Genetic analysis on basmati rice varieties in Indian market using SSR markers

doi:10.21203/rs.3.rs-3887847/v1

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Genetic analysis on basmati rice varieties in Indian market using SSR markers

https://doi.org/10.21203/rs.3.rs-3887847/v1

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In the world of rice, among the thousands of varieties that are available, inarguably Basmati rice occupies the prime position due to its accoladed traits such as grain morphology, quality, fragrance, taste, fluffy texture, and tenderness of cooked rice. Due to these unique traits, Basmati rice that is exported from India and Pakistan earns triplex price compared to domestic non-basmati rice varieties. Among the Basmati rice varieties, the Traditional Basmati rice varieties which are rich in nutrition values and aroma are considered as the premium class and hence most of these rice varieties are being exported to other countries. In the present study, three hundred and fifty (350) Basmati rice varieties were collected from various super markets and retail outlets across India which were evaluated for allelic behavior using 10 SSR markers. According to UK CoP, only 75 sample qualified out of 350 samples. A total of 77.12% of the rice were Basmati varieties including varieties like Pusa Basmati 52%, followed by 28% of Pusa Basmati 1121. Surprisingly, we could not detect any Traditional Basmati rice portions either partially or completely. Very limited number of brands have 100% Basmati rice, without any adulteration in them. Our findings demonstrate the absence of Traditional Basmati rice in the Indian market despite of being the largest producer of the same.

Biological sciences/Genetics

Biological sciences/Molecular biology

Basmati

SSR markers

Adulteration

Pusa 1121 and 1509

Allelic behavior

Rice (Oryza sativa L.) being in the foremost place among the food crops is cultivated around the

world facilitating a major role in the Indian economy. It is a major staple food crop around the world especially in Asia and Africa [1,2]. Consumer quality preferences have always played a vital role besides agro-ecological factors despite of the distinct varietal groups evolved by genetic differentiation. One such varietal group comprising the aromatic pulao biryani rice of the Indian subcontinent known as ‘‘Basmati’’ is the highly priced rice in domestic as well as international markets [3]. Basmati (meaning fragrant in Hindi language) rice commands a special place among all aromatic rice cultivars on account of extra-long slender grain, soft and fluffy texture of the cooked rice, and pleasant distinct aroma.

Rice protein, though small in amount, is of high nutritional value [4]. Basmati rice has a very high nutrition value as it has a metallothionein-like protein and is abundant in cysteine that helps in iron absorption. Due to certain unique characteristics of Basmati rice like its texture of cooker rice, pleasant aroma, palatability, easy digesting, longer shelf life and long grains, Basmati rice is in high demand in the international market. Most of the Basmati varieties are cultivated in the foothills of the Himalayas comprising the North western states of Indian sub-continent like Haryana, Punjab, Uttaranchal, Western Uttar Pradesh, Jammu & Kashmir, Himachal Pradesh and Delhi for decades together.

Since, Basmati rice cultivation is confined to Indo-Gangetic regions of the Indian subcontinent, examples of genuine traditional Basmati (TB) varieties are Taraori Basmati and Dehradun Basmati. TB rice is not only in great demand in the domestic markets, but is also seen in the menu of experts worldwide. However, unsuitability of TB for intensive cultivation prompted the breeders to develop evolved Basmati (EB) varieties (that fall short of the quality traits of TB), leading to the presence of both EB and TB varieties in the market. Traditional Basmati rice is expensive in the market than Evolved Basmati rice and Non-Basmati rice (www.Basmati.com, www.oryza.com). Major challenges are on differentiating these rice varieties through visual basis and conventional methods. Therefore, to distinguish traditional Basmati from evolved Basmati and non-Basmati rice plays a vital role in Basmati trade [6].

Basmati is a high valued and premium rice, quality of rice varieties and price differences in the market create a situation for the adulteration of Basmati rice and increase the profits to the traders [7]. Traders have been found to mix sub-standard rice (long grain and aromatic non-basmati) with approved Basmati rice varieties to increase the profits. Many have indulged in polishing, using chemicals to enhance the look and aroma of basmati rice.

Detection of adulteration in the Basmati Rice and the portion of different Basmati rice varieties has become vital to assure the legitimacy of the products to abide by the regulations of the importing countries. The occasional admixture of non-Basmati rice substitutes in Basmati Rice can happen during the production of Basmati rice and therefore adulteration up to 7% of non-Basmati rice is legal in ‘Basmati rice’ (accepted level of adulteration in the UK Code of Practice). As per the legal rules under food labelling, an admixture of greater than 20% indicates a deliberate substitution which until and unless properly justified is illegal. Hence, accurate quantification of these substitution is an important point in combating such foul practices (www.food.-gov.uk/science/surveillance/fsis2004branch/fsis4704Basmati).

