Expression Analysis of OsGBSSI and OsGS3 Genes, and Investigation of Morphological and Biochemical Characteristics across Diverse Rice (Oryza sativa L.) Genotypes

doi:10.21203/rs.3.rs-4673727/v2

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Research Article

Expression Analysis of OsGBSSI and OsGS3 Genes, and Investigation of Morphological and Biochemical Characteristics across Diverse Rice (Oryza sativa L.) Genotypes

https://doi.org/10.21203/rs.3.rs-4673727/v2

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Background

Quality in the rice value chain encompasses a wide range of features and attributes that are defined by consumers based on their food preferences and habits.

Scope and approach:

we grown two aromatic basmati, one aromatic non-basmati and one non-aromatic rice genotypes during Kharif season 2021–2022 at SVP university research field for grain quality studies. Then we analyzed grain quality based on (i) morphological and physical parameters, (ii) biochemical traits, and (iii) grain quality related gene expression analysis.

Key findings and conclusion:

aromatic basmati genotypes and non-aromatic genotype shared similarities, whereas aromatic non-basmati genotype exhibited dissimilarities in terms of morphological traits. Aromatic basmati genotypes exhibited superior grain quality and yield traits, including elevated panicle length, high 1000-grain weight, extra-long slender grain, high grain elongation ratio (> 1.6), strong aroma, soft gel consistency, high alkali spreading value and intermediate amylose content as compared to both aromatic non-basmati and non-aromatic genotypes. Gene expression analysis revealed that that aromatic basmati genotype (PB-1121) had higher expression level and genotype (NVB-1) exhibited same expression level of GBSSI gene while aromatic basmati genotype showed less expression level of GS3 gene, it was 256 fold lower expression in comparison to control. Aromatic non-basmati genotype had 32 fold lower expression level of GBSSI gene and same expression level of GS3 gene as compared to control.

Plant Molecular Biology and Genetics

Agronomy

grain quality

gene expression

aromatic

basmati

non-aromatic

Rice (Oryza sativa L.) is a valuable energy source for the world population and good source of energy. It is staple food in Asia and used as human consumption (Tong et al., 2019). Rice has significant nutritional value, high carbohydrates, low fat and rich in minerals, vitamins and protein (El-Mowafi et al., 2021). The amount of rice nutrients per 100 g are; carbohydrate of rice is (77.8 g), fiber (1.4 g), protein (6.8 g) and (0.6 g) fat (Verma and Shukla, 2011; Rather et. al., 2016; Tiwari and Pradhan, 2017). Starch content in rice is the primary carbohydrate, ranges from 72 to 75% (Dipnaik and Kokare, 2017). The rice grain comprises a protective outer layer called the husk or hull and inner seed, known as brown rice. Brown rice encompasses the external part such as the seedcoat (testa), pericarp, nucellus, embryo and endosperm. The endosperm consists of the aleurone layer, inner endosperm and the sub-aleurone layer. The aleurone layer encloses the embryo and the polished rice grains are final rice end product. Rice grain quality encompasses all the features and attributes of rice that align with the demands of consumers, especially as living standards improve (Li et al, 2022). There are various natural grain quality characters in rice that are directly or indirectly linked to customer priority (Shamim et al., 2016 and Raghuvanshi et al., 2017).

In general, Rice grain quality is assessed based on its appearance (size and shape), cooking properties and nutritional value (Peng et al., 2014; Tsukaguchi et al., 2016; Zhou et al., 2019). Appearance encompassing size and shape, is a crucial aspect preferred by consumers in the market (Bao, 2014). The primary edible component of rice grain is the endosperm, which comprises 75% starch. The texture quality of cooked rice are largely determined by its starch content, in order that starch is the major part of endosperm. Rice starch consist of amylose and amylopectin polysaccharides. The amylose content has significant influence on rice cooking and eating characteristic. The low amylose in rice make grain tender and glossy after cooking, whereas high amylose in rice grain has more water absorbance and making the grain dry, fluffy and detached after cooking (Julliano, 1971). Rice genotypes are categorized into waxy (0–2%), extremely low (3–9%), minimal (10–19%), moderate (20–25%) and substantial (> 25%) in term of amylose content (Graham, 2002). Mostly rice from Indian subcontinent, Myanmar, Vietnam and Thailand favor rice with high amylose content. The predominant feature of such rice is high volume of expansion and a high degree of flakiness. IRRI survey shows mostly people in the Southeast Asia prefer intermediate amylose content rice (Graham, 2002). Primarily indica rice varieties exhibit moderate to high amylose content (Hu et al., 2014), which is why the renowned Basmati cultivar, characterized by its long-slender grains, possesses moderately to significantly high amylose content. An association between alkali spreading value and cooking attributes of rice reported by (Sirisoontaralak and Noomhorm, 2016) another indicator of cooked rice texture is gel consistency. Rice genotypes containing similar amylose content can differ in their tenderness and glossy. Aroma stands out as a top-quality characteristic of cooked rice, which has consumer susceptibility and high price in the global market (Sirisoontaralak and Noomhorm, 2016; Verma et al., 2018a). The taste and elongation of cooked rice are first and second preferences of consumer, while fragrance or aroma in cooked rice specify as the third and highest desired quality characters (Verma et al., 2018b; Verma and Srivastav, 2018a). Basmati rice is embraced by consumers owing to its delightful fragrance and desirable quality attributes such as its slender grains, lengthwise elongation upon cooking and great cooking properties (Calingacion et al., 2014; Verma et al., 2015; Jayhoon et al., 2023).

