Analysis of Quality of Landrace Rice: AG4 variety in An Giang, Vietnam

Landrace rice genotypes AG4 were evaluated inTri Ton, Tinh Bien, AnGiang Province with three replications in a field experiment during 2019 to 2020. The analysis revealed significant differences among the genotypes against all the characters studied. In general, phenotypic variance is higher than the corresponding genotypic variance for all the characters studied. AG4 rice is considered as the unique landrace varietal group because of its aroma and superior grain quality.To confirm the presence or absence of fragrance in AG 4,a set of 11 lines was phenotypedby using gas chromatographic separation to quantify 2AP content in milled rice samples. KOH tested and PCR method with two directives RM223 and FMU1-2 are recorded to select the lines with the best fragrance followed by line7, 10 ,14 and 20.The shape is determined by the length and width ratio.From shape evaluation, length and width ratio of AG4 are high. Level of chalkiness is. There are mostly low contents on AG4, when doing rice quality analysing. It is the evidence for the deliciousness on rice. Milling quality determines the final yield and fracture rate of milled rice. Recorded line 7 for high milling rate is over 50%. Protein content of rice varieties ranges from 6.9 to 8.6%. Lines 7 and 56 have the highest protein content (8.5-8.6%). Characters like number of panicles per plant, panicle weightg, number of grains per panicle, and grain yield recorded are high. Grain yield analysis revealed significant differences among lines. Selected AG4, lines number 7 can be used in breed selection program in the nearest future to provide the local need. Keywords— aroma, amylose, chalkiness, genotypic, phenotypic, milling quality.


INTRODUCTION
Rice (Oryza sativa L.) is the staple food of more than three billion people, over half the world population. It provides 27% of dietary energy and 20% of dietary protein in the developing world. Rice is cultivated in at least 114 countries, mostly developing, and is the primary source of income and employment of more than 100 million households in Asia and Africa (FAO, 2004). Landraces, traditional varieties or local varieties, form the foundation on which to build better rice plants.Landraces are generally considered to be a rich source of genetic variation. Furthermore, local varieties provide farmers with alternatives, where modern rice varieties are not well adapted in order to contribute to diversity at the field level. However, the number of traditional varieties being planted has reduced, with a few productive and relatively uniform high-yielding varieties dominating the rice landscape. Traditional rice varieties though had been documented to have contributed to the origin of 1,709 modern varieties in Asia which can be traced to 11,592 traditional varieties. The pedigrees of IRRI breeding lines and varieties until 1994 can be traced to hundreds of traditional varieties most from Asian countries (Tran, 2000). Genetic diversity is the basic foundation forspecies survival. The processes of recombination and gene mutation guarantee to the continuous inputs for new variants, as well asthe processes of environmental adaptation and random drift shaping the distribution of genetic diversity in time and space (Brown et al., 1989 . Characterization and evaluation of diversity among traditional varieties will provide plant breeders information that necessary in the identification of initial materials for hybridization to produce varieties with improved productivity and quality.The objective of the study is to evaluate genetic diversity of the traditional rice varieties in HATRI'sgenebank(Vietnamusing morphological characters and microsatellite markers for aroma and yield components AG 4 lines from An Giang province).

Plant Materials
A total of 12 accessions of traditional varieties collected from An Giang province, Vietnam, and conserved in genebank of High Agricultural Technology Research Institute for Mekong delta ( HATRI)in Vietnam.
Theoriginal information of these accessions is presented in Table 1.

Agro-morphology Analysis
11 lines from AG4 traditional varieties were planted in the field at High Agricultural Technology Research Institute for Mekong delta ( HATRI), during the wet season from 2019 to 2020. Seeds were sown in the raised seedbeds, and 30-day old seedlings were transplanted at one seedling per hill. Hills were established at distances of 20 x 20cm. The standard cultural management practices for rice were followed (Bui, 1986).

