Single grain analysis of the complex Basmati rice samples to determine the nature of admixtures and accurate adulteration quantification

Abstract

Adulteration in Basmati rice is a rife phenomenon either due to unavoidable postharvest procedures or intentionally by exporters to gain more profit. SSR marker based multiplex and singleplex assay is being used for detection and quantification of adulteration in Basmati rice using bulked-seed. Identification of different varieties present in the complex Basmati sample which is a mix of more than two varieties is not feasible from analysis on the bulked-seed. Therefore, we have undertaken genotyping using multiplex-PCR of SSR markers on 20 randomly selected single grains from complex samples and the results revealed accurate adulteration levels and presence of different types of admixtures in complex Basmati samples. More importantly this method helped in identifying hybrid (double) profiles for markers indicating outcrossing in Basmati rice.

Introduction

Basmati rice with extra-long grains, soft and fluffy texture of cooked rice, length-wise expansion of grains after cooking and pleasant aroma is indigenous to the Indian subcontinent and is popular all over the world as biryani rice/pulao rice. It is assigned Geographical Indication (GI) status by Intellectual Property Appellate Board (IPAB) as all the traits of the Basmati rice are expressed only when grown in specific geographical regions (Joshi 2018; Siddiq et al. 2012).

Inadvertent mixing of non-Basmati rice in Basmati rice is unavoidable during post-harvest processing. Export of Basmati rice generates three times more income than non-Basmati rice because of its higher price (Siddiq et al. 2012) and seven of the notified Basmati varieties are exempted from import duty by European union (www.riceassociation.org.uk) prompting traders to mix non-Basmati rice in Basmati rice to gain profit. To combat this, the importing countries have set some standards on the acceptable amount of non-Basmati seeds in Basmati rice. European Union rejects the consignments carrying more than 7% of non-Basmati rice in addition to making it mandatory that all the consignments carry the certificate of purity based on DNA test (www.riceassociation.org.uk; (EC)-No. 972/2006).

Until 2013, the Basmati rice samples that were analysed in the APEDA-CDFD Centre for Basmati DNA analysis, PDFS, CDFD were majorly either pure samples or adulterated with one non-Basmati variety. In 2013, 10% of the Basmati 370/Type3 samples were complex in nature which consisted of a mix of three or more varieties with Basmati 370/Type3 as a major variety and the others being mostly non-Basmati varieties. From 2014 onwards, the number of complex samples have increased significantly with either Basmati 370/Type3 or Pusa Basmati 1 as the major variety. In 2019, 23% of samples received were complex, of which 86% were of Basmati 370/Type3 type and Taraori and Pusa Basmati 1 type were of 7% each. The trend remained the same in 2020 also.

The multiplex and singleplex assay, using reported 8 SSR markers (Archak et al. 2007; Vemireddy et al. 2007), which is performed on bulked-seed is very efficient and gives conclusive information on the type of varieties present in the sample only if two varieties with different profiles are present in the sample as each marker gives only two alleles. However, as complex sample is a mix of at least three varieties, all the alleles of all markers (three of RM171, RM55, RM72, RM44 and two of RM202, RM348, RM241 and RM1) are present. Though it is possible to calculate the adulteration percentage based on the unique alleles (Archak et al. 2007; Vemireddy et al. 2007) and non-overlapping alleles of the adulterants present in the sample, it is not possible to analyse the types of these different varieties present. This is because profiles of all Basmati and all potential non-Basmati varieties can be drawn as all the alleles of the markers are amplified making the analysis difficult. A report on analysis with 8 single grains suggested that more number of single grains have to be studied to resolve the complexity of complex samples (Woolfe 2007). In order to delineate the types of different varieties present in the complex sample we have attempted multiplex-PCR on 20 random single grains which helped to understand the nature of complex samples and the results are described in this work.

Materials and methods

Basmati samples that were received at APEDA-CDFD Center of Basmati analysis, PDFS, CDFD were used in this study.

After genomic DNA isolation from bulked-seed using Qiagen DNeasy plant mini kit, multiplex PCR with eight SSR markers using fluorescent flourophores tagged primers, genotyping with capillary based genetic analyser ABI3730 (Applied Biosystems), recording peak-area of alleles and quantification of adulteration were carried out as described (Archak et al. 2007; Vemireddy et al. 2007). If more than one allele comes up even for one marker, singleplex of all eight SSR markers is carried out for accurate determination of adulteration. When the results indicate the sample to be a complex sample, single grain analysis as described below is followed.

