Nongken58S, the first PGMS lines, were found in a conventional japonica inbred lines Nongken58 and its PGMS traits were mainly regulated by pms3 and p/tms12-1 (Ding et al. 2012;Zhou et al. 2012;Zhu and Deng 2012). Pei'ai64S was a derived line bred from Nongken58S, but showed a TGMS trait in indica genetic background. A key single base substitution of C-to G-in p/tms12-1 which resulted into a loss function of a small RNA named as osa-smR5864w was found in Pei'ai64S (Zhou et al. 2012). Tms5 encoded an RNase Z S1 protein processing Ub L40 mRNA was found from an early indica TGMS lines Annong S-1. A nonsense mutation C-to-A mutation at TMS5 gene creating a premature stop codon was the key factor that controls the TGMS trait in AnnongS-1 and its derived lines (Zhou et al. 2014). Different with p/tms12-1 and tms5, tms9-1 mapped on the rice Chromosome 9 is other TGMS gene found in an early indica TGMS lines HengnongS-1. A SNP of C to T at the candidate gene OsMS1 resulting an amino acid substitution regulated the TGMS trait of Hengnong S-1 (Qi et al. 2014). Interestingly, the SNP polymorphism on the different TGMS gene including p/tms12-1, tms5 and tms9-1 is the key factors to control the TGMS trait in TGMS lines and its derived lines. The different thermo-sensitive genic male sterility were determined by those SNP polymorphism, we can convert the SNPs to functional markers (FMs) and used in the mark-assisted breeding program in two-line hybrid rice system. SNPs and Indel are the common mutations in genes that determine many important agronomic traits in rice breeding. The polymorphism of Indel can be identified as co-dominant marker due to the length of PCR production. With respect to genotyping for SNP, various methods have been developed, such as AS-PCR (allele specific PCR), CAPS (cleaved amplified polymorphism sequence), dCAPS (drived CAPS), HRM (high-resolution melting), TS-PCR (temperature switch PCR) are used in rice genotyping study. It is difficult to distinguish the heterozygotes from homozygotes by AS-PCR, as AS-PCR provides a dominant marker results (Gaudet et al. 2009). CAPS and dCAPS rely on the site-specific digestion of PCR products, so it is expensive and time-consuming and difficult to use in a large scale genotyping due to the low efficiency of the enzyme digestion (Chen et al. 2009). HRM is based on the melting curves analysis for different melting temperature to detect SNP alleles, it is need a set of complicated and multiround PCR and easy to lead to false positive . TS-PCR (Temperature Switch PCR) consists of a biphasic PCR with two sets of precisely designed primers. The target sequences enriched by a pair of site-specific primers at the first stage and then DNA containing the SNP allele were amplified and detected by the nested primers at the second stage, so it is time-consuming and laborious (Thanh and Khoo, 2014). With the identification and discovery of large amount SNP on genes that contribute to agronomic traits in rice, the technology for SNP genotyping will provide useful information to breeder for the selection of the parent combination with the desirable trait. KASP (Kompetitive Allele-Specific PCR) is a single-step genotyping technology that is based on terminal fluorescence reading similar to Taqman detection (Patil et al. 2017). Both methods require two fluorescent signals of the end-point by laser scanning to detect the individual samples with different genotypes; different fluorescent signals represent different SNP on the same locus. KASP marker has been used in a wide range of applications, such as germplasm identification, genotyping for SNP, bulked segregant analysis, and MAS, as it has many advantages with high throughput, highly accurate, good genetic stability, high flexibility, locus specificity and low cost (Semagn et al. 2014;Mackay et al. 2014;Cabral et al. 2014;Leal-Bertioli et al. 2015;Wu et al. 2017;Li et al. 2020;Makhoul et al.2020). Functional SNPs and Indels can directly determine the phenotypic trait, and such polymorphism can be developed into functional markers (FMs). Due to the high efficiency and low cost, conversion of those FMs into KASP markers will provide a convenient toolkit for breeder to select the target individual plant from different combinations population.
In this study, SNPs in the two thermo-sensitive genic male sterility genes (p/tms12-1 and tms9-1) was converted into KASP marker and validated into MAS in two-line hybrid rice breeding. The new developed KASP markers can be used to distinguish different PTGMS lines, select the individual plants from different combination progeny, and test the seed purity of two-line hybrid varieties and pyramid two TGMS genes into the same genetic background. KASP assay provide a new reliable alternative method for the high throughput detection in two-line hybrid rice breeding.