Recently, several genes have been shown and different genes have been linked to grain quality attributes including grain appearance, cooking characteristics and eating quality. The major gene which is responsible for amylose biosynthesis is granule bond starch synthase (GBSS) gene. GBSSs are directed to starch granules by GBSS-binding protein, which is analogous to the protein originally identified in Arabidopsis (Seung et al., 2015; Wang et al., 2020). In cereal this gene expressed in endosperm, pollen and embryo sac. GBSS are into two isoforms; GBSSI which is encoded by waxy in cereals which is limited to storage tissue (seed endosperm), while GBSSII is accountable for amylose biosynthesis in transient starch in leaves and non-storage tissues including seedcoat (Li and Liu, 2019). GBSSI gene has an important key role to control of amylose accumulation in caryopsis. Many studies have been shown that the GBSSI gene has considerable influence on the amylose content in rice (Chen et al., 2008; Wang et al., 2018). Grain size 3 (GS3) gene regulate grain length and weight which has high importance in grain yield and quality. This gene located in chromosome 3 in rice and function of this gene results short form grain. GS3 gene is primary gene that control grain size in rice. This gene exhibits significant expression levels in young panicles of both short and long grain genotypes while it not expressed in leaves and panicle post-flowering. This gene linked with enhanced grain length in Oryza sativa though it is absent in other Oryza species (Takanu-Kai et al., 2009). Therefore, the current study was carried out to assessment of grain quality characteristics viz. morphological properties, biochemical properties and expression analysis of OsGBSSI and OsGS3 genes responsible for grain quality characteristics to provide a comprehensive grain quality evaluation on certain diverse rice genotypes.

Totally four rice genotypes (two aromatic Basmati, one aromatic non-Basmati and one non-aromatic) were grown under control condition during Kharif season 2021–2022 at research field of S.V.P University, Meerut, India. The morphological parameters including Days to flowering (DF), Days to maturity (DM), Panicle length (PL), Grain yield per plant (GY/P), Thousand grain weight (1000-GW) Grain length (L), grain breadth (B), grain length breadth ratio (L/B ratio), grain length after cooking (KLaC), and grain elongation ratio (ER) were recorded for all genotypes. Grain length and grain breadth were measured by helping Vernier caliper and the measuring was done for 10 milled rice each for sample the average value were taken. Rice grain appearance quality were classified based on grain length and grain l/b ratio (Table.1). IRRI has provided rice grain classification based on grain length for size and grain length breadth ratio for shape (IRRI, 2002).

Table.1 Rice grain categorization according to (IRRI, 2002) classification

Grain size				Grain shape
Score	Grain type	Length (mm)	Score	Grain type	l/b ratio
1	Extra long	> 7.5	1	Slender	> 3.0
2	Long	6.6–7.5	2	Medium	2.1-3.0
3	Medium	5.51–6.6	3	Bold	1.1-2.0
4	Short	< 5.5	4	Round	< 1.1

Grain aroma test (Sensory test)

A basic laboratory method is employed to assess rice for the presence of aroma IRRI, (1971). Rice grain de-husked for each sample followed by placing 1 gram of milled rice into a test tube. Approximately 20 ml of distilled water is then added, and the tube is covered with aluminum foil. The samples are subsequently immersed in a boiling water bath for a duration of 20 minutes. The cooked samples are kept in room temperature to cool and after cooling tubes were opened, and a panel assesses the presence or absence of aroma by scent. The samples were scored on (1, 2, 3, 4) corresponding to no aroma, trance of aroma, moderate aroma, strong aroma respectively.

