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SSR Marker-based DNA fingerprinting and morphological characterization for varietal identification in popular sorghum varieties of Tamil Nadu

DNA fingerprinting of varieties is mandatory for registration of germplasm and notification of newly released varieties. The present study attempted to develop a DNA fingerprinting profile of newly released varieties of sorghum using publically available SSR markers along with morphological DUS descriptors. Twenty-one SSR markers were used for the identification of unique variety-specific fingerprints in nine varieties/cultures. Of them, 14 primers (66.7%) showed clear and unambiguous amplification which is good enough to identify unique banding patterns for specific cultivars (77.8%). The SSR markers Xtxp024, Xtxp231, Xtxp075 produced unique alleles in CO 32 whereas Xtxp354 produced an unique alleles in K12. The SSR marker Xtxp003, Xtxp201 produced unique alleles in CSV 33 MF which could serve as valid genotype-specific SSR markers in varietal purity test program. The varietal-specific SSR marker will supplement the DUS test and could play a major role in varietal identification, thus resolving disputes during the seed certification process.


INTRoDuCTIoN
Sorghum (Sorghum bicolor (L.) Moench), an annual diploid (2n=2x=20) ranks fifth among global cereal crops next to maize, rice, wheat and barley (FAOSTAT 2015) with a global production of about 57.10 million metric tonnes (USDA, 2019). More than half of world sorghum production is from the semi-arid regions of Africa and Asia and one of the dietary staples of the worlds poorest, especially in the semi-arid tropics. It is notable for its multiple economic uses like food, fodder, biofuel, and other industrial uses.
Elite cultivars coupled with High-quality seeds play an indispensable role in the production. Varietal identification is of prime importance worldwide from the perspective of Plant Variety Protection (PVP). Protection can be granted if the newly evolved cultivar satisfies Distinctiveness, Uniformity, and Stability (DUS) (Prajapati et al.2018).
Since new cultivars were developed through hybridization between members of elite groups of genetically similar parents, the genetic variability among newly evolved cultivars were found to be even smaller (Rahman et al., 2009). This makes the unambiguous distinction of cultivars to be difficult from the rest based on DUS tests which consist of morphological and physiological characteristics. For varietal registration under "Protection of Plant Varieties and Farmer's Rights (PPV&FR) Act, 2001" the newly evolved cultivar must fulfill the DUS test. Elite sorghum genotypes were first selected based on morphological characterization (Beta and Corke, 2001). However, from several studies, it is significant that morphological markers alone are insufficient due to low level of heritability, low level of abundance and is highly influenced by the environment (Seetharam and Ganesamurthy, 2013, Verma et al. 2017, Bhusal et al. 2017, Prajapati et al. 2018. However, Molecular markers based on fingerprinting allows precise, objective and rapid cultivar identification that are challenging to characterize due to the identical morphological characters or indistinct traits (Galovic et al., 2006). It's clear from several studies that several types of molecular markers such as RAPD (Sorghum: Mehmood et al., 2008), AFLP (Wheat: Heun et al., 1997) and SSR marker (Sorghum: Bantte and Mogus, 2016) can be used for genetic fingerprinting.
However, the Simple Sequence Repeat (SSR) represents a high degree of polymorphism, reproducibility, codominance and multi allelic types of variation (Becher et al., 2000) and hence widely used in genetic analysis and cultivar identification. Applicability of SSR markers in cultivar identification has been reported in Rice (Rahman et al., 2009), Grapes (Dangl et al., 2001) and Potato (Coombs et al., 2004).
In recent years, fingerprinting the commercial sorghum cultivars based on molecular markers is of paramount significance for unambiguous and quick identification of similar or closely related varieties which could prevent the disputes arising due to varietal ownership. The applicability of SSR markers in Sorghum Fingerprinting has been reported by Bantte andMogus, 2016 andGangurde et al., 2016. The DNA fingerprinting is developed for the released sorghum varieties of Ethiopia (Bantte and Mogus, 2016). Therefore, this study intended to probe the appliance of the molecular marker in the context of DUS tests to disclose unique variety-specific fingerprints. This varietal fingerprint could be used for various varietal purity test programs of closely related sorghum cultivars and submission of fingerprint data for the crop variety registration.