Quality traits
A total of 12 lines varieties were evaluated (Table 1) and the following quantitative traits were considered: Panicle length (cm), length of panicle at maturity measured from the base of the plant to the tip of the panicle (taken from 10 random selected primary panicles per accession per replication). Panicles per plant (number), the total number of panicles per plant (from 10 random selected primary panicles per accession per replication). 1000-grain weight (g), weight of 1000 welldeveloped grains at 14% moisture content (from 5 random selected primary panicles per accession per replication). Days to maturity, days from seeding when 80% of the grains are fully ripened per replication basis (from 5 random selected primary panicles per accession per replication). Filled grains (number), obtained from counts of total number of filled grains per panicle (from 5 random selected primary panicles per accession per replication). Unfilled grains (number), obtained from counts of total number of unfilled grains per panicle (from 5 random selected primary panicles per accession per replication). Yield obtained from the harvested plants in each replication. Harvested grains were threshed, cleaned, dried, and weighed for each accession per replication. Moisture content per plot was determined immediately after weighing using a moisture meter. Yield = weight of harvested grain(g)/number of hills harvested x number of possible hills x MF (of the harvested grain).

Grain quality analyses
Milled grains underwent assessment of physical traits (grain dimensions, proportion of head rice in milled rice, and chalkiness) and then a test portion of each sample was ground into fine flour(100-mesh) using a Udy Cyclone Sample Mill (model 3010-30, Fort Collins, CO). Reverse osmosis (RO) water and reagent-grade chemicals were used for the chemical analyses.
Physical traits (length, width, and degree of chalkiness) of the milled rice grains were determined using the Cervitec ™ 1625 Grain Inspector (FOSS, Denmark). Grain shape was calculated based on the length-to-width ratio of the grains. The proportion of head rice (%) in the milled rice was determined by measuring the number of grains that are 75% intact after a test portion (100g) of milled rice was sorted using a shaking sieve; the rest are broken grains (%).

Gelatinisation temperature
GT was determined using the alkali digestion test [Little RR et al 1958]. A duplicate set of six whole-milled kernels without cracks was selected and placed in a plastic box (5×5×2.5cm). 10mL of 1.7% (0.3035M) KOH solution was added. The samples were arranged to provide enough space between kernels to allow for spreading. The boxes were covered and incubated for 23h in a 30°C oven. The starchy endosperm was rated visually based on a seven-point numerical spreading scale as a standard evaluation system for rice [IRRI .2013]. According to the ASV score, GT of rice grains can be classified into four groups: high (1-2), high-intermediate (3), intermediate (4)(5), and low (6-7) [Juliano B et al .1985].

Gel consistency
Gel consistency was determined as previously described [34].Rice flour (100mg) was mixed with ethyl alcohol (0.2mL)containing 0.025% thymol blue and 0.2M potassium hydroxide (2mL) and heated in a boiling water bath for 8minutes. After heating,the sample tubes were allowed to cool in an ice-water bath and immediately laid horizontally on the table. Gel consistency was measured by the length of the cold rice paste in the culture tube held horizontally for one hour. Hard, medium, and soft gel standards such Nang Nhen, Khoadawmali105, are respectively included in every set.

