Characterization of little millet (Panicum sumatrense) varieties using morphological descriptors and SSR based DNA fingerprinting

Little millet (Panicum sumatrense) is also known as Indian millet. It is cultivated as a cereal across Nepal, India and Western Myanmar, and its center of origin is West Africa. The wild relative of little millet is P.psilopodium. It forms an important role in tribal agriculture in Eastern Ghats of India. Little millet is grown on temperate and tropical climate. Little millet is an annual tuffed grass with slender culms, soft leaves, inflorescence a panicle with erect hairy branches, spikelets in pairs with two glumes. The discovery of syntenic regions among the cereals which aids to identify useful alleles of important agromorphological traits. An earlier study by Ali et al [2] developed 48 EST-SSR markers among 37 accessions of the little millets. Only limited reports are available on the genetic diversity of little millet germplasm, that too is based on a limited number of DNA markers [3, 4]. Thus the limited sequence information available in the little millets necessitates the search for the available markers from the maize, barnyard millet, and pearl millet due to the cereal millet synteny and also the availability of the SSR markers. The little millet husked grain is cooked as like rice and sometimes made into flour for different types of food preparations. The soft straw is palatable to cattle and the green plant has potentialities as a quick-growing fodder. The present investigation was conducted to carry out the comprehensive characterization of little millet genotypes based on Distinctness, Uniformity and Stability (DUS) characters for Protection of Plant Varieties and Farmers Rights Authority (PPVFRA) registration because of its property to identify the dissimilarity between the newly released and the existing genotypes as well as to distinguish the germplasm. Cereal crops exhibit cross-genera transferability, of DNA markers into many cereals such as maize, Characterization of little millet (Panicum sumatrense) varieties using morphological descriptors and SSR based DNA fingerprinting


INTRODUCTION
Little millet (Panicum sumatrense) is also known as Indian millet. It is cultivated as a cereal across Nepal, India and Western Myanmar, and its center of origin is West Africa. The wild relative of little millet is P.psilopodium. It forms an important role in tribal agriculture in Eastern Ghats of India. Little millet is grown on temperate and tropical climate. Little millet is an annual tuffed grass with slender culms, soft leaves, inflorescence a panicle with erect hairy branches, spikelets in pairs with two glumes. The discovery of syntenic regions among the cereals which aids to identify useful alleles of important agromorphological traits. An earlier study by Ali et al [2] developed 48 EST-SSR markers among 37 accessions of the little millets. Only limited reports are available on the genetic diversity of little millet germplasm, that too is based on a limited number of DNA markers [3,4]. Thus the limited sequence information available in the little millets necessitates the search for the available markers from the maize, barnyard millet, and pearl millet due to the cereal millet synteny and also the availability of the SSR markers. The little millet husked grain is cooked as like rice and sometimes made into flour for different types of food preparations. The soft straw is palatable to cattle and the green plant has potentialities as a quick-growing fodder. The present investigation was conducted to carry out the comprehensive characterization of little millet genotypes based on Distinctness, Uniformity and Stability (DUS) characters for Protection of Plant Varieties and Farmers Rights Authority (PPVFRA) registration because of its property to identify the dissimilarity between the newly released and the existing genotypes as well as to distinguish the germplasm. Cereal crops exhibit cross-genera transferability, of DNA markers into many cereals such as maize, pearl millet, barnyard millet. The present study was conducted with the objectives of fingerprinting the little millet varieties using the cross transferability of maize, pearl millet, barnyard millet genomic SSR markers.

Plant Materials and DNA Extraction
Little millet germplasm was raised in Randomized Block Design (RBD) with two replications at Centre of Excellence in Millets, Athiyanthal during Kharif 2019. The little millet cultures in the advance stage of varietal trail along with released varieties viz., BL6, ATL1, TNPsu176, Co (Samai) 4, Paiyur2 are chosen for the DUS characterization and DNA fingerprinting work. The leaf samples were collected from the Center of Excellence in Millets, Athiyandal. The genomic DNA was isolated from 15 days old seedlings by the modified CTAB method [5]. The genomic DNA quantity and quality was checked in Nanodrop and also using 0.8% agarose gel electrophoresis.

Morphological Characters for DUS Testing
The 15 quantitative traits were recorded from five randomly selected plants from each replication during Kharif 2019 at the Centre of Excellence in millets, Athiyanthal, TNAU. All the five genotypes were characterized for 15 quantitative traits viz., Plant height (cm), Number of basal tillers, Culm branches, Blade length of flag leaf (cm), Blade width of flag leaf (cm), Sheath length of flag leaf (mm), Length of peduncle (mm), Peduncle exsertion (mm), Length of inflorescence (mm), Number of primary inflorescence branches, Number of nodes per primary axis of inflorescence, Number of secondary inflorescence branches, Length of fruit (mm), Width of fruit (mm) and days to flowering (days) were recorded. Observations were also recorded for 7 other qualitative traits viz., Growth habit, culm branches, Ligule pubescence, Degree of lodging at maturity, Inflorescence shape, compactness of inflorescence and Color of the apicules. In addition, nutritional quality, cooking quality and sensory evaluation were done among the three varieties viz., ATL1, Co (Samai) 4, Paiyur2.

