Evaluation of agro-morphological traits, seed characterization and genetic diversity of local rice (Oryza sativa L.) varieties of Pakistan

Broad base genetic diversity is essential for a sustainable crop yield to provide tolerance against biotic and abiotic stresses. In Pakistan, rice is ranked second as a staple food and cultivated under different climatic zones. However, very little is known in terms of rice genetic diversity. The present research was performed to evaluate the genetic diversity among 12 local rice varieties by using 5 Simple sequence repeats (SSR) markers. Furthermore, agro-morphological parameters and seed quality of these varieties were studied. The maximum plant height (204.3 cm) and panicle length (40.1 cm) were observed for Hans-raj. The highest numbers of primary and secondary panicle branches were observed in Ratua-81 (51) and super basmati (62), respectively. Super basmati showed highest number of filled grains (264.3) and total number of grains (315) per panicle. The minimum days to maturity (140) was recorded for ratua-81 and highest 1000 grains weight (27 g) was recorded for Ksk-133. The highest concentration of elements, e.g. zinc (Zn) was observed in Ks-282 (44 µg/g), Iron (Fe) in hansraj (190.3 µg/g), manganese in bamla sufaid (111.3 µg/g), copper (Cu) in hansraj (856.3 µg/g), lead (Pb) in super basmati (3883.3 µg/g), and nickel (Ni) was found in basmati-385 (314.6 ug/g). For the genetic diversity analysis, five SSR markers were used and a total of 60 alleles were amplified with 20% polymorphism where RM28130 showed the highest Polymorphic information content (PIC) value (0.60). The maximum number of alleles (3) were produced by RM28089. Based on secondary panicle branches and total number of filled grains, Super basmati showed best panicle architecture and grain yield, which can be used in breeding program to develop high yielding aromatic rice genotypes. The highest number of filled grains and total number of grains per panicle were observed in super basmati, which indicated the potential adaptation. Basmati-2000, Hansraj, Ks-282, Super basmati and Basmati surkh-161 were found to be highly rich in micronutrients (iron, zinc, copper, lead and manganese). The maximum genetic distance was observed in basmati-2000 and basmati surkh 161 genotypes.

g) was recorded for Ksk-133.The highest concentration of elements, e.g. zinc (Zn) was observed in Ks-282 (44 µg/g), Iron (Fe) in hansraj (190.3 µg/g), manganese in bamla sufaid (111.3 µg/g), copper (Cu) in hansraj (856.3 µg/g), lead (Pb) in super basmati (3883.3 µg/g), and nickel (Ni) was found in basmati-385 (314.6 ug/g). For the genetic diversity analysis, five SSR markers were used and a total of 60 alleles were amplified with 20% polymorphism where RM28130 showed the highest Polymorphic information content (PIC) value (0.60). The maximum number of alleles (3) were produced by RM28089. Based on secondary panicle branches and total number of filled grains, Super basmati showed best panicle architecture and grain yield, which can be used in breeding program to develop high yielding aromatic rice genotypes. The highest number of filled grains and total number of grains per panicle were observed in super basmati, which indicated the potential adaptation. Basmati-2000, Hansraj, Ks-282, Super basmati and Basmati surkh-161 were found to be highly rich in micronutrients (iron, zinc, copper, lead and manganese). The maximum genetic distance was observed in basmati-2000 and basmati surkh 161 genotypes.
Keywords Genetic diversity · SSR markers · Agromorphological traits · Oryza sativa Abstract Broad base genetic diversity is essential for a sustainable crop yield to provide tolerance against biotic and abiotic stresses. In Pakistan, rice is ranked second as a staple food and cultivated under different climatic zones. However, very little is known in terms of rice genetic diversity. The present research was performed to evaluate the genetic diversity among 12 local rice varieties by using 5 Simple sequence repeats (SSR) markers. Furthermore, agro-morphological parameters and seed quality of these varieties were studied. The maximum plant height (204.3 cm) and panicle length (40.1 cm) were observed for Hans-raj. The highest numbers of primary and secondary panicle branches were observed in  and super basmati (62), respectively. Super basmati showed highest number of filled grains (264.3) and total number of grains (315) per panicle. The minimum days to maturity (140) was recorded for ratua-81 and highest 1000 grains weight (27