Aroma
The current definition of aromatic rice is the presence of the volatile compound 2-acetyl-1-pyrroline (2AP). This was quantified at HATRI using gas equipped with a flame ionisation detector [35]. For those rice samples not measured at IRRI, aroma was determinedby smelling and tasting cooked grains.Volatile analysis of aromatic rice by gas chromatographymassspectrometry (GCMS).Volatile compounds in the aromatic rice from Iran, Pakistan,India, and the Greater Mekong Sub-region (GMS) were analysed.Headspace volatile compounds of selected aromatic rice werecollected by solid phase microextraction using a 65mm polydimethylsiloxane-divinylbenzenefibre (Supelco, Bellefonte, USA)and analysed using GC-MS (GC 8000, Fisons Instruments,Cheshire, UK) [36]. GCMS raw data were processed usingMetAlign [37] to extract and align the mass signals, and MSClust[38] to remove signal redundancy per metabolite and reconstructmass spectra. The PCA plot was constructed using SIMCA-P 12.0(Umetrics AB, Umeau, Sweden).The seeds from AG4 plants were manually dehulled. The seedsfrom each line were treated by Satakedehuller. They were milled by testmiller for one hour. Ten seeds from each line plant were individually ground for 10 seconds witha medium speed by Wil grinder. Rice powder of each grain was placed in an individual 5x5cmplastic box. To each box, 500ul of diluted alkali (1.7%) was added and covered immediately. Thetreated samples were placed at room temperature for 30 minutes. The boxes opened one by one, andaroma was scored by smelling. The heterozygotes were recognized based on the presence of aromaticand nonaromatic grains in lines progeny test. When all ten seeds of individual plant werearomatic, the individual was considered as homozygous for aroma. If the ten seeds of individual plant were non-aromatic otherwise, the individual would be considered as homozygous for nonaroma. Presence of aromatic and non-aromatic seeds inlines indicated heterozygous nature of plant. Due toimportance and accuracy of the phenotyping in mapping process, particularly in bulk-segregantanalysis, additional 30 seeds from each homozygous aromatic and homozygous nonaromaticplants were analyzed. It was done to assure the accuracy of phenotyping. Due to the importance and accuracy of phenotying in mapping process, rice leaves were also evaluatedat tillering stage. Ten leaves were sampled from individual plants at tillering and cut into 5mm longpieces. They were put into a capped glassware and stored at -20 o C before aroma evaluation. Onehour was measured from each frozen leaf sample, by putting into a capped test tube, and mixed with5ml of 1.7% KOH solution for 10minutes at 50 o C. Four to five panelists were asked to classify thesamples as either aromatic or non-aromatic by their own smell.

Analysis of variance.
The agro-morphological data collected were initially analyzed through analysis of variance to verify genetic variation in the traits measured. The few traits with insignificant genetic variation, based on the F-test, were not considered for further analyses.

IV. RESULTS AND DISCUSSION
Without understanding of consumer preference for rice grainquality, wide adoption of any newly developed rice variety is notguaranteed. Hence, identifying the grain traits that governacceptance is important to guide a successful breeding program.Quality attributes of the most popular rice varieties consumed inthe countries and provinces in Asia, as well as for some of the ricegrowing countries in other continents have been collected.Currently, rice grain quality is classified in terms of its physical, taste, and visual characteristics. The physical appearance ofthe grain defines its price in the market. Similarly, [Bradburyet al., 2005] identifying the aroma gene between the Badh2 bymolecular markers. Further studies show that the difference between fragrant rice and non-aromatic rice is due to two molecular markers on the gene that encodes betaine dehydrogenase (BADH2). Indeed, after copying based on the map and sequence of the fgr region, it was found that there is a significant difference in the BADH2 gene sequence between fragrant rice and non-fragrant rice, also there is a mutation in fragrant rice in the 7th exon region of the BADH2 gene, which leads to the function of losing BADH2 protein.

Evaluation of yield and components yield AG4 in field
The rice landraces revealed a wide range of phenotypic variation in 8 agronomic traits (Table 1). 1000-grain weight, showed similar at 11 lines .Assessing the yield and components yieldof the AG4 variety in the same field, through a model assessment of 13 elite lines of styling. The AG4 rice variety was also analyzed for aeration properties, the results also showed that the population gave a high genetic purity value on the breed. Particularly, the yieldand panicle numbers per plant , spikelet / hill . are statistically significant, so the conditions of cultivation and conditions of care and fertilizer for the full development of the variety are very important. The promising lines of the AG 4 variety are planted and assess the yield and yield components. The results showed that these lines had more equal and evener length panicle than the opposition, the lines were quite good dust blooms, the number of particles / dusts was quite good, and the ratio of panicle was average. In terms of recorded Evaluation of rice quality appearance Homity in physical characteristics, such as the length and width of the rice sample can play an important role in the willingness of consumers to pay for rice. When analyzing the size of rice grains is evaluated according to the IRRI standard scale.
AG4 seed record size has a long rice grain size fluctuation of 11.22-11.48mm rice grains fluctuation from 7.28-8.67mm. This is a very long group of rice grains. Analysis of the chalkiness ratio of AG 4 lines noted: most lines do not have chalkiness except lines 9 and 10 for a 0-word (99%) chalkiness ratio in order(Table2). Evaluation of rice and rice grain sizes of 12 samples of AG 4 varieties served and 2 types of confrontation. cooked rice, gel durability hardens faster than soft gel properties. The line noted good gel durability soft rice. Lake temperature (GT) determines the absorption of water and time for cooking. GT is the temperature at which starch particles suck up water and begin to bulge . Most lines have special characteristics that were preferred in the polished grain of AG 4 rice: (1) a 'greasy' look without any abdominal white, (2) an entire rice grain, (3) fully developed and uniform kernel, and (4) neither too soft nor too hard when crushed under the teeth. The characeristics of the cooked rice for which it was valuable: (1)individuality of the cooked grain without bursting, and (2) sweetness and a special fragrance of the cooked product.