PCR Amplification with Cereal Genomic SSR Markers
Amplification of little millet genotypes was performed using 16 barnyard millet, 25 maize and pearl millet genomic SSR markers (Table 1). Barnyard millet SSR markers are obtained from a previous study [6] is used to develop EST-SSRs. SSR primers were designed based on the sequences flanking the SSR motifs using the Primer Premier 6.0 software (Premier Biosoft International, Palo Alto, CA). The SSR loci containing ideal repeat units of 2-6 nucleotides only were selected. The standard based on the fixation of minimum SSR length was defined as five reiterations for each repeat unit. Complex SSR types and mononucleotide repeats were omitted. Maize SSR markers were obtained from the Maize GDB database (https:// www.maizegdb.org/data_center/ssr). Pearl millet SSR primer pairs were taken from International Crop Research Institute for Semi-Arid Tropics (IPES, PMSP, ICMP).
The polymerase chain reactions (PCR) were performed in 10μL reaction volume containing 7μL of the 1X Master mix, 0.6μL of primer (forward and reverse), 1.4μL of Sterilized water and about 200ng (1μL) of template DNA. The PCR temperature profile for maize genomic SSRs was initial denaturation at 94 o C for 7 min followed by 35 cycles of denaturation at 94 o C for 30 sec, annealing temperature at 55 o C for 30 sec, extension at 72 o C for 45 sec and a final extension at 72 o C for 7 min and terminated by the hold at 10 o C. The electrophoresis was done at 150V for 1.5 hr at room temperature by using the 3% agarose gel. Gels were added with ethidium bromide and visualized using GELSCAN 1312.

Morphological Variations
Fifteen and seven quantitative and qualitative characters (Tables 2 and 3) were studied and revealed that the varieties selected for the study were significant for the morphological characters. Large variations were recorded in ATL 1 (125-135 cm) for the trait plant height (Figure 1 and Table 3). Whereas other varieties recorded from the range of 105-112cm. Similar results were studied by [7,8].Number of tassel branches were 8-10 which is on par with Paiyur 2 and BL 6 and 10-12 range of number of tassel branches was recorded in CO (Samai) 4 and TNPsu 176. 32-34cm, 1.   (Figure 1). Length of the fruit and Width of the fruit showed a range from 4.2-4.5mm and 3.5-3.7mm for ATL 1 and it is on par with the other varieties. In case of 7 qualitative traits studied the traits, Compactness of inflorescence (Intermediate) (Figure 2), Colour of the apicules (Figure 3) (Yellow), Ligule pubescence (Strongly pubescent), Growth habit (Decumbent), Culm branches (Absent) and Inflorescence shape (Arched) was found distinct for ATL 1 and was different for the other varieties. Similar results in accordance with [9]. In addition characterization of grain quality such as nutritional quality, cooking quality and sensory evaluation sore was conducted among the three varieties viz., ATL 1, CO (Samai) 4 and Paiyur 2 which recorded that the newly released variety ATL 1 has higher characteristics for nutritional quality, cooking quality and sensory evaluation score among the other two varieties (Figure 4). [9]. DUS characterization is the practice done to record the distinct morphological characteristics existed in newly identified varieties and on similar varieties what are called 'common knowledge'. But the availability of DUS characters is limited, hence the certification agency use combination of key distinguishable characters like   plant height, panicle characters at the time of flowering, seed characters at the time of harvesting. Though the little millets had narrow diversity it is difficult to identify a trait specificity exists between the varieties. Hence the molecular markers are employed to fingerprint the DNA along with the DUS characters which could be usefull in varietal identification and also in the seed certification process (Science and Advice for Scottish Agriculture).

Screening of Little Millet Variety using SSR Markers
Amplification of little millet genotypes was performed using 16 barnyard millet, 25 maize and pearl millet genomic SSR markers listed in the (Table 1). Barnyard millet EST-SSR primers showed multiple allelic patterns for the range of annealing temperature 55 o C to 60 o C. Among 25 maize SSR markers, two markers viz., phi213984 and phi295450 showed polymorphism (Figure 4) between the little millet variety under study. The marker phi213984 showed unique 310bp amplicon in ATL 1 whereas, other varieties viz., BL6, TNPsu 176, Co (Samai) 4 and paiyur 2 showed 300bp amplicon size. 600 bp amplicon were uniquely found in ATL 1 and TNPsu 176 which is distinguishable from the other varieties. The maize SSR marker umc2021 and phi087 showed monomorphic with the amplicon size of 140bp and 150bp respectively. Amidst 25 Pearl millet SSR markers, three markers viz., PMSP2216, PMSP2203, PMSP2223 showed monomorphic by the amplicon size of 150bp, 300bp, 190bp respectively. A unique amplicon size with 305bp was recorded in ATL 1 and BL 6 which is distinguishable from the other three varieties under study ( Figure 5). These are in accordance with [10,11].
Utilization of three microsatellite markers in the analysis of varieties revealed a high level of genetic polymorphism which allowed unique banding pattern. Microsatellites are considered appropriate for variety identification because of their ability to detect large numbers of discrete alleles repeatedly, accurately and efficiently [12]. In this study, three SSR markers distinguished the ATL 1 from other ruling varieties. The set of SSR markers used here provides a positive assessment of the ability to produce unique DNA profiles of little millet germplasm. Maize and little millet might have originated from a common ancestor in Poaceae species [13], and hence the variation was introduced in the structural and regulatory sequences after species during speciation. Previously Ali et al, 2017 demonstrated the validation of switchgrass EST-SSR markers in assessing the relationship of the little millet genomic. Thus, this study could be utilized for identification of the diverse germplasms and also for fingerprinting in the future.

CONCLUSION
The morphological DUS descriptors can be effectively used for identification, documentation and grouping of varieties for registration of varieties under the PPVFRA. The morphological descriptors sometimes seem difficult to differentiate the closely related varieties and hence the DNA fingerprint data is given along with plant variety notification proposal which would provide great help to the plant breeders for registration of germplasm in NBPGR. Although DUS testing has been done precisely to provide diversity assessment at morphological level there remains a further scope to validate the findings through DNA markers.