Molecular Profiling of Pakistani Selected Advance Lines of Rice for Amylose Content

Full Length Research Article

Molecular Profiling of Pakistani Selected Advance Lines of Rice for Amylose Content

Shah Faisal1, Shafee Ur Rehman2, Hassan Sher3, Khushi Muhammad1, Rahmat Ali1, Shahid Ali4, Zahid Hussain4, Wajid Khan4, Arshad Iqbal4, Murad Ali Rahat4*

Adv. life sci., vol. 9, no. 4, pp. 560-566, December 2022
*- Corresponding Author: Murad Ali Rahat (Email: rahatmurad32@yahoo.com)
Authors' Affiliations

 1. Department of Biotechnology and Genetic Engineering Hazara University, Mansehra- Pakistan
2. International Islamic University School and College Matta, Swat- Pakistan
3. Centre for Plant Sciences and Biodiversity, University of Swat, Swat- Pakistan
4. Centre for Biotechnology and Microbiology, University of Swat, Swat- Pakistan 
 
[Date Received: 11/09/2022; Date Revised: 27/11/2022; Date Published: 31/12/2022]

Abstractaa download_button
Introduction
Methods
Results
Discussion
References 

Abstract

Background: Pakistani rice is well-known for its quality. Its consumption increases with the increase in population. The gel consistency (GC) amylose content (AC) and gelatinization temperature (GT) are the most important rice characters, which are associated directly to eating and cooking attributes. But for its good taste and eating quality depends on its endosperm starch quality and quantity. Amylose, a chief determinant of rice attribute, is principally synthesized and controlled by a major gene (Waxy gene) encoding an enzyme called granule bound starch synthase (GBBS).

Methods: Current investigation was carried out to characterize advance lines of rice by both conventional and molecular approaches. In present study Waxy gene was identified in advance lines of rice.

Results: Show that out of 17 advanced lines, 9 lines were waxy or low amylose, and 1 line was non waxy or high amylose rice because of the presence of 425 bp fragment and 225 bp fragment of Wx gene respectively. For morphological data 14 morphological quantitative traits were studied.

Conclusion: Advance lines of rice analyzed during the present investigation showed better grain quality. A number of advance lines contain extra-long and medium slender grains which have intermediate to high gelatinization temperatures. Thus these advance lines are appropriate for the improvement of saline rice. Except one advance line 19 that showed Hard gel consistency and the majority of advance lines fall in the category of soft gel consistency and thus are of excellent quality.

Keywords: Rice; Amylose;  Gelatinization; Wx genes       

Introduction6th button-01

Rice is one of the most important staple crops that provide food for more than half of the world’s population [1]. As a major cereal crop, it is one of the most diversified crop species due to its adaptation to a wide range of geographical, ecological, and climatic regions [1]. It is a vital food of the enormous proportion of population of Pakistan, but it is economically major crop of this country. Rice stands next to cotton in exports and contributes major part of the total export’s earnings. More than 90% of rice is consumed and produced in Asia. The world‘s population is estimated to be over 9 billion by 2050, as large in universal raise food production will be needed [2]. (FAO, 2009). The increase in rice yield had beginning to slow by the start of the present century [1, 3]. Through genetic development through the green revolution in the previous half century rice production dramatically improved along with application of a greater quantity of chemical fertilizer [4], it is a regular Asian diet generally used as an entire grain after cooking and contribute of the total calorie intake for 40% to 80% [5, 6, 7, 8]. The priority of rice cooking and eating traits within a definite region and culture may not be accepted by other cultures. In general, the Japanese like better little grain, sticky rice that is normally used in creation sushi. On the other hand, Basmati rice is well-accepted in Pakistan, and India, the Middle East, because to its aroma and it‘s lengthens, dry grains when cooked [9].

