Identification of Thermotolerant Rice Genotypes with Allele Coding at Seedling Stage

Rice-The most important plant in the world to ensure food security. Heat is one of the main factors that greatly limit rice production. With the increasing global warming, industrialization there is a great effect on climate change which requires us to see various alternatives for strains that are more tolerant to heat so that some techniques are developed to filter a large number of genotypes for high temperature tolerance. Here we report the standardization of Temperature Induction Response (TIR) technique to identify thermotolerant rice genotypes. The phenotypic characteristics of Rice due to high temperature is calculated with germination (%), growth of the seedling and molecular analysis is also considered. The heat stress is provided to the plants with the help of TIR protocol with the adjustment of temperature to lethal (55°C) and sub-lethal levels (38-55°C) in a TIR chamber with alterations in humidity. Of the 74 genotypes screened, 14 showed thermo tolerance caused by high temperatures. Both tolerant and sensitive genotypes were separated based on their survival percentages. The tolerant class are selected based on the growth and development of genotypes having high survival percentage and also their shoot and root lengths, fresh and dry weights are compared to the heat tolerant checks N22, Dular and Nipponbare. These genotypes have intrinsic heat tolerance and thus can be explored as a source of donors in breeding programs intended for global warming. The molecular markers which are identified to be linked with heat tolerant class through allele code are quite helpful and can be used in marker assisted breeding approach to attain heat tolerance in cultivated varieties.


INTRODUCTION
Rice is the most significant and essential cereal food grain domesticated all through the world particularly in Asia and Africa (Krishnan et al., 2011). The name wild rice is generally utilized for types of the genera Zizania and Porteresia, both wild and tamed, albeit the term may likewise be utilized for crude or crude assortments of Oryza (Lafarge et al., 2017). Rice, a monocot, is regularly developed as a yearly plant, albeit in tropical regions it can get by as a perennial and can deliver a ratoon crop for up to 30 years (Huang et al., 2012). Rice development is appropriate to nations and areas with low work expenses and high precipitation, as it is work concentrated to develop and requires sufficient water. The rice plant can develop to 1-1.8 m (3 ft 3 in-5 ft 11 in) tall, every so often relying upon the assortment and soil ripeness.
Rice enhancement for wetland rice fields is acknowledged to be answerable for 11% of the anthropogenic methane outpourings. Methane conveyed is achieved by long stretch flooding of rice fields cuts the earth off from ecological oxygen and causes anaerobic maturing of regular matter in the soil . Methane creation from rice improvement contributes ~1.5% of complete anthropogenic nursery gases. Methane is on numerous occasions more impressive an ozone-exhausting substance than carbon dioxide (Yu et al., 2014).
A new report found that, due to rising temperatures and lessening sun-based radiation during the later extended lengths of the 20th century, the rice yield improvement rate has decreased in various bits of Asia, standing out from what may have been seen had the temperature and sun arranged radiation designs not happened (Kumar and Kaushik, 2021;Malhi et al., 2021). The yield improvement rate had fallen 10-20% at specific regions (Raza et al., 2019;Jain et al., 2018). The examination relied upon records from 227 farms in Thailand, Vietnam, Nepal, India, China, Bangladesh, and Pakistan (Jain et al., 2019). The instrument of this falling yield was not palatable, anyway may incorporate extended breath during warm nights, which devours energy without having the choice to photosynthesize (Fu et al., 2019).
In view of expanding temperatures by an unnatural weather change, plants are defenceless to intermittent warmth and dry spell pressure that generally influences the growth and development. Plants adjust to high temperature pressures with basal level resilience innate and can acquire resistance to serious temperature stress (Kim et al., 2011). Thermo tolerance acquired is very quick and has been demonstrated to be initiated during the phone's acclimation until the temperature time frame is very high. The temperature influences the expansive range of cell and digestion parts, and outrageous temperatures force the seriousness of factors relying upon the degree of progress in temperature, power, and length . The capacity to hold and change in accordance with the supra-ideal temperature results from both warmth harm avoidance and warmth touchy fix segments (Yufang et al., 2021). Seeds presented to the temperature of the sub-deadly prior to testing with weighty temperatures have a preferred development recuperation over the seeds tested straightforwardly to extreme temperatures (Satishraj et al., 2016). Both outrageous conditions (dry seasons and floods), on the off chance that they surpass certain basic periods, will have significant ramifications for rice and can cause complete disappointment of rice plants during delicate stages either as water deficiencies or exorbitant splashing (Chaturvedi et al., 2017). So, there is a need to receive a diverse methodology while considering the effect of high temperature stress, likewise zeroing in on other ecological pressing factors, which might be similarly adverse to rice usefulness (Priyanka et al., 2021b(Priyanka et al., , 2021a. Resistance acquired for certain abiotic stress has been displayed to give cross security to different pressing factors like saltiness, cold temperatures, and dryness. Along these lines, assessing the overall exhibition of rice genotypes for high temperature resistance utilizing TIR approach (Liu et al., 2013).
The best temperature for rice germination is some place in the scope of 28 and 30 °C.
High temperature impacts essentially all advancement periods of rice from germination to developing (Shah et al., 2011). The cut-off temperature at the seedling stage has been perceived as 35 °C; the essential sign of warmth stress is the vulnerable turn of events. By thinking about its significance, accessibility, uses and need for creation, heat pressure has acquired a substantial significance because of winning expanded temperatures (Burke et al., 2011).
Warmth stress majorly affects every one of the phases of rice. By taking every single detail into thought, our investigation is focused on heat pressure lenient genotypes in rice accomplished by TIR convention so these genotypes can be additionally used to deliver heat open minded assortments either by reproducing program or by quality articulation examination (Sato et al., 2016).