Plant materials and fertility identification
Eighteen TGMS lines that were widely used in two-line hybrid rice production from the South of China were selected to test the KASP assay for TGMS genes. According to the previous reports and the source of TGMS genes, the TGMS trait of Hua201S and Pei'ai64S were regulated by p/tms12-1 gene, and that of HengnongS-1were regulated by tms9-1 gene, others were regulated by tms5 gene (Table 1) Zhang et al. 2015). In order to convert the SNPs in tms9-1 and p/tms12-1 genes, two sets of KASP markers were designed according to SNP allele (Table 2). To test the validation of KASP assay for tms9-1 gene, one cross combination with HengnongS-1×Minghui63 was used to detect the SNP allele of tms9-1 gene in F 2 population. A total of 164 individual plants from the F 2 population were selected and performed the KASP assay. For the validation of KASP assay for p/tms12-1, two hundred seeds of hybrid rice varieties with Liangyoupeijiu and Hualiangyou1206 were selected to test the seed purity respectively. Liangyoupeijiu and Hualiangyou1206 were bred with Pei'ai64S and Hua201S as female parent conferring p/tms12-1 gene, respectively. In order to pyramid p/tms12-1 and The temperature of PCR mixture was declined into room temperature, the read for fluorescence signal was performed at the Pherastar scanning machine (LGC Limited, UK). Once there have no clear results, an additional 3 cycles of amplification (94°C for 20 s, 55°C for 60 s) can be added to further amplify.
All KASP assays were applied at the Shanghai Baygene Biotechnologies Limited Company, Shanghai, China. Genotyping data were collected and viewed by SNP viewer 2 (LGC Limited, UK).

Identification of TGMS lines and genetic analysis for tms9-1 gene by KASP assay
According to previous study, the G/T and C/T polymorphism located at the noncoding small RNA and the third exon of OsMS1 gene, which is responsible for the regulation of the trait of TGMS by p/tms12-1 and tms9-1 gene, respectively (Zhou et al. 2012;Qi et al. 2014). Those functional SNPs can discriminate different genotypes such as G/G, G/C, and C/C on p/tms12-1 gene and C/C, C/T and T/T on tms9-1 gene. KASP assay showed that two TGMS lines (Hua201S and Pei'ai64S) were classified as homozygous G/G genotypes, and other sixteen TGMS lines were determined to have homozygous C/C genotype on p/tms12-1 gene. One TGMS line (HengnongS-1) was classified as homozygous C/C genotype, but others were determined to have homozygous T/T genotype on tms9-1 gene (Fig 2). In order to validate the KASP marker on tms9-1 gene, one cross combination with HengnongS-1×Minghui63 was prepared, and 164 individual plants from this F 2 population were selected to genotype analysis for tms9-1 gene by the KASP assay. The results showed that 31 individual plants were classified as homozygous C/C genotype which represented as the TGMS trait, 82 individual plants were classified as heterozygous C/T genotype and 51 individual plants were classified as homozygous T/T genotype which all represented as non-TGMS trait (Fig 3). Chi squared analyses for genotype in the F 2 population showed that the segregation ratio of three genotypes (C/C, C/T, T/T) in the F 2 populations was at 1: 2.65: 1.65 (χ 2 0.05 = 4.89 < 5.99, df = 2), which fitted to 1:2:1, indicating that the genetic model of tms9-1 was a simple recessive Mendalian trait that is consistent with our previous study (Qi et al. 2014).
KASP assay for the seed purity of two-line hybrid rice varieties To validate the KASP assay used in the two-line hybrid rice varieties, two two-line hybrid rice varieties including Liangyoupeijiu and Hualiangyou1206 which were widely planted in the south of China were selected to measure the seed purity of the variety. 200 F 1 individual plants seeds of them were randomly selected to detect the seed purity by the KASP marker. The KASP assay showed that the genotype of 196 individual plant of Liangyoupeijiu was heterozygous C/G genotype with green signal, and three plants was homozygous G/G genotype with blue signal same with female parent, but only one plant was homozygous C/C genotype with red signal same with male parent or other conventional plants (Fig 4). The pollen fertility of individual plant showed that the plants with blue signal were male sterile, whereas the plants with green signal and red signal were male fertile. The KASP assay also showed that the genotype of all 200 F 1 plants of Hualiangyou1206 was heterozygous C/G genotype with green signal (Fig 4), which is consistent with our previous study tested by one dCAPS marker (Qi et al. 2017).
