Molecular Profiling in Relation to Grain Zinc Accumulation in Rice

Abstract

A study was carried out using a set of four candidate gene markers for grain zinc accumulation and genetic polymorphism was examined in twelve genotypes of rice. The objective was to examine the nature of genetic variation at molecular level for characterization of these genotype. Putative candidate gene based primer directed amplification was successfully achieved by all the primer pairs. Polymorphism was recognized among the genotype in the form of absence of bands. Analysis of polymorphism pattern based on amplification profiles clearly indicated that nucleotide sequence variations at the primer binding sites within the putative candidate genes was the most probable casual factor for differential grain zinc accumulation rice genotypes. Candidate gene based amplification assay dependent detection of non-alleles in some genotypes basically provided the basis to relate nucleotide variation at primer binding sites with differential grain zinc accumulation in rice genotype.

Introduction

Rice is a cereal grain from the grass species (Oryza sativa L.) of Poaceae family. It provides 20 percent per capita energy and its protein contribution is 13 percent. It is staple food for one third of the population of the world with a per capita consumption of 62 to 190 kg/year .It is the main source of income for small scale farmers. It is cultivated over 150 hectare of land mainly by flooding method to avoid weed and pest infection.

As per the data analysis done in 2014,China is the largest producer of rice all over the world with a production of 206.5 million tons while India ranks as second largest rice producing country with a production 157.2 million tones and when we talk about Bihar ,it contributes about 6356.7 MT. Developing countries have 95 percent of rice production and the whole rice production over the world is 741.5 million tones which is the source of living of more than half of the population .While if we analyze data of 2016 then world contributes 446.0 million tons of production and India at the same point contributes about 104 of production and India at the same point contributes about 104.32 MT.

Rice is having a high mineral and nutrients content which is important for plant growth and development .If we come to zinc, rice bran contains 50.3 ppm of Zinc concentration as compared to 8.6 ppm in endosperm .Zinc act as a cofactor of more than 300 enzyme .It also plays an important role in transpiration ,DNA replication ,protein synthesis, etc. It helps plant to produce chlorophyll .Genes containing Zn concentration in rice are OsNAS3, OsNAS1, OsNAAT1, OsYSL, MTP1, etc. Its deficiency causes many disorders in plant like chlorosis that is discoloration of leaves and in rice it causes dusty brown spots on upper leaves of stunted plants, increased spikelet sterility in rice ,leaf blade size is reduced and white lines appears sometimes along the leaf midrib while it causes diseases like pneumonia, jaundice, impairment of brain etc. About 71 percent of population is suffering from Zn deficiency that is about 2 billion people around world are having this problem because of decreasing concentration of zinc in rice crops.

Some actions are taken to deal with the Zn deficiency like breeding crops producing high yield and accumulate minerals, selection of germplasm with greater quantity of Zn etc. But the most effective remedy is biofortification in which bioavailable nutrients content is increased within the plant.

The present study was conducted to examine the polymorphism pattern and nature of differentiation and divergence in relation to differential grain zinc accumulation among rice varieties using candidate genes based primers.

Review of the Literature

Murray et al. (1980) presented a method for rapid isolation of high molecular weight plant DNA (50,000 base pairs or more in length) which was free of contaminants which interfere with the complete digestion by restriction endonucleas.

Meyerhans et al. (1990) had done PCR co-amplification of two distinct HIV1 tat gene sequence that had led to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 54% of all amplified molecules, can be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time.

Chakravarti et al. (2006) stated that all the primers showed distinct polymorphism among the cultivars studied indicating the robust nature of microsatellites in revealing polymorphism.

Padmalatha et al. (2006) presented the optimization of DNA isolation and PCR conditions for RAPD analysis of selected medicinal and aromatic plant. The yeid of DNA ranged from 1-2 ug/ul per gram of the leaf tissue and the purity was between 1.6-1.7 indicating minimal level of contaminating metabolites.