Introduction
Rice (Oryza sativa L.) is classified in the family Graminae and sub family Oryzoidea, which is an essential cereal crop with nutritional and agronomic values. Rice is the staple food and major source of daily calories for more than half of the world's population (Saski and Burr 2000). Rice genome is the smallest and well characterized compared to other major cereal crops with 400 to 430 Mb size. Other cereal crops like sorghum has 750 to 770 Mb genome sizes, while wheat genome is about 37 times larger than the rice at around 16,000 Mb (Arumuganathan and Earle 1991). In rice there is a vast range of variation in their genotypes among and between landraces (Singh et al. 2018). Rice is cultivated worldwide, that includes Asia, North and South America, throughout Europe, Middle East and Africa (Aljumaili et al. 2018). Twenty-two species of rice have been introduced so far, but among them the most cultivated are: Oryza sativa or Asian rice and Oryza glaberrima or African rice (Chang 1976).
The rice crop is cultivated worldwide under hot and humid climatic condition . Almost 11% of the global arable land is planted annually to the rice crop and it ranked second after wheat. It is cultivated on 163 million hectares, produces approximately 491 million tons of rice. Asia comes on the first in cultivation and usage of rice in the world that contains more than quarter population of the world. Approximately 90% of the rice is produced in Asia around the world (Nachimuthu et al. 2015). An essential metallothionein like protein formed by the Basmati rice that are rich in sulfur. At the international level basmati rice demand is also high because of the good quality, different and pleasing aroma, its fluffy texture and high-volume enlargement when cooked (Salgotra et al. 2015). In Pakistan basmati or aromatic rice are considered of high quality and an essential crop to export. About two million ha of the land covers by the rice in Pakistan which is almost 10% of the cultivated land (Ranjan et al. 2022). During the last 25 years the rice production become doubled because of the technological advances like introducing genetically improved high yielding varieties and appropriate crop management practices. Furthermore, to study grass genetics, rice is counted as an example due to its diploid and small genome size, genetic polymorphism, large collection of genetically diverse and conserved genetic material of about 100,000 accessions and availability of compatible wild species (Naaz et al. 2022;Mccouch 1998;Wang et al. 1992).
The Asiatic rice, Oryza sativa is domesticated from O. rufipogon Griff. the common wild ancestor. To understand domestication and genetic diversity in crops, rice as a model species, holds a unique position due to the sequenced genomes of Indica and Japonica (Chang 1976;Ishikawa et al. 2020;Cheng et al. 2003;Yamanaka et al. 2004). The aromatic rice also called basmati rice, is long grain rice that is used for the improvement of novel varieties of rice as it has more diversity and great potential. Pakistan counts in the world's countries for the export of aromatic rice (Ashfaq and Khan 2012). The good quality of basmati rice is due to its aroma, nice texture and flavor. Like Pakistan, Nepal, India and Iran also grow aromatic rice, which possess high value (Islam et al. 2018).
Micronutrients are not only crucial for plants growth but also a basic need for both the human and animal health. Iron helps in the cellular process that includes photosynthetic electron transport, respiration and chlorophyll biosynthesis. Zinc is also an important type of element that is involved in cellular processes as well, for the metabolism of lipids, proteins and carbohydrates. The deficiency of zinc in soil badly affects the nutritional quality of the grains and the yield of crop. Manganese act as a cofactor to activate the enzymes with functional (Zulfiqar et al. 2020;Pooniya and Shivay 2013). Copper is among the trace metals and is one of the redox active elements that is needed for the plant growth. But in soil its high concentration is harmful for the growth of plants like it inhibits the growth and producing the reactive oxygen radicals that cause oxidative damage (Cui et al. 2019). Among the heavy metals nickel is also an important microelement for humans which acts as a cofactor for some of the enzyme's proteins that contains metal ions and in humans it helps in the iron metabolism (Cao et al. 2017).
During the last 20 years, vast progress has been observed in the molecular mechanisms implicit in the significant agronomic features. To achieve high yield for a crop, flowering time is a captious trait through the use of maximum sunlight and temperature. Others agronomical traits that contribute to crop production are plant height, tiller number and panicle architecture. Panicle number is determined by the growth of tiller that has a direct effect on the yield of crop. Tiller quality make up the foundation of lodging tolerance and significance of cereal plant fiber as animal food (Wang and Li 2019). Multiple genes control the most agronomic traits in rice which show complicated and numerical inheritance. Molecular breeding is beneficial to recognize the QTLs associated with the desired traits, as QTLs controlling agronomic traits are labeled by DNA markers and introduced into the elite cultivars by Marker Assisted Selection ((MAS). The benefits of SSR markers are accessible and potent in breeding new rice cultivars to improve important agronomic traits, like plant height, maturity, seed shattering, amylose content, yield and resistance to disease and environmental stresses (Xuan et al. 2019).
Genes that are linked to drought stress in rice have been identified through the characterization and screening of rice genotypes genetically and morphologically, to determine the genetic variation in them. Through molecular marker technology quantitative traits are now possible to be identified (Sahebi et al. 2018). Molecular markers help to identify polymorphism in rice genomes and are very useful to study the diversity at genetic level (Salam et al. 2017). In order to identify the enhanced genotypes for the quantitative and qualitative traits the study of Agro-morphological diversity is very important for the germplasm of local and exotic varieties. It is very helpful to determine the best genotypes that can be promoted further for the yield purposes (Xuan et al. 2019).
Presently in plant genetics and breeding studies, microsatellites or SSR are widely used and chosen markers after Single Nucleotide Polymorphism (SNPs), due to their large genome coverage, comparative abundance, high reproducibility, co-dominant inheritance, able to differentiate both inter-and within-species variations, and simple to analyze them (Li et al. 2004;Liu et al. 2013). There are several applications for which the SSRs markers are being used that includes; (i) genome mapping, (ii) to assess genetic diversity and linkage between different cultivars in aromatic and non-aromatic rice, (iii) for the estimation and testing of purity in varieties, (iv) to determine genetic relatedness among several sub species (Ashfaq et al. 2022). For rice, SSR markers for any genome part or region are easily available and the candidate gene markers can easily be developed. The molecular markers are very important to the rice cultivars for the purpose of improvement and possess so many applications in the field of agriculture (Ramchander et al. 2022). The objectives of this study are to examine the genetic diversity through SSR markers, seed characterization and agro-morphological study amongst twelve rice genotypes.