500 grains were randomly picked from properly mixed Basmati sample (based on the fact that 460 grains are required to detect 1% adulteration at 99% confidence (Armitage 2003) and placed one seed in each eppendorf tube that are serially numbered from 1 to 500. Twenty random numbers between 1 and 500 were generated using random number generator of random.org website (www.random.org) and grains present in eppendorf with these numbers were used for analysis. DNA was isolated from the powder of each single grain using modified CTAB method (Saghai-Maroof et al. 1984) and multiplex PCR with eight SSR markers (Archak et al. 2007) was set up with 10 ng of genomic DNA, 1 × buffer, 100 μM dNTPs, 2 mM MgCl2, 0.5 U Amplitaq Gold DNA polymerase (Applied Biosystems) following PCR reaction conditions comprising of an initial denaturation at 95°C for 10 min followed by 30 cycles of denaturation at 95°C for 45 s, annealing at 55°C for 90 s, and extension at 72°C for 60 s, with a final extension at 60°C for 30 min. Genotyping using ABI3730 and recording peaks using Genemapper software version 4 was performed as described (Archak et al. 2007).

Results and discussion

Purity testing of Basmati rice samples that are received at APEDA-CDFD Centre for Basmati DNA analysis, PDFS, CDFD is performed with eight SSR markers on DNA isolated from bulked-seed.

The presence of 322 bp allele of RM171, 106 bp allele of RM1 indicates the presence of adulteration in Basmati rice since they are present in frequent adulterants such as Sharbati and Terricot. While few recently notified Basmati varieties like Malaviya Basmati, Vallabh Basmati 21, Pant Basmati 1 and 2 also carries these alleles (IS 16538: 2019), none of the pure samples tested at CDFD so far have been identified to be any of these varieties. This suggests that these varieties are not yet getting exported and thus allows the usage of these alleles in detecting and quantitating adulteration.

When the Basmati sample is pure without any adulterant in it, eight peaks corresponding to one allele for each marker are observed. However, when Basmati sample is adulterated with one non-Basmati variety, for instance if Basmati370 variety is adulterated with non-Basmati variety Sharbati (Table 1, Sr.No.1), since the profiles of these two varieties with these eight SSR markers are completely different two alleles per marker are amplified. The relative fluorescence units (rfu) for the peaks of both Basmati alleles and adulterant alleles are used to calculate peak-area ratios to determine the amount of adulterant PCR in the total PCR product for each marker. Finally, the peak-area ratios of all the markers are averaged to determine the final adulteration percentage. However, some Basmati varieties and adulterants have same alleles for markers RM55 (228 bp), RM72 (159 bp) RM348 (131 bp), and RM241 (130 bp) and these are considered as overlapping alleles. In such cases (Table 1, Sr.No.2), peak-area ratios of these alleles are not considered while determining adulteration percentage.

Table 1.

Profiles^a of the Basmati samples generated from genotyping on bulked-seed samples

S. nos	RM171	RM55	RM202	RM72	RM348	RM241	RM 44	RM1	Variety Name	Adulteration percentage
1	335	233	182	174	140	143	108	74	Basmati370/Type3
	322	228	162	159	130	130	103	106	Sharbati
2	343	228	182	159	131	130	112	74	Pusa Basmati 1
	322	228	162	159	130	130	103	106	Sharbati
3	335(73.6%)	233(77.3%)	182	174(61.6%)	140(69.7%)	141(44%)	108(67.3%)	74(90.2%)	CS1	17.7%
	322(12.5%)	218(9.5%)		159(18.5%)	131(30.3%)	130(56%)	103(29.5)	106(9.8%)
	343(13.9%)	228(13.3%)		150(20%)			112(3.2%)
4	335(94%)	233(91.3%)	182	174(92.3%)	140(94.3%)	141(48%)	108(87%)	74(98.9%)	CS2	5.2%
	322(2.8%)	228(8.7%)		159(4.1%)	131(5.7%)	130(52%)	103(13%)	106(1.1%)
	343(3.2%)			150(3.6%)
5	335(92.4%)	233(94.3%)	182	174(90.1%)	140(94.3%)	141(49%)	108(92%)	74(97.2%)	CS3	5%
	322(4.2%)	228(5.7%)		159(4.6%)	131(5.7%)	130(51%)	103(8%)	106(2.8%)
	343(3.4%)			150(5.3%)
6	343(83.9%)	228	182	159	131	130	112(66.5%)	74(86.8%)	CS4	20.4%
	322(14.6%)						103(33.5)	106(13.2)
	335(1.5%)
7	335(84.1%)	233(77.5%)	182	174(74.2%)	140(81.6%)	141(69%)	108(83.2%)	74(94%)	CS5	10%
	322(7.2%)	228(9.7%)		159(11.1%)	131(18.4%)	130(31%)	103(16.8%)	106(6%)
	343(8.7%)	218(12.8%)		150(14.7%)