Аlkali Spreаding Vаlue test (АSV)

Аlkali Spreаding Vаlue (ASV) scores ranging 1–7 were assessed as per Little et al., (1958). Sixed rice kernels of every genotype were placed in a petri dishes having 10 ml of potassium hydroxide (19.54 g of KOH dissolved in 1 litter of distilled water). The samples were incubated for 23 hours at room temperature (27–30 0C) for spreading. Samples were evaluated on a scale of 1 to 7, indicating the degree of damage: (1) unaffected kernels, (2) swollen kernels, (3) swollen kernels with incomplete or narrow collars, (4) swollen kernels with complete and wide collars, (5) split or segmented kernels with complete and wide collars, (6) dispersed kernels with merging collars, and (7) completely dispersed and intermingled kernels (Bhattacharya and Sowbhagya, 1972). This test was done in triplicates to ensure accuracy of the results.

Gel Consistency (GC)

Determination of gel consistency was followed with the protocol of Cagampang et al., (1973) with minor modification. For each genotype 100 ± 2 mg rice flour were taken and place in test tube (13x100 mm) then rice flour wetted with 200µg of ethanol having 0.30% Thymol blue. Shakes tubes with vertex shaker for suspending the rice flour. Immediately 2.0 ml of KOH solution added to the tube and for 2–3 min shaking vigorously. The samples were initially immersed in boiling water for 10 minutes for refluxing. Following this, they were taken out of the water bath and left to cool for 5 minutes at room temperature. Subsequently, the samples were transferred to an ice bath and kept there for 20 minutes. Test tubes containing the samples were then placed horizontally for 30 minutes on graph paper marked with millimeter graduations. After an hour, the gel length was measured from the bottom of the tube to the gel front using a gel consistency test. The gel was then categorized as soft (> 61 mm), medium (41–60 mm), or hard (< 40 mm). To ensure precision, the tests were conducted in triplicate.

Amylose content (AC)

Starch-Iodine-colorimetric method was used for measuring of amylose content in each sample. 100 ± 2 mg of meshed rice flour of each sample were weigh and put in a round bottom flask. Added 1.0 ml ethyl alcohol followed by 9.0 ml 1.0N NaOH. Samples mixed well by vertex shaker. Then for 8.0 min heated in boiling water bath and allowed to cool to room temperature. Each sample was then diluted to a volume of 100 ml with distilled water. In and new flask 5.0 ml of diluted sample were taken and acidify with 1.0 ml of 1.0N acetic acid. After that 2.0 ml Iodine solution (I2 + KI) was added and the volume was adjusted to 100 ml with distilled water. The samples were left at room temperature (27 ± 2°C) for 20 minutes. The color of each sample was measured in triplicate using a spectrophotometer at 620 nm, and the mean value of amylose content was calculated relative to the standard curve of Perez and Juliano (Perez and Juliano, 1978). All rice genotypes classification were categorized based on Julliano (1992) classification, (1–2%) for waxy rice, (3 − 1%) for very low amylose, (10–19%) for low amylose,(20–25%) fore intermediate amylose, (26–30%) for high amylose and (> 30%) for very high amylose content. To ensure accuracy and validity of the results, test was done in triplicates and Iodine solution were added between each sample in 1 min gap.

Statistical analysis

In this study, the results were find out using IBM SPSS statistic 20 software and spread excel sheet. The analyzed data were showed in tables among morphological and biochemical characteristics. The average morphological data was used to generate a distance matrix using EuclidIan Coefficient. The data of distance matrix were used to construct Dendrogram uses Ward’s method (Ward, 1963).

Gene expression studies related to grain quality characters

The gene expression study materials was conducted under controlled field grown of two aromatic Basmati, one aromatic non-Basmati and one non-aromatic rice genotypes for expression analysis of grain quality characteristics responsible genes (GBSSI and GS3 genes) through semi-quantitative PCR (Table.2).

Table.2 Rice genotypes selected for gene expression analysis

Table 2 is available upon request from the corresponding author.