Rice is a staple starchy food, which gives a large portion (~ 90% in Asia) of nutritional energy. Starch is the main component of rice grain, thus the starch- synthesizing capability of endosperm is mainly determined by the capacity of grain filling. Therefore Starch properties are a significant factor to determine the grain quality. Milled and Brown rice contains about 75-85% and 90% carbohydrates, respectively. The starch properties are mainly determined from Eating and cooking characteristics that creates up 90% of milled rice. The significant starch properties that influence eating and cooking properties are Gelatinization temperature, amylose content, and gel consistency. The most important forecaster is considered amylose of sensory attribute in rice [10]. Starch is consisting of two polysaccharides: amylose and amylopectin, which is primarily influenced by Rice quality. The amylose is linked glucose polymer by_ (1,4)– a relatively less branched while in amylopectin the branching enzyme generates 1,6 linkages. The percentage of amylose on, apparent Amylose Content (AAC ) as measured, is the important determinant of cooking rice properties. High amylose content variety like risotto varieties are becoming dry and separated of grains after cooking, whereas low amylose cultivars (cvs) after cooking are glossy and tender cohesive or waxy. Lesser amylose rice is preferable than (over 20%) higher amylose rice because when they cooked it does not become dry and hard. Recommended classification on the basis of amylose content milled rice is classified in to the following five classes such as high (25-33%) intermediate (20-25%) low (12-20%) very low (5- 12%) waxy ((0-5%), even considering that commercially amylose content by rice is classified as either low (below than 20% amylose), medium (21–25%) and high (26–33%) [11, 12].

Waxy gene is responsible for amylose synthesis in rice, which encodes granule- bound starch synthase I (OsGBSSI). While amylopectin is synthesized by the action of four classes of enzyme: starch synthase, branching enzyme, ADP-glucose pyrophosphorylase, and debranching enzyme [13, 14]. In non- waxy rice cultivars GBSS quantity depend on the amylose content accumulating during grain filling process, in the Wax locus two functional alleles are present, Wxa and Wxb [15]. Therefore, present study is proposed for evaluation of Pakistani selected rice advanced lines for physical grain quality, yield and yield attributing traits and identification of waxy gene.

Methods6th button-01

Plant materials

Plant materials were included of seventeen genotypes of rice varieties that have been developed at Hazara University, Mansehra, Pakistan. These following genotypes were growing at experimental field NTHRI, Shinkiari, Mansehra (Table 1 and Figure

Morphological characterization

Seventeen genotypes of rice were morphologically evaluated. All the genotypes were grown in the research fields of Hazara University, Mansehra. Environmental conditions were same for all the genotypes under investigation. Morphological traits studied in these genotypes of rice were categorized as quantitative traits. Total 14 morphological traits were investigated, such as number of tillers per plant, flag leaf length, grain length, grain chalkiness, plant height, flag leaf width, panicle length, number of branches per panicle, number of filled grains per panicle, unfilled grains, yield per plant, grain width, grain thickness and 100 grains weight.

Identification and extraction of genomic DNA for waxy gene

Extraction of Genomic DNA

Genomic DNA was extracted from new seeds of 17 genotypes of rice by using CTAB technique, with small alterations high quality of genomic DNA was obtained. With the help of spectrophotometer the concentration of DNA samples was maintain from 20 to 50ng/µl (Figure 2).

Furthermore, the waxy gene were further confirmed in the gnomic DNA of selected advance lines of rice by PCR using WxF and WxR primers. Moreover the amplified product was further confirmed by 1.5% Agarose gel electrophoresis.

Data analysis

The collected data from the selected lines of rice was further analysed by using Statistical software while the waxy gene presence was confirmed by PCR and Agarose gel electrophoresis.

Results6th button-01

Morphological characterization

Total 14 morphological traits were investigated, such as number of tillers per plant, flag leaf length, grain length, grain chalkiness, plant height, flag leaf width, panicle length, number of branches per panicle, number of filled grains per panicle, unfilled grains, yield per plant, grain width, grain thickness and 100 grains weight.

There is variation in plant height and the range recorded was 77.33 to 134.33 cm. Minimum height (77.33) was recorded for line 16, while maximum height (134.33) was recorded for line 65-66. There are variations in the number of productive tillers per plants the range was recorded from 4.66 to 25.33. Minimum tillers (4.66) were recorded for line 61, 62. While maximum tillers (25.33) were recorded for line12. Length of flag leaf ranged from 19.16 cm to 48.66 cm. The smallest value (19.16 cm) was recorded for line 21 and the maximum value (48.66 cm) was recorded for line 62. Width of flag leaf ranged from 1.10 cm to 2.36 cm. least value (1.10 cm) was recorded for line 11and extreme value (2.36 cm) were calculated for line 22. Ranged of Panicle length from 21.66 cm to line 29 and the maximum value (53.33 cm) was calculated for line 62. Branches per panicle were ranged from 9.33 to 18.66. Least value (9.33) was recorded for line 11 while the maximum value (18.66) was calculated for line 62 respectively (Table 2 and 3).