^aWhenever more than one allele is amplified, contribution of each allele in total PCR product is expressed in percentage in parenthesis next to the allele

The multiplex and singleplex analysis of complex samples revealed the presence of all three alleles for the markers RM171, RM55, RM72, and RM44 indicating the presence of at least three varieties in the sample. Moreover overlapping alleles of RM348, RM241 and RM55 are also present and hence the alleles of RM171 (322 bp), RM72 (159/150 bp), RM44 (103 bp), and RM1 (106 bp) have been used to determine the adulteration (Table 1) as these are the alleles specific to the adulterants (Archak et al. 2007; IS 16538: 2019). Though the above alleles are used, it is possible that adulterants with profiles other than these alleles are present in the sample and as it is difficult to predict them and actual adulteration present in the sample might have been underestimated. Therefore, in order to elucidate the amount of adulteration present and the number and kind of different varieties present in the complex sample, 20 random single grains were selected and multiplex assay with eight SSR markers was run on all the 20 single grains. The results on five representative complex samples are presented here.

The samples described here were either received from Export Inspection Council (EIC) or private exporters to CDFD for analysis and their details are kept anonymous and labelled as CS1, CS2, CS3, CS4, and CS5 (Tables 1 and 2).

Table 2.

Profiles of the complex Basmati samples generated from genotyping on randomly selected twenty single grains

S. nos	Complex sample	Single grain No.	RM171	RM55	RM202	RM72	RM348	RM241	RM 44	RM1	Variety name	Adulteration percentage
1	CS1	SG1, SG3-5, SG7-9,SG11-15, SG17-18 and SG20	335	233	182	174	140	141	108	74	Basmati370/Type3
		SG10	335	218,233	182	174	140	141	108	74	Variant of Basmati370/Type3
		SG2	343,322	228	182	159,150	131	130	103	106	Adulterant
		SG6	343	228	182	159	131	130	103	106	Adulterant
		SG16	343	233	186	159	131	130	103	106	Adulterant
		SG19	347	228	186	159	131	130	103	106	Adulterant
												20%
2	CS2	SG1 to SG14,SG16-SG18 and SG20	335	233	182	174	140	141	108	74	Basmati370/Type3
		SG15	335	233	174	174	140	141	108	74	Adulterant
		SG19	343	233	182	174	131	130	108	74	Adulterant
												10%
3	CS3	SG1 to SG4, SG6-SG9, SG11-SG12, and SG14-SG20	335	233	182	174	140	141	108	74	Basmati370/Type3
		SG5	322	228	182	159	131	130	103	106	Adulterant
		SG10	322,343	228	182	159,150	131	130	103	106	Adulterant
		SG13	322,343	228	182	150	131	130	103	106	Adulterant
												15%
4	CS4	SG2,SG6,SG8-SG12, SG16-SG20	343	228	182	159	131	130	112	74	Pusa Basmati 1
		SG1,SG5,SG7, SG13, SG14	322	229	182	159	131	130	103	74	Terricot-Adulterant
		SG3, SG15	322	229	162	159	131	130	103	74	Sharbati-Adulterant
												35%
5	CS5	SG1	322	228,233	182	174,159	140,131	130	108	106	Adulterant
		SG2	335	233,218	182	174,159	140,131	141,130	108	74	Variant of Basmati370/Type3
		SG3,SG4	335	233	182	174,159	140,131	141,130	108	74	Variant of Basmati370/Type3
		SG5	335	233	182	174	140,131	141,130	108	74,106	Variant of Basmati370/Type3
		SG6	335	233	182	174,159	140,131	141,130	112	74	Variant of Basmati370/Type3
		SG7	335	233	182	174,159	140,131	141,130	108	74	Variant of Basmati370/Type3
		SG8	335	233,218	182	174,159	140,131	141,130	112	74	Variant of Basmati370/Type3
		SG9	335	233	182	174	140	141,130	108	74	Variant of Basmati370/Type3
		SG10,SG11,SG14,SG15,SG19,SG20	335	233	182	174,159	140,131	141,130	108	74	Variant of Basmati370/Type3
		SG12	335	233,218	182	174	140,131	141,130	108	74,106	Variant of Basmati370/Type3
		SG13	343,335	233,218	182	174	140,131	141,130	108	74,106	Variant of Basmati370/Type3
		SG16	335	233	182	174,159	140	141,130	108	74,106	Variant of Basmati370/Type3
		SG17,SG18	335	233,218	182	174,159	140,131	141,130	108	74,106	Variant of Basmati370/Type3
												100%