Sample collection and extraction of total RNA

The samples were collected from freshly harvested young panicle at milking stage from each rice genotypes from field grown rice genotypes. RNA was extracted from panicle samples at the milking stage utilizing TRIzol™ reagent (Thermo Scientific, USA), followed method (Rio et al., 2010) and manufacture’s guidance. The RNA pellet was washed with pre-chilled RNase-free 75% ethanol, followed by DNase I treatment to eliminate DNA contamination.

cDNA synthesis and normalization

First-strand cDNA was synthesized using oligo dT primers according to the following protocol: 3 µl of total RNA was used to prepare the cDNA template. The reaction was carried out in a total volume of 20 µl with high-fidelity M-MLV reverse transcriptase enzyme (Himedia). The reaction mix for cDNA synthesis included 4.0 µl of 5X RT buffer, 1 µl of 10 mM dNTPs, 1 µl of oligo dT, 0.5 µl of RNase inhibitor, 1 µl of reverse transcriptase, 3 µl of RNA, and 9.5 µl of nuclease-free water. The synthesized cDNA was stored at -20°C for regular use. The cDNA synthesized from total RNA of various samples were normalized with OsACTIN serving as the housekeeping gene in rice (Wang et al., 2021).

Semi-quantitative PCR various components

To carry the expression profile of different grain quality genes in rice using semi-quantitative PCR, we utilized 2µg of first strand cDNA as template followed by adding 2 µl of the forward primer, 2 µl of the reverse primer, 5 µl of 10X buffer, 1 µl of dNTPs, and 0.6 µl of Taq polymerase to the reaction mix. Then, adjust the final volume to 50 µl with nuclease-free water.

Primers identification

The target gene was amplified with specific primers designed for each genes: I. OsGS3 forward sequence 5ʹ- GATTTTGGTGGTGTCCAACC-3ʹ (T_m 56°C), reverse sequence 5ʹ-GAAACAGCAGGCTGGCTTAG-3ʹ (T_m 58°C). II. OsGBSSI forward sequence 5ʹ- AACGTGGCTGCTCCTTGAA-3ʹ (T_m 59°C), reverse sequence 5ʹ- TTGGCAATAAGCCACACACA-3ʹ (T_m 56°C) and III. OsACTIN forward sequence 5ʹ- CGAGCATGGTATTGTTAG-3ʹ (T_m 49⁰C), reverse sequence 5ʹ- AGGGCATATCCTTCATAG-3ʹ (T_m 49°C). The annealing temperature was calculated 51°C, 49°C and 47°C respectively.

PCR Cycle Steps and PCR’s amplification Products

The semi–quantitative PCR program included an initial denaturation at 94°C for 3 minutes (1 cycle), followed by denaturation at 94°C for 1 minute, annealing at 51°C for 55 seconds, extension at 72°C for 55 seconds, and a final extension at 72°C for 10 minutes. 10µl PCR product was taken from each cycles (21, 24, 27, 30, 35) to assess the expression level of the rice grain quality related genes. The PCR products were then loaded onto a 2% agarose gel along with 100bp DNA ladder, and the amplified products were documented using a gel documentation system (Alpha Inotech, UVI Tech). The expression level of different genes was estimated by comparing them to the non-aromatic (IR-64) genotype as the control.

Result and Discussion

The results of morphological, physical, biochemical parameters and expression analysis of genes related to grain quality are determined as follow:

