Novel FISSR-PCR primers and methods of identifying genotyping diverse genomes of plant and animal systems including rice varieties, a kit thereof

#The present invention relates to a set of inter-simple sequence repeats (ISSR)-PCR primers of SEQ ID Nos. 1 to 37 for genotyping eukaryotes and a method of genotyping diverse genomes of plant and animal systems using FISSR-PCR primers and SSR markers; more particularly, a FISSR and SSR method of distinguishing Basmati rice varieties from Non-Basmati (NB) rice varieties, and Traditional Basmati (TB) rice varieties from Evolved Basmati (NB) rice varieties, and also, a method for determining adulteration of Basmati rice with other rice varieties and a kit thereof.

#Rice is the staple food for more than half of the world's population. Its rich genetic diversity in the form of thousands of land races and progenitor species besides its economic significance have aroused unending interest among scientists for several decades. In the evolution of rice and its genetic differentiation into distinct varietal groups, consumer quality preferences have played a significant role besides agro-ecological factors. One such varietal group comprising of aromatic pulao/biryani rice of Indian sub-continent known as ‘Basmati’ is the highly priced rice in the domestic as well as international markets. Originated in the foothills of the Himalayas Basmati rice is characterized by extra long slender grain, pleasant and distinct aroma and soft and fluffy texture of cooked rice. These unique features of Basmati said to be the culmination of centuries of selection and cultivation by farmers, are well preserved and maintained in their purest form in the traditional Basmati (TB) varieties.

#The historical and archeological findings infer that the varieties with such unique morphological and quality attributes are not present in traditional rice growing areas anywhere in the world1. A number of undesirable traits of Basmati such as tall stature, low yield, sensitivity to photoperiod and poor response to fertilizer application prompted breeders to develop ‘elite’ Basmati varieties by making use of the high yielding semi-dwarf non-Basmati (NB) rice varieties. Such ‘elite’ evolved lines of Basmati (EB), however, fall short of the quality features of traditional varieties. Difficulty in recovering desirable recombinants from crosses involving NB and TB varieties, and reversion often to parental types in the backcross generations, suggest that probably indica and Basmati types are phylogenetically divergent2. A study on Asian rice varieties using isozyme markers clustered Basmati varieties in the group V gene pool, which is well separated from groups I and VI comprising of indica and japonica types, respectively3. Further evidence of high degree of divergence of Basmati from other indica varieties comes from high percentage of hybrid sterility4. The difficulties experienced in evolving ‘elite’ Basmati varieties combining all the desirable traits of TB and NB varieties have retained the preeminent status of TB varieties in the rice industry.

#FIG. 1 shows Fluorescence based ISSR profiles of TB, EB and NB rice varieties for three representative primers: a) SEQ ID NO. 5; b) SEQ ID NO. 3; c) SEQ ID NO. 26. Arrows and arrowheads indicate the markers, which differentiate Basmati and non-Basmati rice varieties respectively.

#FIG. 2 shows Fluorescence based SSR polymorphisms detected by 8 representative SSR loci in TB, EB and NB rice varieties. Arrows and arrowheads indicate the markers, which differentiate Basmati and non-Basmati rice varieties respectively. The asterisk indicates the duplication of locus 330 only in NB and some of the EB varieties. The first 7 loci are GENESCAN images and the last locus (RM 234) is 3.5% Metaphor agarose run ethidium bromide stained image.

#FIG. 3 shows Dendrogram derived from a UPGMA cluster analysis using Nei and Li Coefficients based on (a) ISSR markers; (b) SSR markers. Numbers on the nodes indicate the number of times a particular branch was recorded per 100 bootstrap replications following 1000 replications.

#In still another embodiment of the present invention, wherein evaluation of ISSR-PCR markers. The fluorescence based ISSR-PCR markers could be clearly resolved on an ABI automated sequencing gels. All the 12 anchored SSR motifs employed in the present study produced varying number of DNA fragments in different size range (Table 2, FIG. 1). The average number of bands produced by ISSR primers with different repeat motifs was negatively correlated with the number of nucleotides in the repeat unit of the motif. For example, among the 5′ anchored primers, the dinucleotide-based primers produced more number of bands (43.8±9.82) than tri- (37.6±7.89) and tetranucleotide (32) primers. Although we have not used similar number of primers in each repeat class of primers for such a comparison, the number of products amplified in different repeat length class reflected the frequency of different repeat motifs distributed in the rice genome. The 12 primers produced a total of 481 PCR products, of which 389 (80.9%) were polymorphic in all the 24 rice varieties. There appeared to be no correlation between the number of bands amplified and the degree of polymorphisms. For example, the primers R(TG)7 and Y(TG)7 generated 42 and 33 bands respectively, out of which 73.8% and 78.8% were polymorphic, on the other hand 98% of the 50 bands amplified by T3(ATT)4 primer were polymorphic. Among the 12 primers, two 5′ anchored, T3(ATT)4 and T(GT)9 and one 3′ anchored primer, (GT)8R generated more than 90% scorable polymorphisms.

#In still another embodiment of the present invention, wherein since our efforts were directed towards comparative genetic analysis of TB, EB and NB rice varieties, we evaluated the polymorphisms in these three groups. The degree of polymorphisms differed substantially among the three groups. The TB varieties showed very low level of polymorphism as compared to EB and NB varieties. Out of 340 bands scored in the TB varieties, only 115 (33.8%) were polymorphic (Table 2). On the other hand, 360 (77.4%) out of 465 bands were polymorphic in the EB and 166 (51.7%) out of 321 bands were polymorphic in NB varieties. The primer T3(ATT)4 was found to produce the most polymorphic bands in all the three rice groups: TB (91.4%), EB (92%) and NB (91.66) whereas RA(GCT)6 generated the least degree of polymorphisms (Table 2).

#In still another embodiment of the present invention, wherein the genetic diversity was calculated for each of the ISSR primers in all the three rice groups (Table 2). The difference in diversity values was as striking as the degree of polymorphism. The two primers, T3(ATT)4 and RA(GCT)6 which recorded the highest and the lowest polymorphisms respectively, in the three groups also recorded the highest and the lowest diversity values. The 12 primers had an average diversity value of 0.23±0.132 with a range of 0.07 to 0.6 for TB. The diversity values ranged from 0.37 to 0.72 averaging 0.57±0.10 for the EB, and 0.19 to 0.65 with a mean of 0.34±0.15 for the NB varieties. The difference was significant (p<0.001, between TB and EB; p<0.10 between TB and NB groups). Out of the 12 ISSR-PCR primers, 5 revealed 12 PCR products, a combination of which could distinguish Basmati from NB varieties (data not shown). On the other hand 21 specific PCR products generated by 9 ISSR-PCR primers could distinguish EB from NB varieties (FIGS. 1a to 1c). By making use of 2 to 3 informative primers the TB from the EB and NB varieties could be unambiguously distinguished. For example, by using two primers SEQ ID NO. 5, and SEQ ID NO. 26 (FIGS. 1a-and 1c) one can make a decision whether a given variety is a TB or an EB rice variety.