Filled grains per panicle ranged from 93.33 to 277. The lowest amount of filled grains for each panicle (93.33) was recorded for line 29 whereas the maximum quantity of filled grains of each panicle (277) was recorded for line 19. Unfilled grains per panicle ranged from 12.33 to 322.

The minimum value (12.33) was recorded for line 16 and the maximum value (322) was recorded in line 62. Yield per plant ranged from 22.33 to 69.33g. The minimum yield value was (22.33g) recorded in advance line 15 and the maximum yield value (69.33g) was calculated in line28 (Table 4 and 5).

100 grain weight ranged from 1.86 g to 3.30 g. Minimum value (1.86 g) was recorded for line 12 and maximum value (3.30 g) was recorded for line 61. Grain length ranged from 5.36 mm 8.20 mm. Minimum value (5.36 mm) was recorded for line 19 and 25 while the maximum value (8.20mm) was recorded for line 66. Grain width recorded from 1.50 mm to 2.30 mm. The smallest value (1.50 mm) was recorded for line 29 and the maximum value (2.30 mm) was calculated for line 61. Grain Length and breadth ratio ranged from 2.62 mm to 4.24 mm. the least value (2.62mm) was recorded for line 28 and the maximum value (4.24) was recorded for line 16. Grain chalkiness recorded as 26.03 mm to 66.97 mm. the minimum value (26.03 mm) was recorded for line 11 and the maximum value (66.97 mm) was recorded for line22 (Table 6 and 7).

Molecular analysis of waxy genes

Waxy gene expressed differentially in various rice cultivars as its role is to control amylose synthesis and amylose content variation in rice endosperm. In the present study waxy gene was identified in the advanced lines of rice. The amplified PCR products of 17 samples were 228 bp and 425 bp in length. Products of PCR were generated by using the verified primers WxF (forward) and WxR (reverse). The PCR reaction was executed in 10 μl volume (Figure 3). According to the PCR results, out of 17 advanced lines, only 10 advance lines showed results. 1 showed 425bp of Wx gene and 9 showed 228bp of Wx gene. It indicates that varieties with 425bp of Wx gene have high expression level for amylose content, while varieties with 228bp have low expression level for amylose content. Therefore varieties with high amylose content in their endosperms are known as non- waxy rice varieties and varieties with low amylose content in their endosperm are known as low amylose or waxy rice varieties. Conferring the above results, investigated advanced lines 60, 61, 62, 65, 66, 11, 12, 15, 16, are considered waxy rice varieties having Waxy gene and because of it contain low amylose in their endosperms and advance line19 showed high expression level of amylose content are considered non-waxy rice variety because of high amylose content in their endosperm (Table. 8).

Gelatinization temperature

Advanced lines of rice show the following gelatinization value of lines. 62, 65, 66, 11, 16, 19, 22, 28, 29, 31 showed high gelatinization temperature (GT).While line 60 and line 61- showed partial Intermediate gelatinization temperature while line12-and line 15 show high intermediate gelatinization temperature (GT) and line21- line25 show low gelatinization temperature (Table 9).

Gel consistency

Advance lines 60, 61, 62, 65, 66, 11, 12, 15, 16, 20, 21, 22, 25, 28, 29, 31 showed above than 60mm length of blue gel and thus have soft gel consistency (GC). Advance line line-19 show less than 40 mm of blue gel length and thus have hard gel consistency (GC) (Table 9).

 

Figures & Tables

   T7 T8 T9          

 

 

 