Morphological characteristics

The mean value for days to flowering for all genotypes were 102.412 days (Table.3). The morphological characters such as panicle length, grain yield per plant, thousand grain weight, days to flowering, days to maturity are important for plant yield and quality (Rawte et al., 2018; Shrestha et al., 2021). Maximum days to flowering recorded for aromatic non-basmati genotype (124.33 days) and minimum days to flowering recorded for non-aromatic genotype (92 days). Days to flowering for aromatic basmati genotypes were (94.66 days) for PB-1121 and for NVB-1 (98 days). The mean value of days to maturity across all genotypes were 128.33 days. Maximum days to maturity recorded for aromatic non-basmati genotype (153.00 days) and minimum days to maturity recorded for aromatic basmati (PB-1121) genotype (114.33 days). Days to maturity for aromatic basmati (NVB-1) genotype was (129.66 days) and non-aromatic genotype recorded (116.33 days) days to maturity. Similarly Singh et al., (2020) reported that aromatic non-basmati (Kalanamak) rice genotype had maximum 50% DF (121 days) and maximum DM (159 days) and Mackill et al., (2018) reported that non-aromatic (IR-64) had days to flowering (91 days) and days to maturity (114 days). Days to flowering and days to maturity and other morphological parameters are different in different genotypes (Mollier et al., 2023). Panicle length mean value for all genotypes were 27.955 cm. Maximum panicle length recorded for aromatic basmati (NVB-1) genotype (30.43 cm) and minimum panicle length recorded for aromatic non-basmati genotype (24.40 cm). Panicle length for aromatic basmati (PB-1121) genotype was (29.23 cm) and non-aromatic genotype recorded (27.76 cm). Calingacion et al., (2014) reported greater uniformity of grain filling within panicles is linked more closely to panicle lengths exceeding 22% of plant height, as the ultimate yield is predominantly influenced by both panicle length and quantity (Shrestha et al., 2021). The mean value for grain yield per plant for all genotypes were 17.852 gm. Maximum GY/P recorded for aromatic basmati (NVB-1) genotype (19.36 g) and minimum GY/P recorded for aromatic non-basmati genotype (16.06 g). Grain yield per plant for aromatic basmati (PB-1121) genotype was (16.76 g) and non-aromatic genotype recorded (19.23 g). Too many tillers causes poor grain filling and reduce grain quality and yield (Peng et al., 2019). The mean value for 1000-Grain weight was 22.630 for all genotypes. Maximum 1000-GW recorded for aromatic basmati (PB-1121) genotype (25.13 g) and minimum 1000-GW recorded for aromatic non-Basmati genotype (19.33 g). 1000-GW for aromatic basmati (NVB-1) genotype was (24.0 g) and non-aromatic genotype recorded (22.06 g). According to Rather et al., (2016) values below 20 g for thousand grain weight shows presence of unfiled grain or immature damage. Also Shijagurumayum et al., (2018) reported 1000-grain weight of different rice varieties ranged from 21.53 g to 34.96 g.

Table.3 Mean performance of morphological and physical characteristics of four diverse rice genotypes

Figure 3 is available upon request from the corresponding author.

The mean value for grain length for all genotypes were 6.942 mm (Table.3). Maximum grain length recorded for aromatic basmati (PB-1121) genotype (8.65 mm) and minimum grain length recorded for aromatic non-basmati genotype (4.80 mm). Grain length for aromatic basmati (NVB-1) genotype was (8.02 mm) and non-aromatic genotype recorded (6.30 mm). The mean value of grain breadth for all genotypes were 1.682 mm. Maximum grain breadth recorded for aromatic non-basmati genotype (2.05 mm) and minimum grain breadth recorded for aromatic basmati (NVB-1) genotype (1.43 mm). Grain breadth for aromatic basmati (PB-1121) genotype was (1.44 mm) and non-aromatic genotype recorded (1.81 mm). According to Peng et al., (2019).

The mean value of L/B ratio for all genotypes were 4.35. L/B ratio or grain shape is an important characters for grain quality (Rather et al., 2016). The L/B ratio serves as a criterion for categorizing rice shapes, where a lower ratio suggests round or bold shapes, an intermediate ratio indicates a medium shape, and a higher ratio signifies slender shapes. (Kumoro and Noprastika, 2008). Maximum l/b ratio recorded for aromatic basmati (PB-1121) genotype (6.0) and minimum recorded for aromatic non-basmati genotype (2.34). L/B ratio for aromatic basmati (NVB-1) genotype was (5.60) and non-aromatic genotype recorded (3.46). Similarly, high L/B ratio for Pusa Basmati-1121 was reported by (Khurana and Kumar, 2023) and Kaur et al., (2011) reported grain length varying between 6.77 mm to 8.23 mm, grain breadth 1.54 mm to 1.75 mm and L/B ratio 3.86 to 5.01 for different basmati rice genotypes. The mean value of KLaC for all genotypes were 11.505 mm. Maximum KLaC recorded for aromatic basmati (PB-1121) genotype (20.02 mm) and minimum recorded for aromatic non-basmati genotype (5.68 mm). KLaC for aromatic basmati (NVB-1) genotype was (13.18 mm) and non-aromatic genotype recorded (7.14 mm). The mean value of ER for all genotypes were 1.565. Maximum ER recorded for aromatic basmati (PB-1121) genotype (2.31). Present investigation verifies the results of Singh et al., (2018) reported that Pus Basmati-1121 has an exceptional high cooked grain length of more than 20 mm. minimum ER recorded for non-aromatic genotype (1.13). ER for aromatic basmati (NVB-1) genotype was (1.64) and aromatic non-basmati genotype recorded (1.18). Grain elongation ratio is one of the exceptional characteristics of basmati rice genotypes (Khurana and Kumar, 2023). ER is crucial to assess the cooking qualities of rice (Bao et al., 2014). Grain size and shape classifications were determined for all genotypes, utilizing grain length and the length-to-breadth ratio (l/b ratio) (IRRI, 2002). Aromatic basmati genotypes classified into extra-long slender grain. Basmati rice are classified in slender grain but it classified into extra-long slender grain when the L/B ratio is ≥ 3 (Khurana and Kumar, 2023), aromatic non-basmati genotype classified into short medium grain and non-aromatic genotype classified into medium slender grain. Grain size and grain shape are strong rice quality criterion which is seen firstly by consumers (Jallow et al., 2023), Grain size is defined by the length of each grain, while grain shape is defined by the proportion between grain length and breadth (Li et al., 2023).