Experiment design
The area selected for the experiment is Phenotypic lab at Institute of Frontier Technology, Regional Agricultural Research Station, Tirupati, using TIR (Temperature Induction Response) protocol. The experimental material includes 74 diverse rice genotypes taken from Nellore, Maruteru, several land races and African lines (NERICA) including proven varieties for heat tolerance such as N22, Dular and Nipponbare which were used as genotype checks to choose tolerant sets (Prashanth et al., 2012). These TIR approaches involve first identification of challenging temperature and induction temperatures and then they are standardized before using the germplasms for intrinsic tolerance. Phenotyping of rice genotypes for thermo tolerance utilizing TIR protocol was set up in this lab and the same technique was utilized in this investigation.

Treatments
Rice seeds were washed with distilled water 2-3 times and are stored for germination at room temperature. After 42 hours, the seedlings that attain 0.5 cm uniformly were selected and planted in an aluminium tray containing blotter paper moistened with water (Bado et al., 2016). Plates with these seeds are liable to sub-deadly (sub-lethal) temperatures (expansion in slow temperatures for each half an hour from 38°C to 55°C for 4 hours in this atmosphere -'Labline' -(Humidity controlled chamber). Then these seeds are exposed to a deadly (lethal) temperature (55 o C) (induced) for 2 hours. Sub-set of other seeds are exposed directly to the deadly temperature (non-induced). The seeds of the rice induced and non-induced are permitted to recover at room temperature for one week. A control tray is maintained at room temperature, which are not exposed to the temperature of the sub-lethal and deadly (Liu et al., 2017) conditions. The treatment for the just sprouted seedlings is carried out in a special chamber called TIR (Thermal Induction Response) chamber where the plates are arranged according to the treatment. The chamber contains temperature adjustment along with humidity maintenance. The treatment varieties are tested along with the check varieties to compare the heat tolerance capacity in different varieties.

Phenotypic Analysis
Highly vigour seedlings are selected as tolerant types because certain seedlings do not respond properly towards heat stress. The susceptible varieties did not germinate even under suitable conditions. Along with the germination the shoot and root lengths of seedlings are taken into consideration to select tolerant varieties. The maximum root length and shoot lengths of the growing seedlings are compared with each other and the highly tolerant , medium tolerant and susceptible varieties are segregated.

Molecular Analysis
Out of 74, a set of 14 genotypes each under tolerant and control conditions were selected based on their survival percentage under sub-lethal conditions and are allowed for Selective line genotyping by comparing with the three checks i.e., N22, Dular and Nipponbare.

DNA isolation and quantification
The tender, sprouted seedlings were maintained for 15 days at room temperature thereafter, the complete genomic DNA content of the seedlings was extracted using the CTAB method. The purified DNA pellet which was obtained through CTAB method is air-dried and

Selection of primers
In General, 51 primers were chosen out of which 43 were SSRs reported for heat resilience and 8 were genic markers out of which 4 were brought to light from rice database www.gramene .org, and rest were designed from Primer 3 software. The microsatellite region of candidate genes were identified using SSRIT tool (http://archive.gramene.org/ db/markers/ssrtool) and then primer designing was done using primer-3 v  Table S1 and Table   S2.

PCR amplification and product isolation
The PCR amplification was performed using an Eppendorf Master cycler to understand the polymorphism. The reactions were performed with standard temperatures and are repeated for 35 times. Agarose gel was prepared to understand the detailed amplification with a permanent marker of 50bp or 100bp which was loaded along with the samples (Xie et al., 2014). For gel perception UV trans-illuminator was utilized and Alpha Innotech Multi Imager gel documentation framework program from Alpha Innotech, California, USA was used for photography (Vallejos et al., 2007).