Grinding Quality Assessment -Analysis of Milling
Rate: A review of the percentage of raw rice shows that line 7 has a fairly high percentage of raw rice 52.1%, quite good compared to the control variety of 45.7%. The percentage of raw rice is also affected by post-harvest treatment, storage time and conditions, and milling. Nutritional Qualities Assessment -Protein Analysis: The quantity and type of protein are important factors in rice nutrition. Various factors affect the content of rice proteins: climate and environment, and the number of fertilizers applied, the duration of maturity, the degree of milling, and the characteristics of the breed. The protein content of rice varieties ranges from 6.7 to 8.6%. Lines 7 and 56 have the highest protein content (8.5-8.6%) in order.

V. DISCUSSION
Preference for grain size and shape vary from one group of consumers to the other. Some ethnic groups prefer short bold grains, some prefer medium long grains, and long slender grains are highly priced by others. In general, long grains are preferred in Vietnam subcontinent,but in Southeast Asia, the demand is for medium-to-medium long rice. In temperate areas, short grain varieties are prevalent.
There is a strong demand for long grain rice for the international market (Singh et al 2000).
Usually, the quality of rice grains is evaluated through consumers and is therefore used as some first choice criteria in  Rice can be classified into three groups based on GC: hard rice (≤ 40mm), medium (41-60mm), and soft (> 61mm) [Graham, and ctv 2002]. Analysis of 8 AG 4 lines recorded shows that most belong to the rice soft group.
On the other hand, milled rice/brown rice kernel appearance and dimensions determine the price in the market. Milling quality determines the final yield and fracture rate of milled rice, which is a concern of consumers and farmers. The three main parameters, the recovery of brown rice (the ratio of brown rice to raw rice), the recovery of ground rice (the ratio of ground rice to raw rice) and the recovery of raw rice (the ratio of raw rice to raw rice) are used to assess the quality and effectiveness of the milling process. Line 7 gives a high percentage of rice over 50%. Pure lines of selection from AG 4 submited at NCBI with sequences were determined directly using thedideoxynucleotide chain-termination method with a DNA Sequencer (ABI PRISM 3130xl; Applied Biosystems/) and BigDye Terminator (version 3.1) cycle sequencing kit (RR-100, Applied Biosystems), according to the manufacturer's instructions. Obtained rbcL gene sequence was submitted to NCBI /GenBank database(Accession no. MT177967:AG4).

VI. CONCLUSION
Through aroma analysis on 11 lines from AG 4 series recorded all three evaluation methods for aroma: rice reaction test with KOH, calculated 2-acetyl1-pyrroline (2AP) and PCR method with two markers RM223 and FMU1-2 recorded to select the best fragrant line, which is line 7 followed by line 10 and line 48. When calculating qualities through shape evaluation, the length and width of AG 4 are high. As well as a good level chakiness. Rice quality analysis recording amylose content is recorded that most of the low content on AG4 lines. This proves the delicious lines of rice. Milling quality determines the final yield and fracture rate of milled rice. Recorded lines of 7,10,14, and 20 for high milling rates above 50%. Protein content of rice varieties ranges from 6.7 to 8.6%. Lines 7 and 56 have the highest protein content (8.5-8.6%) inrespectively. The protein content of rice varieties ranges from 6.7 to 8.6%. Lines 7 and 56 have the highest protein content (8.5-8.6%) in respectively. -Analyzing yield and yield component, line 9 gives the highest yield (4.47 tons / ha, next is line 7 (4.62t / ha). VII.