With CS1, adulteration percentage after genotyping on bulked-seed based on adulterant alleles mentioned above was around 18% (Table 1, Sr.No. 3). When the single grain analysis was performed, the profiles of four grains (SG2, SG6, SG16, SG19) out of 20 grains did not match with any of the notified Basmati varieties (Table 2, Sr.No. 1). Though the adulteration percentage calculated based on bulked-seed and single grain analysis are very close, it turns out that all the adulterants had 343 bp allele of RM171 which is usually not considered for adulteration calculation on bulked-seed analysis as it is also present in Basmati varieties. Single grain 2 (SG2) has hybrid peaks (343 bp, 322 bp) for RM171 marker and probably 322 bp fragment is present in all adulterants but did not get amplified and this is corroborating with the identical peak-area ratios obtained for 343 bp and 322 bp peaks on bulked-seed. Since SG2 also has hybrid peaks (159 bp, 150 bp) for RM72 marker, average peak-areas ratios of 150 bp and 159 bp was used for calculation of adulteration percentage in complex samples whenever 150 bp allele was present as this is not present in most of the Basmati varieties (IS 16538: 2019).

With CS2 (Table 1, Sr.No.4 and Table 2, Sr.No.2) the profiles of adulterants SG15 and SG19 are different from the profiles of adulterants observed in CS1. In addition, the adulteration percentage calculated based on analysis on bulked-seed is 5.2% while that of single grain analysis is 10%. This is because adulterants detected have 335 bp allele of RM171 which cannot be used for determining adulteration from analysis on bulked-seed. Further it also suggests that this complex sample also has adulterants with 322 bp allele which did not get picked up in single grain analysis since its contribution is very less. This shows that there are more than three different types of adulterants in this sample. Similarly, with CS3 (Table 1, Sr.No.5 and Table 2, Sr.No.3) adulteration percentages from single grain analysis is higher than that from bulked-seed. Despite the peak-area ratios of all the alleles from multiplex analysis in CS2 and CS3 are very similar, the profiles of adulterant obtained are different. CS4 has Pusa Basmati 1 as the major variety which is adulterated with two adulterants Sharbati and Terricot (Table 1, Sr.No.6 and Table 2, Sr.No.4). While analysis on bulked-seed has detected 20% adulteration, single grain analysis revealed adulteration as 35%. The above results indicate that analysis on bulked-seed does not determine actual amount of adulteration present in the complex sample. In addition, because of the diverse nature of adulterants getting mixed up in Basmati sample, it is difficult to choose the alleles to use in calculating adulteration percentage.

All the Traditional Basmati varieties are expected to be homozygous at all the loci as they are being cultivated with their selected traits for more than 200 years (Siddiq et al. 2012). In complex sample CS1 we observed a variant of Basmati370/Type3 grain (SG10) with hybrid peaks in RM171 (218, 233), suggesting that some level of crossing is occuring. The above suggestion has been corroborated with the analysis of complex sample CS5. The profile of CS5 and contribution of alleles of all the markers from analysis on bulked-seed are similar to CS1, CS2 and CS3 (Table 1). However, the single grain analysis of this sample revealed that all the grains have hybrid (double) profiles at more than three markers (Table 2, Sr.No.5). These observations indicate that the Basmati370/Type3 analysed in the study has been crossed with other variety and therefore more likely that the alleles of genes governing quality traits are also either crossed out or are in heterozygous state leading to deprivation of quality traits of Basmati rice and therefore all the single grains possessing double peaks should be considered as adulterants. Apart from this sample, we received only one such sample where all the 20 single grains had hybrid peaks for at least three markers.

If analysis based on only bulked-seed was considered, then CS2 and CS3 would have been exported even though the actual adulteration percentage is more than 7%. Moreover, single grain analysis has helped to delineate the type of admixtures present in each complex sample and also if there are any hybrid peaks in the profiles of Basmati samples. However, limitations of single grain analysis are that analysis cost is 20 times higher than conventional analysis on bulked-seed and in 10% of analysed samples grains with adulterant profiles were not picked up indicating that in such cases more number of grains are needed to be tested. From these results it is better to take up single grain analysis of complex samples when the contribution of RM171 (322 bp), RM1 (106 bp), RM72(159/150 bp) and RM44 (103 bp) in complex samples is equal to or more than 5% by conventional analysis on bulked-seed.

Conclusion

Multiplex analysis on 20 single grains of complex samples is robust in identifying different types of Basmati and non-Basmati varieties present in the sample. This work has thrown light on the extent of adulteration in the complex Basmati samples and also revealed incidence of cross breeding with other rice varieties which can affect the Basmati quality traits.

Author’s contribution

RLV carried out the experiments; VVS conceived, KA conceived, supervised the work and wrote the MS.

Funding

The study was supported from the core grants of CDFD.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare no conflict of interest.

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