RESULTS
It was noted that all the 74 genotypes which were involved in sub-lethal temperatures were considered a major importance involving both tolerant and sensitive classes ( Table 2).
For all these genotypes the survival percentage was seen as a primary target, where all the 74 genotypes were segregated depending upon their survival ability and growth ( Table 2). The survival percentages of all the genotypes were checked by taking their germination and growth into consideration. The survival percentage was calculated in two major classes i.e., SP between 80-100% and another one 60 % or less than 60% ( Table 2). The genotypes that fall between the SP range of 80-100% were chosen as heat tolerant class and genotypes other than tolerant class were considered as heat sensitive class, depending upon their germination and growth after heat treatment in TIR chamber ( Table 2).    (Table 3).   (Table 4).

Heat tolerant genotypes
After checking for the survival percentage of the selected genotypes i.e., 43  conditions. In comparison of both the root and shoot lengths with control and sublethal the relativity is calculated i.e., relative lengths of root and shoot in which relative root length is maximum for FR 13A which is around 62.53 and it is minimum or very less for the check Nipponbare which is around -31.59 (Table 4). Also, the relative shoot length is maximum for FR 13A which is around 106.20 cm and very less for the check Nipponbare which is -59.11 proving that FR 13A is giving good results similar to that of the checks and compared with all the 14 varieties (Table 4). The relative root and shoot lengths were calculated using maximum root and shoot lengths of control and sublethal. Out of all the three checks taken relative root and absolute shoot length is maximum in case of Dular whereas the relative root length and the relative shoot length of FR 13 A is maximum i.e., 62.53 and 106.20 respectively compared to other selected and tolerant varieties. Interestingly the values of FR13A were more than that of check variety Dular proving it to be highly tolerant.
Most of the primers were standardized at 59ºC temperatures but very few were observed at 57ºC which include RM10115, RM10469, RM15087, RM282, RM17270 and RM17296 (Table 5). It was also shown that the product size is maximum for RM19715 which is 350 bp and it was observed that the product size of the primer is less for RM16216 which is around 90-110 bp (Table 5). We designed a set of 8 primers with respect to heat tolerance in rice using Primer 3 software (Table 6). These primers were designed from different HSP families and TT 1 (Thermal tolerance gene) gene targeted markers. We chose four polymorphic primers namely, RM16216, RM 17271, RM 5687 and a TT 1 gene targeted marker, TTC/ TTM primers in the study (Table 5 and Table 6) for screening all the 14 genotypes each from heat tolerant and sensitive groups to understand the allelic patterns.  The allelic patterns of the polymorphic markers were observed between checks, tolerant and sensitive genotypes (Fig 3). Several genotypes exhibited allelic patterns between 80-120 bp in case of tolerant group but not 120bp (Fig 3) with the primer RM5687.  with polymorphic patterns, and also the distribution of genotypes between checks using these primers was understood ( Table 7). The three major varieties like Basmati 386, FR13A, Swarna Sub1A were concluded to have high heat tolerance not only due to their phenotypic characters but also their polymorphic patterns were also considered with the checks. Basmati 386 gave less growth compared to the other two varieties when considered along with the phenotypic characters ( Table 7).
The TTC/TTM primer of TT 1 gene showed the polymorphic patterns as the other primers obtained from the literature. Mostly all the genotypes gave good results showing heat tolerance because alleles are seen near to above 350 bp and few are less than that i.e., near 100 bp alleles (Fig.4). The alleles that were obtained by different polymorphic markers were not clearly in distinguishing the tolerant set from sensitive set of genotypes. Among the reported genotypes also the allele sizes were varied except for the marker TTC/TTM, as they were from different sub groups of rice (Fig. 4). N22 and Dular comes under Aus group, whereas Nipponbare comes under japonica group. To understand this clearly allele codes were assigned for each primer.