Cluster analysis based morphological parameters using Ward’s method

The average value of all morphological parameters used to generate a distance matrix using EuclidIan Coefficient and the data of distance matrix were used to construct Dendrogram uses Ward’s method to show dissimilarity (Ward, 1963). Sathish et al., (2017) also generated Dendrogram using Ward’s method and cluster analyzing within four diverse rice genotypes to indicate the similarity or dissimilarity (Figure.1). Cluster I is contains 3 genotypes includes aromatic basmati (NVB-1, PB-1121) genotypes and non-aromatic (IR-64) genotypes with similarity. PB-1121 had 18.164% dissimilarity with NVB-1 and 23.142% similarity with IR-64. Cluster II contains one genotype such as aromatic non-basmati (Kalanamak) genotype. Aromatic non-basmati genotype showed dissimilarity with aromatic basmati and non-aromatic genotypes. Aromatic non-basmati had 54.44% dissimilarity with aromatic basmati PB-1121genotype, 41.815% dissimilarity with aromatic basmati NVB-1 genotype and 48.978% dissimilarity with non-aromatic genotype. Similarly Singh et al., (2020) cluster analysis revealed that aromatic non-basmati (Kalanamak) genotype had dissimilarity based on morphological characters with basmati rice genotypes.

Figure 1 is available upon request from the corresponding author.

Figure.1 Dissimilarity Dendrogram using Ward’s method

Bio-chemical grain quality characteristics

Based on aroma, aromatic basmati and aromatic non-basmati genotypes were strongly scented aroma with high score (4) and non-aromatic genotype was non-scented aroma with score (1) (Table.4). The results is similar to finding of Giri et al., (2020) which mostly basmati genotypes and Kalanamak are strong in aroma. Aroma is the mean key quality characters that directly impact the market price of rice (Hui et al., 2022). Kalanamak variety is the highest scented aroma which grown in northeastern of Uttar Pradesh (Singh et al., 2020) and genotype IR-64 is non-scented aroma genotype reported by (Hinge et al., 2016; Ndikuryavo et al., 2023). The primary indicator of starch consumption, cooking, and processing in rice is the alkali spreading value (Sirisoontaralak and Noomhorm, 2016). Based on alkali spreading value (Figure.2), aromatic non-basmati and aromatic basmati (PB-1121) genotypes had high ASV with score (7), The high ASV genotypes exhibit a low gelatinization temperature, making them economically significant as they require shorter cooking times, thus leading to savings on fuel costs (Chemutai et al., 2016). Aromatic basmati (NVB-1) genotype had low ASV value with score (2). Genotypes that had low ASV has high GT or less grain affected due to the existence of long amylopectin chain (Tuano et al., 2018). Non-aromatic genotype had intermediate ASV with score (4) which are mostly preferred globally for having good cooking quality such as softness after cooling, water absorption and moistness (Sthapit et al., 2015).

Figure 2 is available upon request from the corresponding author.

Figure.2 Alkali Spreading Value of diverse rice genotypes

1-Aromatic basmati (PB-1121), 2-Aromatic basmati (NVB-1), 3-Aromatic non-basmati, 4-Non-aromatic

Gel consistency is the indirect screening of cooking rice for hardiness (Chemutai et al., 2016). Based on Gel consistency (Figure.3) aromatic basmati (PB-1121) genotype was soft gel with gel length (74.0 mm), aromatic basmati (NVB-1) genotype was soft gel with gel length (73.0 mm),

Figure 3 is available upon request from the corresponding author.