Plant material and experimental design
The current research was carried out at the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar, Pakistan. The research was performed on local rice genotypes including ten basmati and two non-basmati varieties ( Table 1). The selected varieties were evaluated for agro-morphological traits and genetic diversity. The rice varieties included ten aromatic and two nonaromatic rice genotypes under the Randomized Complete Block Design. The rice seeds were collected from Plant Genetic Resources Institute (PGRI), National Agriculture Research Center (NARC), Islamabad, Pakistan. Seeds were grown in pots in screen house at IBGE under normal growth condition. As soon the nursery was established, uniform seedlings were transferred to the field.

Agro-morphological traits
The data were recorded on plant height (cm), number of panicles plant −1 , main panicle length (cm), primary and secondary branches panicle −1 , filled grains panicle −1 , total number of seeds panicle −1 , days to maturity, seed morphology and quality, 1000 grains weight (gm) of rice genotypes.
Determination of seed quality The heavy metal contents in samples of rice grains were measured after acid digestion (wet digestion) by following the protocol (Benton Jones 1991). Dried seedst sample (0.5 g) was taken in 250 ml calibrated Pyrex flask with 10 ml of HNO3 solution and kept overnight. Per chloric acid (4 ml) solution was added and the mixture was heated on hot plate under fume hood until the digest became cleared. After heating, distilled water was added to make the volume up to 100 ml. Heavy metal and micronutrients concentration in seeds extract was measured by atomic absorption spectrophotometer.