There are very few skilled technicians who are capable of differentiating the Basmati rice varieties with the proportionate of mixture as these are very closely correlated. Few varieties of Basmati rice cultivated in Indian and Pakistan have been classified as as ‘‘Basmati’’ by European Union which is sold at a very high price in the market as compared to the common rice (a ratio of 4:1) [4]. These rice are exported in huge amounts to USA and Europe. Traditionally used methods like morphological and chemical parameters have not been found enough to be discriminate, deserving more precise techniques. Several molecular techniques are available for detecting genetic differences within and among cultivars [7-12]. Currently, DNA-based methods proved handy and robust enough for unambiguous detection and quantification of adulteration [13-14].

Despite of the health benefits associated with Basmati Rice like lower arsenic and enriched in vitamins and minerals, instead of consuming Basmati Rice in India, due to high demand and attractive prices, India exported around 4.5 million metric tons of basmati rice in 2022/23 (source: APEDA). Since, Basmati is one of the major exports in rice varieties, it is indeed necessary to bring an awareness regarding the varieties that are consumed in India.

In the present study, we have tried to evaluate the Basmati varieties available in Indian market in the form of Branded and non-branded packages by using the microsatellite SSR marker based detection following the British code of practice. The current data and findings can give a clear insight about the Basmati varieties that are consumed in India.

Sampling and DNA isolation:

All the 350 different types of Basmati Rice from branded as well as non-branded suppliers were properly homogenized and processed for DNA isolation. A high quality and good yield of DNA (3-4µg) was obtained from all the samples.

Post PCR and capillary electrophoresis, successfully generated allelic information from 3730xl Genetic Analyser. Capillary electrophoresis (3730xl) being the gold standard analysis for the authentication of Basmati Rice is believed to be accurate and with least error.

In Gene mapper analysis minimum signal strength of 2000rfu has been obtained for alleles. Alleles of shortest and longest were determined and labelled as V-Z according to letter code specified by FSA (24), and considered the highest fluorescence intensity among stutter peaks for the evaluation. Each character defines a DNA fragment of a distinct length for each SSR marker. Interestingly, markers such as RM1, RM55, RM171 and RM229 expressed multiple allelic behavior for the studied samples which indicates huge adulteration in present sample. Allele patterns of different Basmati rice varieties approved according the CoP of 2005, 2011 and 2017 and based on 10 SSR markers have mentioned in Table 2 [16, 17].

With the reference from the above SSR marker allelic pattern, we have analysed all the 350 varieties of Basmati Rice using 10 SSR markers and compared the profiles.

SSR Marker Analysis:

After a detailed analysis of the 350 samples from different outlets and stores in India, we have observed that only 75 samples fall under the compliance of UK Code of Practice. We have obtained only 77.12% of basmati portions and 22.88% of Non-Basmati portions among the entire samples (Fig. 1). These 77.12% of the Basmati samples comprises of 52% of Pusa Basmati, 28% of Pusa Basmati 1121, 16% of Pusa Basmati 1509 and remaining portions of Pusa Basmati 1718 and Pusa Basmati 1609 (Fig. 2)

Basmati rice is superior rice variety characterized by its unique qualities differ from all other traditional rice varieties [16], Basmati is considered as a higher ranking rice variety around the globe. Over the course of times, due to improved living standards, Basmati rice has earned an attractive price in both the domestic and international markets. In the recent past 5 years, the area under Basmati cultivation in India has been increased to manifold. Keeping the increased demand, finest price and low water consumption into consideration from European Union, there is a huge scope to increase the area under Basmati cultivation and to increase the economy by increasing the profitability of farmers, forex earnings, and export volumes [17].

Not only the commercial values but also the health benefits that are associated with Basmati rice consumption makes it a premium choice all over the map. Basmati rice is rich in iron, zinc, phosphorus, magnesium, and many more essential amino acids [18] and also low glycemic index [19]. High in vitamin B1 (thiamine) and fibre supports optimal brain function as well as helps to control appetite and overall calorie consumption. Low glycemic index helps to maintain the stable blood sugar levels, Antioxidants in brown Basmati rice reduce cancer risk, magnesium and potassium promote healthy blood pressure.

Barcode Biosciences had collected and analysed (at genetic level) 350 samples which are available in Indian market and detected the mixing. The impurity accounted is up to 20%. To deceive buyers, traders are known to have even sprayed non-basmati rice with aroma extract from grass. Adulteration, either intentionally or not, is a global source of economic fraud to consumers and also to all stake holders involved in rice production and distribution. Because most counterfeiting incidents do not cause health harm, most of them remain undetected. Also, Basmati rice adulteration is the question of deceiving the consumers.

The Basmati varieties used in the present study covers most of the brands available at local markets and big grocery stores in India. According to UK Code of Practice (UK CoP), either Traditional, Evolved or New Basmati rice portion which are listed in CoP, should contain 93% or more Basmati portion, then the particular rice is considered as Approved. In the present study, out of 350 sample, only 75 sample qualified the criteria of UK Code of Practice (UK CoP). Surprisingly, no Traditional Basmati rice portions are observed either partially or completely. Traditional Basmati rice, which are cultivating in India and exporting to other countries in a great extent but end up with no sale in India market.