Figure.3 Gel consistency of diverse rice genotypes

1-Aromatic basmati (PB-1121), 2-Aromatic basmati (NVB-1), 3- Aromatic non-basmati, 4-non-aromatic

aromatic non-basmati genotype was soft gel with gel length (89.53 mm) and non-aromatic genotype was soft gel with gel length (89.33 mm) this study was concurrent with the finding of (Chemutai et al., 2016).Amylose content has important function in determination of eating quality and cooking characteristics of rice genotypes (Ojha et al., 2018). Based on amylose content intermediate AC genotype was aromatic basmati (NVB-1) genotype (23.63%) and aromatic basmati (PB-1121) genotype (22.92%). According to IRRI survey, intermediate AC rice is mostly liked by south-east Asia people (Graham, 2002). Also Garcia et al., (2011) reported that intermediate AC rice has higher expansion volume, become harder after cooling and non-sticky. Low amylose content genotype were aromatic non-basmati genotype (15.55%) and non-aromatic genotype (13.46%). Low amylose content rice is good for eating due to sticky texture and soft after cooking (Ojha et al., 2018).

Table.4 Grain biochemical quality characteristics in diverse genotypes

S, No.	Genotypes	Aroma		Alkali Spread Value		Gel consistency		Amylose percentage
S, No.	Genotypes	Range	Aroma	Range	Category	GC mm	Category	Amy%	Category
1	Aromatic basmati (PB-1121)	4	Strong aroma	7	High	74.00	Soft	22.92	Intermediate
2	Aromatic basmati (NVB-1)	4	Strong aroma	2	Low	73.00	Soft	23.63	Intermediate
3	Aromatic non-basmati	4	Strong aroma	7	High	89.53	Soft	15.55	Low
4	Non-aromatic	1	Non-aroma	4	Medium	89.33	Soft	13.46	Low

Expression analysis of genes belonging to grain quality in diverse rice genotypes

The expression analysis of genes belonging to grain quality studied in certain diverse rice genotypes involving two basmati premium varieties (I. Pusa Basmati-1121 II. Nagina Vallabh Basmati-1, one aromatic non-basmati (Kalanamak) and one non-aromatic (IR-64) rice genotypes. The genes (OsGBSSI, and OsGS3) related to rice grain quality expression analysis come out from the cDNA (synthesized from panicle at milking stage samples total RNA) using semi-quantitative-PCR at different PCR cycles to estimate the amount of transcript in the form of fold change as compared to the control in aromatic basmati genotypes and aromatic non-basmati genotypes. Actin (housekeeping gene) was used for the normalization of cDNA which synthesized from young panicle tissue total RNA of divers rice genotypes (Figure.4).

Figure 4 is available upon request from the corresponding author.

Figure.4 Normalization of cDNA synthesized from total RNA of Panicle sample using OsACTIN as housekeeping gene in aromatic basmati, aromatic non-basmati and non-aromatic genotypes. L-100 bp Ladder, amplification at PCR cycle 21–35. (I) Aromatic basmati PB-1121, (II) Aromatic basmati NVB-1, (III) Aromatic non-basmati, (IV) non-aromatic.

OsGBSSI (Granule bond starch synthase I) gene expression analysis

Granule bond starch synthase I (GBSSI) gene play an important role to control amylose content in rice. Huang et al., (2020) proposed that granule bond starch synthase I (GBSSI) gene controls the starch properties in rice. The expression analysis of OsGBSSI gene was carried out using semi-quantitative-PCR in aromatic basmati, aromatic non-basmati and non-aromatic rice genotypes and non-aromatic rice genotype was used for check as control (Figure.5).

The GBSSI gene expression analysis through semi-quantitative-PCR revealed that aromatic basmati PB-1121 genotype had higher expression level of GBSSI gene, Aromatic basmati NVB-1 genotype had same expression level of GBSSI gene in comparison to control. Aromatic non-basmati genotype showed 32 fold lower expression level of GBSSI gene as compared to control. Oko et al., (2017) suggested that GBSSI gene usually has high expression level in amylose rich genotypes and lower expression level in poor amylose genotypes.

Figure 5 is available upon request from the corresponding author.

Figure.5 Semi-quantitative expression analysis of GBSSI gene in aromatic Basmati, aromatic non-basmati and non-aromatic genotypes. Aromatic basmati PB-1121 had higher expression level of OsGBSSI gene, aromatic basmati NVB-1 genotype had same expression level of OsGBSSI gene and aromatic non-basmati genotype had 32 fold lower expression level of OsGBSSI gene in comparison to control (non-aromatic genotype). L-100 bp DNA Ladder, amplification at PCR cycle 21–35. (A) Aromatic basmati PB-1121, (B) Aromatic basmati NVB-1, (C) Aromatic non-basmati, (D) non-aromatic

Oryza sativa GS3 gene expression analysis

GS3 (Grain size 3) gene encodes membrane protein by controlling cell proliferation that regulate grain size (grain length and weight) in rice. The GS3 gene expression analysis was carried out using semi-quantitative-PCR in aromatic basmati, aromatic non-basmati genotypes and non-aromatic rice genotype was used as control.