Discussion6th button-01

Pakistan has been traditionally an agricultural country. Rice is an important food crop of Pakistan. Pakistan rice holds second position in consumption after wheat. For better economy, export and consumption it is necessary to improve the grain quality and yield. For this aim it is necessary to improve plant both morphologically and genetically. Improving architecture of plant can support to protect the plant from lodging, salinity and any type of biotic and abiotic stresses. Investigation conducted on 17 advanced lines of rice designated differences in morphology and level of amylose due to the presence and absence of waxy gene. According to the morphological analysis, maximum and minimum values were recorded among these lines of rice. Maximum plant height 134.33 cm, number of productive tillers 25.33, yield per plant 69.33, and length of flag leaf 48.66 cm, width of flag leaf 2.36 cm and panicle length 35.33 was recorded in lines 65, 12, 28, 62, 22, and 62, respectively. Maximum numbers of primary branches 23.33 were recorded in lines 25 correspondingly. Maximum value for grain length 8.20 mm, and grain width 2.30 mm was recorded in lines 66, 61, respectively. Maximum filled grains 277, unfilled grains 322 and 100 grains weight 3.30g was recorded in lines 19, 62 and 61, respectively. Advance Line 62 and 66 have a good grain quality because of extra-long grain and maximum grain width. Variety 28 significantly exhibits high yield. The gel consistency (GC) amylose content (AC) and gelatinization temperature (GT) are main rice character, which are directly associated to cooking and eating quality . Advanced lines of rice analyzed during the present study showed better grain quality. The majority of the advance lines contained long slender grains and intermediate to high gelatinization temperatures. Thus these advance lines are appropriate for the development of high quality saline rice. Except one advance line that showed hard gel consistency and the majority of advance lines fall in the category of soft gel consistency and thus are of excellent quality. Gel consistency is a good directory of cooked rice texture, particularly among rice of high amylose content. Rice differs in gel consistency from soft to hard [7, 8]. Cooked rice with hard gel consistency solidifies earlier than those with a soft one. Rice with soft gel consistency cook rapidly or tender and stay soft even upon cooling [11]. Soft gel consistency rice is favored by most rice consumer. Breeder is therefore demanding to develop high yielding varieties with soft gel consistency [16]. Amylose content is the single core vigorous feature for rice eating and cooking quality. Classifying rice varieties amylose is considered as the significant and best indicator [11]. Rice varieties are categorized as high, intermediate, low and waxy rice varieties with amylose concentration >25%, 20 – 25%, 11 – 19%, 3 – 10%, and 0 – 2%, respectively [17]. Amylose is synthesized by an enzyme called the starch synthase (GBSS) which is the product of waxy gene. Variation in amylose content is because of high and low expression of Waxy gene. G to T point mutation in the first intron of the waxy gene alleles interrupts the formation of mature mRNA which causes low synthesis of amylose in the cultivated rice varieties [18, 19].

Using SDS – PAGE, Sano (1984) first studied the accumulation of rice waxy protein in rice seeds and illustrated its molecular weight i-e 60 kDa. Two alleles Wxa and Wxb of Wx locus were recognized by RFLP analysis (Sano et al., 1986). Information regarding genetic diversity in a crop plant is important for selection of parental lines and for construction of populations for various purposes. To evaluate genetic diversity of different crop species DNA markers were used [19]. In present study Waxy gene was recognized in advanced lines of rice. Variations in amylose content mainly depend on particular rice varieties and it is also proposed that oscillations in cold weather cause higher amylose content in the similar variety [20, 21]. Amylose variation was also observed in advanced lines because two different fragments of 425bp and 228 bp were appeared after a PCR reaction. Varieties with high amylose content in their endosperms are known as non-waxy rice varieties and varieties with low amylose content in their endosperm are known as low amylose or waxy rice varieties. Varieties having 425bp length fragments are non- waxy because of having negative results for Waxy gene and these includes line 19 while variety having 228 bp length fragments are waxy because of having positive results for waxy gene this include 60, 61, 62, 65, 66, 11, 12, 15 ,16, which showed low amylose content in their endosperm.

Pakistani rice is one of the best quality rice. For improved yield rice grain should be healthy and for good cooking and flavor quality an excellent amount of starch should be present in rice grain. High amylose content variety are becoming dry and separated of grains after cooking whereas low amylose cultivars (cvs) after cooking are glossy and tender, cohesive or waxy. Lesser amylose rice is preferable than (over 20%) higher amylose rice because when they cooked it does not become dry and hard. For better economy, export and consumption it is necessary to advance the grain quality and yield. Genotypes 62 and 66 have a good quality grain because of extra-long grain and a maximum grain width. Variety 28 significantly exhibits high yield. A gene that controls the amylose is known as Waxy gene and in present study out of 17 advanced lines 9 (nine) lines ,( 1) 60, (2) 61-(3) 62-, (4) 65, (5)66, (6)11 , (7)12 , (8) 15, (9) 16,) exhibited the Waxy gene. All the good quality grain varieties also contain a Waxy gene. So these varieties can be further used for improving better grain quality and yield of low yielding rice cultivars with low grain quality hence improving and increasing the country‘s economy and export values.

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Author Contributions

SF, SUR, ZH, WA, AI, Performed experiment, MAR, SA write the manuscript, HS and RA performed Analysis, KM review the manuscript.

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Conflict of Interest

The authors have declare that there is no conflict of interest.

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