MATeRIAlS AND MeThoDS
A total of eight elite sorghum cultivars were studied. These genotypes were chosen based on their significant role in the seed production system and are categorized based on their use in agricultural practices: grain sorghum (K8, K12, CO(S) 28, CO 30, CO 32), fodder sorghum (CSV 33MF) and pre-release cultures which are in the advanced stage of yield trails like MLT and ART (TNS 660, TNS 661) ( Table 1). The popular sorghum varieties along with cultures were evaluated for varies agro-morphological traits based on 31 DUS descriptors provided by Protection of Plant Variety and Farmers Right Act (PPV&FRA, 2007).
A set of twenty-one informative SSR markers were used for identification of unique variety-specific fingerprint. These SSR markers were picked based on their maximum genome coverage. The SSR markers include Xtxp003, Xtxp024, Xtxp027, Xtxp030, Xtxp031, Xtxp038, Xtxp043, Xtxp051, Xtxp058, Xtxp075, Xtxp088, Xtxp145, Xtxp201, Xtxp231, Xtxp274, Xtxp285, Xtxp286, Xtxp287, Xtxp297, Xtxp312 and Xtxp354. The SSR markers PCR conditions validated by Shehzad et al 2008, Kong et al., 1999 is used in the present study (Table 2). Seedlings were raised under the greenhouse during November 2018. Genomic DNA from each cultivar was obtained from fresh leaf tissues of two-week-old seedlings based on the modified CTAB method (Grewal et al., 2013). DNA quality and quantity were obtained photometrically by Bio-Spectrometer, Kinetic (Eppendorf, Germany) and visually by agarose gel electrophoresis (0.8% agarose gel).

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for 1 min, Extension at 72°C for 1 min. In the last cycle, the Final extension was provided at 72°C for 7 min. For the separation of PCR product, electrophoresis was carried out on a 3% agarose gel containing Ethidium bromide using 1X TBE buffer (pH 8.0). The amplified products were visualized under UV light source (Bio-Rad, CA and USA). Only clear and unambiguous SSR alleles were scored based on base pair (bp) size in each genotype.

ReSulT AND DISCuSSIoN
Morphological markers have been frequently used in the genus Sorghum for descriptive purposes and are used in plant variety protection for distinguishing the individual varieties based on its distinctness, uniformity, and stability (DUS) test (Prajapati et al.2018). Characterization and evaluation of the accessions are the pre-requisites for the utilization of the available diversity in breeding perspective. Hence, the sorghum varieties are characterized to identify cultivar specific traits (Table 3) and cultivar specific SSR fingerprint which could be used for variety identification.
The medium-sized anther type varieties were further differentiated using the SSR marker. The SSR marker Xtxp145 differentiated K12 (210bp) and CO 30 (220bp) from CO (S) 28 and CO 32 by the presence of unique alleles whereas, Xtxp354 and Xtxp024 can be used for further differentiation of K12 (210bp) and CO 32 (160bp). Henceforth the SSR markers Xtxp354 and Xtxp024 can be used as a genotype-specific marker in varietal purity testing program. Absence of lemma arista formation (awn) reduced the evapotranspiration rate (Ayana and Bekele, 1998) which is noticed in all the tested varieties except CSV 33 MF. The presence of awn in CSV 33 MF acts as a defensive mechanism as bird scarers. Darker Glume color was found to be associated with grain mould resistance (Audilakshmi et al., 1999) in many of the sorghum varieties tested. The corneous endosperm was found to be correlated with grain mould resistance (Jambunathan et al.1992 andMukuru, 1992). The genotypes K12, CO (S) 28, CO 30, TNS 660, TNS 661 and CO 32 grains had 75% corneous endosperm. Hence, the glume color and endosperm texture can be used as a morphological marker for selecting parents in resistance breeding program. In addition to this, CSV 33 MF can be easily differentiated from the rest based on seed characters like caryopsis color after threshing (greyed orange), size of mark of germ (small) and color of vitreous albumen (greyed orange).
The discriminating morphological and DUS criteria can be efficiently used for varietal identification and grouping of varieties/cultures as grain or forage type. The SSR marker profile of Xtxp24, Xtxp231, Xtxp075, Xtxp354, Xtxp003 and Xtxp201 can be used for identification of specific cultivar. The unique variety-specific fingerprint obtained from the study can be used for varietal registration under the PPV and FR Act for obtaining plant varietal protection. This will also be used in varietal identification for consumer protection and resolving disputes in seed certification.