Analysing the informativeness of polymorphic markers
Each genotype was assigned an allele code generated by respective polymorphic marker based on their allele size and colour coded as shown in Figure 6. As many as a highest number of 100bp alleles were identified by TTC/TTM in the range of 100 to 350bp (Table 7). It was understood that the marker RM17270 was amplified at 190bp in N22, but no other genotype showed similar allele size to N22. The same marker was amplified at 195bp in Dular and Nipponbare genotypes, wherein Basmathi386, FR13A and Swarna Sub1A were also shown similar amplification pattern like Dular and Nipponbare ( Table 7). The marker RM16216 showed three kinds of alleles i.e. 90bp, 100bp and 110bp. Of which, 90bp and 100bp are major alleles (Table 7). By considering the allele patterns and allele codes it was proved that all the 14 genotypes from the tolerant set showed maximum polymorphic alleles (Table 7). Hence, the identified list of tolerant genotypes from the study, especially FR13A, Swarna Sub1A, Sathya, Vajram, VLDhan16, Basmathi386 and Jagannadh can be used for further gene identification studies apart from targeted breeding programmes (Table 7).      (Lin et al., 2012). Day by day temperatures, higher than 30ºC or every day greatest temperatures higher than 35ºC during the blossoming time frame will bring about helpless anther dehiscence.
Plants' heat response is highly complex (Hedhly et al., 2011). In current days, some reputed techniques like gene editing are being used to address the heat tolerant/induced functional basis of the genotype. From the recent experiments conducted, it was proven that high temperature decreases the grain filling period in basmati rice from 32 to 26 days, reduced yield by 6%, and caused a decrease in absolute starch (3.1%) and amylose content (22%).
Quantifiable exercises of key chemicals associated with sucrose to starch change, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and dissolvable starch synthase in endosperms created at 32°C were lower than those at 22°C contrasted and comparable aging stage on an endosperm premise. Specifically, granule-bound starch synthase (GBSS) action was fundamentally lower than comparing action in endosperms creating at 22°C during every formative stage (Ahmed et al., 2015).
The genotypes selected for the study include genotypes from Indica, Japonica Javanicas, Aus groups and wild relatives of rice. These genotypes are selected and are allowed to grow under sub-lethal conditions. The genotypes giving good performance i.e., survivability, maximum root and shoot lengths are selected and are separated from the entire genotype sets (Alberio et al., 2018). High temperatures are induced by using TIR protocol in which temperatures to a maximum level and control in humidity can be done (Sudhakar et al., 2013). TIR technique relies on genotype's acquired tolerance, to determine their heat tolerance, wherein a gradual exposure of genotypes to a temperature regime over a period was used. Using TIR technique, it was proved that sufficient genetic variability was present among rice genotypes for high temperature tolerance. The percent survival of seedlings varied from 0 to 100 % with a mean of 80.33%. The percent reduction in root growth varied from 0 to 73 % with a mean of 20.89 %. Four genotypes namely NLR 34242, NLR40066, NLR40059, NLR40050 also exhibited higher thermotolerance without reduction in root but slight reduction in seedling survival by 10%. Similar studies were also conducted by Vijaylakshmi et al. (2015) Later molecular analysis was carried out to check the polymorphic patterns obtained by heat tolerance specific SSR primers. A total of 51 primers were selected out of which 43 are There has been developing interest with the use of marker-based models in recent years. In the studies conducted by using these models, descriptions of the effect of allele coding system on inference and computations are often absent or missing (Lv et al., 2017). Also, several other common allele coding alters these regression coefficients by deducting a value from each marker such that the mean of regression coefficients is zero within each marker. It is also known as centered allele coding (Stranden et al., 2011). This kind of coding is highly useful to identify the genotypes that fall within similar allele size ranges for a major number of loci, to identify like allele pattern with proven genotypes (heat tolerant) and to group them under a class (Shim et al., 2020).
Thus, keeping in lieu of the above identified potently reported polymorphic set of markers, irrespective of their allelic differences among the genotypes it can be concluded that the efficiency of selective line genotyping technique used in the current study is remarkable in associating effective markers to the trait (Sharma et al., 2019;. Further the efficiency of the technique is well explored with limited genotyping panel to arrive at small number of useful markers out of huge tests, for further studies (Gouda et al., 2020). The above outcomes propose that the TIR strategy is an amazing and productive method to recognize hereditary changeability in high temperature resistance in rice inside a brief timeframe and it is appropriate for screening an enormous number of genotypes (Hsuan et al., 2019).

CONCLUSION
Rice occupies 23% of the total area under cereal production in the world. It is the staple nourishment for the greater part of the total populace with Asia addressing the biggest producer and consumer region. For the most part, rice is unfavourably influenced by high temperature in the lower heights of the tropical regions. Under molecular analysis carried out in the present study, a set of 51 reported and as well new SSR and genic markers were employed to assess the genetic diversity among the 74 rice genotypes. These markers gave good amplification with prominent alleles at 57˚C and 59˚C. By employing Selective line genotyping approach; both of each fourteen selected Tolerant and Sensitive genotypes along with three reported genotypes (N22, Dular and Nipponbare) were subjected for genotyping with a total of 51 markers. Out of 51 markers 92% were monomorphic between heat tolerant and sensitive classes, as well as compared to checks. Four markers i.e., RM17270, RM16216, RM5687, TTC/TTM, were polymorphic. Despite the fact that, allele coding is a viable strategy the alleles produced from various polymorphic markers were not unmistakably recognized lenient set from touchy arrangement of genotypes like TIR procedure. The recognized 14 genotypes of rice can be utilized as contributor hotspot for growing high temperature lenient rice genotypes to oppose worldwide ascent temperature.