Most of the premium brands of Basmati rice has a combination of Pusa Basmati 1121 and Pusa Basmati 1509, both varieties comes under Evolved Basmati category. Whereas the unbranded has more non-Basmati varieties that has aroma and long grains but not categorized as Basmati rice due to the absence of other nutritional factors. Very limited brands have absolute Basmati rice although evolved basmati rice, without any adulteration in them.

Therefore, from the current study it is evident that even though India is the largest exporter and cultivator of Traditional Basmati rice, majority of the Basmati varieties being sold in Indian markets are either purely Evolved Basmati and/or its admixtures. Most of the Traditional Basmati is exported to other countries due to its high economic demand, premium tag, price and number of buyers in global markets.

Sampling and DNA isolation

A total of three hundred and fifty varieties of Basmati rice were collected from various super markets and retail outlets across India. These samples from different brands were subjected to grinding and made 20g of fine powder using coffee-bean grinder (Wonderchef Nutri-blend Mixer, Grinder & Blender). 200mg of rice flour taken for DNA isolation and genomic DNA was isolated from 3 aliquots of individual rice sample using NucleoSpin® food kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. The quality and quantity of DNA was measured using Biotek Epoch (Agilent Technologies Inc, USA) and further estimated by using 1% agarose gel electrophoresis and good quality of genomic DNA was used for subsequent process.

PCR amplification/DNA finger printing

Ten (10) universal SSR markers named RM1, RM223, RM348, RM202, RM44, RM201, RM241, RM229, RM55 and RM171 were used for PCR amplification [15], SSR markers for amplification (Table 1) were labeled with fluorescent dyes such as FAM (6-carboxylfluorescein), JOE (6-carboxy-4, 5-dichloro-2, 7-dimethoxy-fluorescein) or TAMRA (carboxy-tetramethyl-rhodamine) at the 5’-end by Barcode biosciences pvt ltd, Bangalore, India. The extracted DNA was served as the template for PCR amplification for SSR marker analysis and PCR reaction was performed with a total volume contain 25μL of 60 ng of DNA, 0.1 μg of forward and Reverse primers, 200 mM of dNTPs, 2.5 μl of 10 x buffer with MgCl2, and 2 unit of Amplitaq Gold DNA polymerase (Thermo Fisher Scientific, USA). PCR thermal cycler was programmed as initial denaturation at 94°C for 5min, followed by 35 cycles of 94°C for 1min, 55°C for 1min, 72°C for 1min and final cycle of 45 min at 60°C using Veriti™ 96 well Thermal cycler (Thermo Fisher Scientific, USA).

Capillary electrophoresis

The fluorescent labelled amplified fragments were suspended in 10 ul of Hi-Di formamide (Thermo Fisher Scientific Inc, USA) and proceeded for fragment separation. Fluorescent fragments were mixed with 0.2 ul of Internal lane standard 600 (Promega Inc, USA) and subjected to capillary electrophoresis using ABI3730XL Genetic Analyzer with POP7 polymers on 50-cm capillary arrays (Applied Biosystems Inc, USA). Fragment Length polymorphisms were analysed using Gene Mapper software v6.0 software. The Genotype profile (allelic table) with the labelled marker names were compared with the standard to determine the variety and percentage.

SSR marker analysis

To find the allelic polymorphism among all Basmati rice were performed by using Capillary electrophoresis based microsatellite SSR analysis. Allelic profile of the test samples were analyzed using Gene Mapper software v6.0 (Life Technologies, USA). Tandem repeats of alleles with 10 microsatellite markers represent as V, W, X, Y and Z, where V stands for shortest and Z for longest amplicon (allele) size as per survey on Basmati rice. The test experiments were performed in triplicates with standard deviation of three samples.

Acknowledgement

We are grateful to thank Barcode Biosciences Management for providing fund to collect sample and infrastructure to perform complete study.

Author’s contributions

RPK involved in designing the experiment and analysis of results. MM participated in revision to prepare the final version of the manuscript. PT and MV performed the experiment, KRG involved in the review of manuscript. TS involved in preparation of part of manuscript.

Data Availability Statement:

All the scientific data for 350 samples collected across India is available with the corresponding author and can be produced upon request.

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

Figure Legend:

Plant Guideline statement

Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation.

Figure 1: A doughnut plot describing the total percentage of Basmati and Non-Basmati rice out of 350 specimens collected across Indian outlets.

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Table 2 is available in the Supplementary Files section

Table 1 Characteristics of 10 microsatellite SSR markers. PCR amplification.

Markers	Repeat motif	Fluorescent Labelled	Allele size
RM 1	(GA)₂₆	FAM	65-120
RM 223	(CT)₂₅	JOE	140-180
RM 16	(CAG)₇	TAMRA	120-150
RM 202	(CT)₃₀	FAM	140-180
RM 44	(GA)₁₆	TAMRA	90-120
RM 201	(CT)₁₇	FAM	130-160
RM 229	(TC)₁₁ (GA)₅C₃ (CT)₅	JOE	105-120
RM 241	(CT)₃₁	JOE	120-140
RM 171	(GATG)₅	FAM	300-350
RM 55	(GA)₁₇	TAMRA	210-240

No competing interests reported.

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Genetic analysis on basmati rice varieties in Indian market using SSR markers

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