The GS3 gene expression analysis through semi-quantitative-PCR revealed that aromatic basmati genotypes had lower expression level of GS3 gene among other genotypes, it was 256 fold lower expression in comparison to control. Aromatic non-basmati genotype had same expression level of GS3 gene as compared to control (Figure.6). Similarly, Takano-Kai et al., (2009) results revealed that GS3 gene was expressed more in short grain varieties such as Asominori and Nipponbare. Grain size has impact on weight, which is measured with grain length and grain width (Xu et al., 2002). Promotion of cell division and grain filling are associated with increased expression of GS3 gene, leading to favorable outcomes for grain width and yield in rice. Conversely, a slender grain phenotype indicative of visual quality, is linked to a mutation occurring in the second exon of GS3 gene in Basmati rice (Wang et al., 2018). GS3 mRNA was detected in panicles and was not detected in leaf tissues (Takano-Kai et al., 2009).

Figure 6 is available upon request from the corresponding author.

Figure.6 Semi-quantitative expression analysis of GS3 gene in aromatic basmati, aromatic non-basmati and non-aromatic genotypes. Aromatic basmati genotypes had less expression level of GS3 gene among other genotypes, it was 256 fold lower expression in comparison to control (non-aromatic genotype). Aromatic non-basmati genotype had same expression level of GS3 gene as compared to control. L-100 bp Ladder, amplification at PCR cycle 21–35. (A) Aromatic basmati PB-1121, (B) Aromatic basmati NVB-1, (C) Aromatic non-basmati, (D) non-aromatic

In the present investigation based on morphological parameters, aromatic non-basmati genotypes had maximum days to flowering and days to maturity, high grain breadth and short medium grain physical appearance. Aromatic basmati genotypes had high panicle length, high grain yield per plant, high 1000-seed weight, high grain length after cooking, acceptable grain elongation ratio more than 1.6 and extra-long slender grain physical appearance. Non-aromatic genotype had minimum DF and DM, high grain yield per plant and medium slender grain physical appearance. Based on biochemical traits, Aromatic non-basmati genotype had strong aroma, high alkali spreading value, soft gel consistency and low amylose content. Aromatic basmati genotypes had strong aroma, high alkali spreading value for genotype (PB-1121), soft gel consistency and intermediate amylose content. Non-aromatic genotype had no aroma, intermediate alkali spreading value, soft gel consistency and low amylose content. Gene expression analysis revealed that aromatic basmati genotype (PB-1121) had higher expression level and genotype (NVB-1) exhibited same expression level of GBSSI gene while aromatic basmati genotype showed less expression level of GS3 gene, it was 256 fold lower expression in comparison to control. Aromatic non-basmati genotype had 32 fold lower expression level of GBSSI gene and same expression level of GS3 gene as compared to control.

Declaration of interest

I declare that author doesn’t have conflict of interest

Acknowledgment:

The author (Ali Sina Jayhoon) would like to express gratitude to the Department of Agricultural Biotechnology, S.V.P.U & T, Meerut, India, for providing research facilities in both field and laboratory settings.

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Expression Analysis of OsGBSSI and OsGS3 Genes, and Investigation of Morphological and Biochemical Characteristics across Diverse Rice (Oryza sativa L.) Genotypes

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Abstract

Background

Scope and approach:

Key findings and conclusion:

Figures

Introduction

Material and method

Grain aroma test (Sensory test)

Аlkali Spreаding Vаlue test (АSV)

Amylose content (AC)

Statistical analysis

Gene expression studies related to grain quality characters

Sample collection and extraction of total RNA

cDNA synthesis and normalization

Semi-quantitative PCR various components

Primers identification

PCR Cycle Steps and PCR’s amplification Products

Result and Discussion

Morphological characteristics

Cluster analysis based morphological parameters using Ward’s method

Bio-chemical grain quality characteristics

Expression analysis of genes belonging to grain quality in diverse rice genotypes

Conclusion

Declarations

Declaration of interest

Acknowledgment:

References

Additional Declarations

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