DNA extraction and PCR amplification
Genomic DNA extraction from Rice cultivar was carried out by cetyltrimethylammmonium bromide (CTAB) method described by Healey et al. (2014). A fresh young leaves of 1 g was taken and ground to powder form in liquid nitrogen. Sample was transferred to 1.5 ml Eppendorf tube and 500 μl of CTAB buffer was added. Sample was incubated in water bath at 60 °C for 30 min and was vortexed after every 5 min. Tubes were centrifuged at 12,000 rpm for 15 min and supernatant was transferred to a new 1.5 ml Eppendorf tube and 500 μl of Phenol:Chloroform: Isoamylalcohol (25:24:1) solution was added. After centrifugation at 12,000 rpm for 10 min, the upper aqueous phase was transferred to a new tube. After that 600 μl ice cold isopropanol was added for the purpose of precipitation. Sample was centrifuged again at 12,000 rpm for 2 min and supernatant was discarded without disturbing the pellet. The pellet was washed twice with 500 μl of 70% ethanol. Tubes were dried at room temperature. Sample was resuspended in 50 μl TE buffer. The extracted DNA quality and optical density were measured at a wavelength of 260/280 nm by using Nanodrop and was stored at −20 °C for future use.

Molecular diversity screening
To study the genetic diversity a total set of five SSR markers were selected from Gramene Markers Database (https:// archi ve. grame ne. org/ marke rs/) and were used to determine the genetic diversity among the selected rice genotypes as shown in (Table 1).

Polymerase chain reaction (PCR)
The PCR reaction was performed as described (Prasad et al. 2000). A toral of 10 μl reaction mixture containing 1 μl DNA template, 1 μl of each forward and reverse primer, 1.8 μl of nuclease free water, 0.2 μl of Taq DNA polymerase and 5 μl of PCR master mix. PCR conditions was set with an initial denaturation step of 5 min at 94 °C followed by 35 cycles, each comprises of a denaturation at 94 °C for 30 s, annealing at an appropriate temperature for 30 s and elongation step of 1 min/ kb for 72 °C, and final extension at 72 °C for 10 min. The BIO-RAD T100 thermal cycler was used (Table 2).

Gel electrophoresis
The amplified PCR products were confirmed using 2% Agarose gel. The agarose gel was prepared in 1X TBE buffer that was mixed and heated for 2 min in microwave oven. The gel was solidified by cooling for 20 min and then transferred to gel tank. The product size of different genotypes was determined by comparing with 100 bp DNA ladder. The gel was run for 45 to 50 min at 110 V. The PCR products were visualized by using 2ul of Ethidium Bromide and checked under UV light Trans illuminator and documented.

Data analysis
Molecular and morphological data was compiled in MS Excel and Popgene 3.5 version was used to find out Allelic Frequency, PIC, Heterozygosity and Homozygosity and Genetic diversity. The Unweight Pair Group Method with Arithmetic Means (UPGMA) procedure was used to construct dendrogram using the Popgene software.

Evaluation of rice cultivars for agro-morphological traits
Morphological traits of twelve Pakistani rice cultivars were evaluated and data was taken after harvesting time. The highest plant height (204.3 cm) and panicle length (40.1 cm) were observed for Hans-raj, whereas the maximum number of panicles was noted for dilrosh-97(47.3). The maximum number of primary and secondary panicle branches were observed in Ratua-81 (15) and super basmati (62), respectively. Super basmati showed highest number of filled grains (264.3) and total number of grains per panicle (315). The minimum days to maturity was exhibit by ratua-81 (140 days), and highest 1000 grains weight was recorded for Ksk-133 (27 g) which exhibited highly significant variation for agro-morphological parameters among the rice varieties (Table 3 and Fig. 1).

Grains characterization of rice genotypes
The seed quality analysis including, minerals content of the rice seeds, including iron, zinc and manganese and heavy metals, including lead, copper and nickel were evaluated (Fig. 2).