Narayanan et al. (2007) analyzed the expression of the metal related genes in flag and non-flag leaves of four different rice cultivar during the period of the mid grain fill. Genes (24 of 36) exhibited low non-detectable signals the microarray, while 12 genes were found to be highly expressed in both flag and non-flag leaves of all the 4 cultivars.

Carrigg cora et al. (2007) by doing analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community generated.

Zhang et al. (2008) stated that in the pot trails, the optimum application of N alone on the rice crop could increase the concentration of Fe in the polished rice.

Garcia-Oliveria et al. (2009) detected a total of 31 putative quantitative trait loci (QTLs) for Fe, Zn, Mn, Cu, Ca, Mg, P and K by single point analysis. Wild rice (O.ruflpogon) contributed favorable alleles for most of the QTLs (26 QTLs), and chromosomes 1,9 and 12 exhibited 14 QTLs (45%) for these traits while the largest amount of phenotypic variation (11% - 19%) was observed near the SSR marker RM152 on chromosome 8.

Arnold et al. (2010) showed with a mathematical model that, for realistic rates of secretion of the phytosiderophore deoxymugineic acid (DMA) by rice, and effect of DMA in soil, solubilization and uptake by the proper mechanism is necessary and sufficient to account for the measured Zn uptake and the difference between genotypes.

Usharani et al. (2012) identified three polymorphic markers SC 120, SC 128 and SC 129 which were unlinked and hence single marker analysis was done to check the association of the marker with the trait. SC129 showed highest significant variation with both iron and zinc at the tune of R2=13.09% and R2=19.51%, respectively.

Sperotio et al. (2013) stated that flag leaves play a major role in synthesis and translocation of photoassimilates to the rice seeds, affecting grain yield.

Impa et al. (2013) stated that a protocol was developed for growing in ager nutrient solution (ANS), with optimum Zn- sufficient growth achieved at 1.5 uH ZnSo4.7H2O. The redox potential in ANS showed a decrease from +350mV to -200mV, mimicking the reduced condition of flooded paddy soils.

Brar et al. (2014) studied the genetic diversity among fourteen genotypes, using 50 microsatellite markers uniformly distributed across the rice genome. A total of 257 alleles were detected that ranged from 1 to 9 with average of 5.14 alleles per locus. The overall size of amplified PCR products ranged from 73 to 585bp.The Polymorphic Information Content (PIC) value ranged from 0 to 0.862 with an n average of 0.66.

Wang et al. (2014) stated that as compared to CF, the AWD regime significantly increased grain yield and Zn concentration in both brown rice and polished rice. Grain yield of genotype (Nipponbare and Jiaxing27), on the average, was increased by 11.4% and the grain Zinc concentration by 3.9% when compared to that of the CF regimes.

Wang et al. (2014) observed that entophytic strains successfully colonized rice roots after 72 h. Improved root morphology and plant growth of rice was observed after inoculation with strains especially SaMR 12 and SaCS20.

Suma et al. (2015) stated that out of 50 SSR primers and CGs pairs studied, 47 showed polymorphism on screening in 12 rice genotypes. The trend of diversity was estimated based on the number of allele, Nei’s genetic diversity index and polymorphism information content (PIC).

Mia et al. (2015) observed that three markers (Rm23, RM35, and RM217) showed effective polymorphism in DNA band appearance for Zn content out of 10 SSR markers.

Johnson- Beebout et al. (2016) stated that Zn uptake behavior of a rice genotype determines the fate of Zn from soil to grain. This has implications on overcoming Zn translocation barriers between vegetative parts and grains, and achieving grain zinc biofortification target (30.0 ug/).

Mori et al. (2016) observed that tolerant genotypes produced more crown roots per plant and had greater uptake rates per unit root surface area; the latter was at least as important as root number to overall tolerance.

Affholder et al. (2017) stated that differences in venting of soil Co2 through root aerenchyma were responsible for the genotype and planting density effects.