Pyramiding of tms9-1 and p/tms12-1 gene by KASP assay To generate the new TGMS lines with different TGMS genes including tms9-1 and p/tms12-1, a total of 92 F 4 individual plants with TGMS trait from one combination cross with HengnongS-1 ×Hua201S were prepared to detect TGMS gene by KASP marker. KASP assay for tms9-1 gene showed that the genotype of 77 individual plants was homozygous C/C genotype with red signal same with Hengnongs-1, and that of 13 individual plants was heterozygous C/T genotype with green signal, but two individual plants cannot be detected. KASP assay for p/tms12-1 gene showed that the genotype of 76 individual plants was homozygous G/G genotype with blue signal same with Hua201S, and that of 15 individual plants was heterozygous C/G genotype with green signal, but only one individual plant cannot be detected. So, the genotype of 74 individual plants was homozygous C/C for tms9-1 gene and G/G for p/tms12-1 gene (Fig 5). Different TGMS genes were pyramided into the same genetic background by MAS with KASP assay.

Discussion
SNPs are one-base variation in a single nucleotide allele that is widely distributed on the different rice accessions with high density of genetic variants which can occur in coding and non-coding regions.
With the development of next-generation sequencing (NGS) technology, a large scale of SNPs were generated in diverse rice genome (Kumar et al. 2012). As the functional SNPs can directly reflect natural genetic variability of rice accession, a high-throughput genotyping technology for SNPs is need, and it will facilitates breeders to select the parents for the improvement of desirable traits.
Pariasca-Tanaka et al (2015) reported that a total of 2,015 KASP markers were found and 1,890 existing KASP can be applicable to indica rice. Some SNPs and Indel that determine the rice eating and cooking quality have been converted into KASP markers and used to improve the rice quality by the molecular marker assisted selection (MAS) combined with conventional breeding approach (Yang et al.2019).
Two-line hybrid rice is widely planted in the south of China due to its higher yield and other advantages than three-line hybrid rice (Si et al. 2012;Qi et al. 2014). Thermo sensitive genic male sterile line is the most important genetic resource in two-line hybrid rice breeding, and the conversion of its pollen fertility were regulated by TGMS genes under different environmental conditions. To date, many TGMS genes were identified and cloned on different rice Chromosome, but only tms5, p/tms12-1 and tms9-1 genes have been used in the two-line hybrid breeding in China (Zhou et al. 2014;Zhou et al.2012;Qi et al. 2014). The pollen fertility of different TGMS lines was regulated by the SNPs on the TGMS genes. In the TGMS line including Pei'ai64S and Hua201S, a point mutation from G to C on the lncRNAs of p/tms12-1 gene controls the pollen fertility under different environmental conditions.
Meanwhile, a point mutation from C to T on the third exon of tms9-1 gene controls the conversion of pollen fertility in HengnongS-1 line (Zhou et al.2012;Qi et al. 2014). In this study, we successfully convert the SNPs on p/tms12-1 and tms9-1 to KASP marker and used in two-line hybrid rice breeding.
These functional co-dominant KASP assay can be used to distinguish different TGMS lines conferring the TGMS genes such as p/tms12-1 or tms9-1 through different fluorescent signal. These visual markers can also be used to analyze the genetic analysis for tms9-1 gene and detect the seed purity of two-line hybrid rice varieties in our study. The results of genetic analysis for tms9-1 gene is consistent with our previous study (Qi et al. 2014), which indicated that the KASP assay for tms9-1 gene will be validated to make use in two-line hybrid breeding by MAS. The test of seed purity for two-line hybrid rice varieties is also consistent with our previous study in which a co-dominant dCAPS marker was used (Qi et al. 2017). However, it is little difficult and high cost for high-throughput genotyping and detection by dCASP marker. So, the KASP marker has the distinct advantages compared to the previously published marker especially in large scale detection Qi et al. 2017). It will facilitate breeder to precisely identify the genotype of different TGMS genes with low cost and save time in two-line hybrid rice breeding combined with conventional breeding. In our study, we use the KASP marker to pyramid tms9-1 and p/tms12-1 into the same genetic background and generate the new TGMS lines in which the TGMS trait of them was regulated by tms9-1 and p/tms12-1. Utilization of KASP assay for p/tms12-1 and tms9-1 gene will overcome the difficulty that we cannot identify the TGMS genes in the p/tms12-1/tms9-1 TGMS lines by the regular phenotype analysis for pollen fertility.   Fig. 2 Eighteen TGMS lines were performed by KASP assay for p/tms12-1 and tms9-1 gene a: KASP assay of the TGMS lines for p/tms12-1 gene, Hua201S and Pei'ai64S were homozygous G/G genotype with blue signal; and other TGMS lines were homozygous C/C genotype with red signal. b: KASP assay for the TGMS lines with KASP assay for tms9-1 gene, HengnongS-1 were homozygous C/C genotype with red signal; and other TGMS lines were homozygous T/T genotype with blue signal.  b: KASP assay for p/tms12-1 gene in F 4 population.
Note: the individual plants with homozygous G/G and C/C were selected as the TGMS plant pyramiding of tms9-1 and p/tms12-1 gene.