Genetic diversity based on SSR markers
Genetic diversity among twelve rice cultivars were detected utilizing 5 SSR markers (Table 2) with a toral of 60 allelic band with highly polymorphism were recorded. RM28099 primer exhibited 12 allelic bands with two patterns of polymorphism. The allelic frequency observed for allele A was 0.7500 and for allele B, it was 0.2500. PIC value for RM28099 was recorded as 0.29 (Fig. 3). RM28089 produced a total 12 allelic bands were amplified, which comprising 3 band patterns (Fig. 3). The allelic frequencies recorded for allele A, B and C was 0.2917, 0.5417 and 0.1667, respectively. PIC value for RM28099 was recorded to be 0.34. RM315 produced 12 allelic bands and 2 band patterns. The allelic frequency for allele A was recorded as 0.7273, while for allele B, it was 0.2727. PIC value for RM315 was recorded as 0.27 (Fig. 3). RM28130 primers showed overall 12 allelic bands and 2 band patterns were observed. The allelic frequency recorded for allele A, among 12 rice varieties were 0.6667 and for allele B, showed 0.3333. PIC value for RM28130 was recorded as 0.60 (Fig. 3). Marker RM211 amplified 12 allelic bands with 2 band patterns. The allelic frequency recorded for allele A and B was 0.7500 and 0.2500,  3  3  20  RM315  2  2  20  RM28130  2  2  20  RM211  2  2  20  Total  11  11  20 respectively. PIC value for RM211 was recorded as 0.29 (Table 6).
Overall allelic frequency In current study, RM28099 exhibited two alleles A, B was observed with highest frequency of 0.7500 and lowest frequency of 0.2500, respectively. For RM28089, three alleles were observed with highest frequency for allele B (0.5417), followed by allele A (0.2917) and the lowest frequency was observed for allele C (0.1667). For RM315 the highest frequency was noted for allele A (0.7273) and lowest frequency was recorded for allele B (0.2727). For RM28130, the  highest frequency was observed for allele A (0.6667) and lowest frequency was noted for allele B (0.3333). For RM211 the highest allelic frequency was recorded for allele A (0.7500) and lowest allelic frequency was observed for allele B (0.2500) (Tables 5 and 6).

Genetic relationship of the rice germplasm
Dendrogram constructed based on similarity coefficient for the estimation of genetic relationship using UPGMA cluster analysis, which are based on Nei's Genetic distance Modified from NEIGHBOR procedure of PHYLIP Version 3.5 Pop-Gene software. All the genotypes were divided into two main groups A and B. Group A was further divided into sub groups A1 and A2. Group A1 included the varieties dilrosh-97 and sara sella. Group A2 divided into further subgroups A2a, A2b and A2c. The A2a group included ksk-133, A2b included ratua-81 and super basmati varieties. While the A2c contained basmati-385, bamla sufaid-121and basmati  KS-282, and BASMATI-2000, and no genetic distance (0.000%) was observed between the varieties Basmati-385, Bamla sufaid, and basmati surkh-161 as they belonged to the same origin of genotype ( Fig. 4 and Tables 7-8).

Discussion
Pakistan is the 11th largest producer and 4th largest exporter of rice, which is cultivated under different climatic zones; however, very little is known in terms of rice genetic diversity in Pakistan with respect to global germplasm (Shahzadi et al. 2018). A broad Fig. 4 Dendrogram constructed for 12 rice genotypes by Popgene version 3.5 based on genetic similarities using set of five SSR markers base genetic diversity having a wide range of genetic variations in genome is crucial to provide tolerance against biotic and abiotic stresses. Germplasm serves as the main source of genetic variation that could be utilized by the breeders in the future for the enhancement of crops through breeding programs or hybridization. For the improvement and development of any variety the main source for crop breeding programs is the germplasm due to the conserved characters needed for any good crop yield production, tolerance against environmental stresses and for enhanced crop quality. To monitor and evaluate the diversity and some essential genes among the crops, the assessment of germplasms collections is very indispensable (Zulfiqar et al. 2022). Germplasms plays a vital role for the development of novel varieties and to secure our food. The developed varieties contain a distinct genic trait responsible for combating against all the diseases, insects and have improved nutritional value. Rice germplasm including landraces, traditional varieties, breeding lines and elite genotypes. Worldwide, around 4,500,000 accessions of plants have been collected and specifically in rice about 400,000 accessions are reported (Fao 1998;Gaballah et al. 2022).
For the estimation of rice germplasm, IRRI has presented more than 1000 genotypes yearly (Joshi BK 2017). In the present the qualitative and quantitate agro-phenotypic characteristics has been showed highly phenotypic variation included plant height, number of panicles per plant, main panicle length, primary and secondary branches per panicle, filled grains per panicle, total number of seeds per panicle, days to maturity, seed quality and 1000 grains weight. Super basmati showed maximum number of secondary branches per panicle (62), maximum number of filled grains (264.3] and total grains per panicle (315). The crucial and main agronomic character to achieve the improved rice production is the grain yield under favorable and unfavorable environments (Gaballah et al. 2022). A previous study displayed similar results with high significant variation in all the 14 traits of rice germplasms (Gaballah et al. 2022;Karimah et al. 2021).
Researchers investigated the copper concentration in rice genotypes and observed high content in rice root (Cui et al. 2019). In the current study the quality analysis of rice grains of twelve rice varieties ware analyzed, total six micronutrients included iron, zinc, manganese, copper, lead and nickel. The highest content of iron was found in seeds of Hansraj, the highest Zn concentration was found in Ks-282, highest manganese and lead were found in super basmati, highest concentration of copper was recorded in Hansraj, and highest content of nickle was recorded in basmati −385. Highly significant variation was recorded for elements in all the genotypes of the rice. The results of this study are consisted with previous work (Cao et al. 2017) where they worked on nickel metal concentration in milled rice grain and found the significant variation. Moreover, our results are also in line with the study of (Sakina et al. 2022), who analyzed eleven aromatic and one non aromatic rice genotypes for micronutrients (Zn, Mn, Fe, Cu). (Cao et al. 2017) investigated lead content in rice grains. It is prominent to explore the genetic variation within a rice population, for the management and utilization of rice germplasm. That could be further used for the improvement and development of new rice varieties (Ming et al. 2015). Traditionally the use of morphological or physiological traits were used to evaluate the genetic diversity in plants but this technique was not trustworthy as this procedure was dependent upon the environment due to the gene expression influenced by it. The scientists approached an alternative way to figure out this problem and they introduced the use of molecular markers. Molecular markers can be used to assess the genetic diversity among the varieties of rice, as well as the quantitative and inherited traits can also be analyzed through it (Beena et al. 2021). In the present study total of five SSR primers were used in order to identify the genetic variation among the 12 local rice genotypes. The highest number of polymorphic alleles (three alleles) was observed for RM28089. While the remaining markers showed less number of alleles i.e. 2 polymorphic alleles each. These markers resulted in 20% polymorphism among all the rice genotypes. (Gao and Innan 2008) identified the genetic variation and characterized 15 varieties using 30 different SSR primers that revealed distinct polymorphism between the studied varieties. The number of band pattern in the current study corresponded well with the study (Karimah et al. 2021). The PIC values varied from 0.27 to 0.60 with average value of 0.358. The highest PIC value (0.60) was observed for RM28130. In a previous study (Ramchander et al. 2022) has shown similar results with PIC values ranged from 0.0476 to 0.5993 with average of 0.3785 using 24 SSR markers. PIC value shows the variation, diversity and frequency among the genotypes of the varieties or cultivars (Ramchander et al. 2022). The 12 rice genotypes were divided in to mainly two groups, i.e. A and B based on UPGMA cluster analysis. The highest genetic distance 41.4% was recorded among the varieties of basmati-2000 and basmti surkh-161 having same geographical origin.

Conclusion
The current study observed highly significant difference for the agro-morphological traits including plant height, number of panicles per plant, main panicle length, primary and secondary panicle branches, filled grains per panicle, total number of seeds per panicle, days to maturity, and 1000 grains weight. The highest number of filled grains and total number of grains per panicle was noted in super basmati, which indicated the potential adaptation of this variety to the prevailing high temperature during the growth period. Basmati Hansraj, Ks-282, Super basmati and Basmati surkh-161 were found to be highly rich in micronutrients (iron, zinc, copper, lead and manganese). Based on the molecular markers assessment, the 12 local varieties cluster into two groups. The maximum genetic distance was observed in the varieties of basmati-2000 and basmati surkh 161. RM28310 was highly informative having maximum PIC value, whereas highest polymorphic alleles (three) were observed for RM28089. Based on current research study it is recommended that broad genetic background especially highly yield contributing components, super basmati is recommended to be included in breeding program. Rice varieties may further be characterized for nutrients uptake ability and its underlying mechanism.
Funding This research was supported by Higher Education Comission of Pakistan under startup research grant program (Project No. 1932).

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or fifinancial relationships that could be construed as a potential conflflict of interest.
Ethical approval This article does not contains any study with human participants or animals performed by any of the authors.