Available online at www.sciencedirect.com

ScienceDirect Rice Science, 2015, 22(2): 53−64

Characterization of Myanmar Paw San Hmwe Accessions Using Functional Genetic Markers

Kyaw Swar OO1, 2, 3, Alisa KONGJAIMUN4, Srisawat KHANTHONG2, 5, Myint YI1, Tin Tin MYINT1, Siriporn KORINSAK2, Jonaliza Lanceras SIANGLIW2, Khin Myo MYINT6, Apichart VANAVICHIT2, 3, 7, Chanate MALUMPONG3, Theerayut TOOJINDA2 (1Rice Division, Department of Agricultural Research, Yezin, Nay Pyi Taw 15013, Myanmar; 2Rice Gene Discovery Unit, National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand; 3Agronomy Department, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand; 4Center of Excellence on Agricultural Biotechnology, Office of Higher Education Commission, Ministry of Education, Bangkok 10900, Thailand; 5Plant Breeding, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand; 6Plant Biotechnology Center, Myanmar Agriculture Service, Yangon 11021, Myanmar; 7Rice Science Center, Kasetsart University, Kamphaeng Saen, Nakhon Pathom 73140, Thailand)

Abstract: Paw San Hmwe (PSM) rice has been cultivated in many areas of Myanmar for a long time. Strong aroma, good taste and its elongation during cooking are the key characteristics of PSM rice. Thirty-one PSM accessions were genotypically characterized, and their physical grain and cooking quality traits were studied. We used specific gene markers associated with aroma, apparent amylose content (AAC) and alkali spreading value to determine the alleles carried by different PSM accessions. The results revealed that six PSM accessions (PSM10, PSM12, PSM13, PSM21, PSM22 and PSM30) had a 3-bp insertion in Os2AP gene. Gel consistency (GC) allele was predominant among the PSM accessions for gelatinization temperature (GT), however, the phenotype observed was between low and intermediate GT because of the combination of the GC allele with the presence of low GT allele at heterozygous state from the other loci of the SSIIa gene. Intermediate to high AAC was observed among the PSM accessions corresponding to the haplotype identified for the single nucleotide polymorphism G/T and the (CT)n repeat in the Wx gene. The characterization and grouping data of PSM accessions posted benefits to Myanmar seed banks, and our results will help in maintaining the integrity of PSM rice variety. Key words: Paw San Hmwe; rice; aroma; apparent amylose content; gel consistency; gelatinization temperature; alkali spreading value; functional genetic marker; grain quality; haplotype analysis

Quality traits are important characteristics of rice affecting the market value and consumer acceptance. The demand for high-quality rice varieties has been increasing because of the recent changes in consumer preferences and market requirements. The quality of rice grain is determined by many factors, such as grain appearance, nutritional value, cooking and taste properties (Juliano et al, 1990). Physical properties of

the grain, including size, shape, color, uniformity, general appearance, chalkiness, milling and head rice recovery, are the most important traits determining the overall quality (Dela Cruz and Khush, 2000). Cooking characteristics include apprent amylose content (AAC) (Webb, 1980; Juliano, 1985), gelatinization temperature (GT) (Little et al, 1958) and gel consistency (GC) (Cagampang et al, 1973). Good fragrance is another

Received: 28 August 2014; Accepted: 29 December 2014 Corresponding author: Theerayut TOOJINDA (toojindatheerayut@gmail.com) Copyright © 2015, China National Rice Research Institute. Hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of China National Rice Research Institute. http://dx.doi.org/10.1016/S1672-6308(14)60285-7

Copyright © 2015, China National Rice Research Institute. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer review under responsibility of China National Rice Research Institutehttp://dx.doi.org/10.1016/j.rsci.2015.05.004

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54 Rice Science, Vol. 22, No. 2, 2015

important feature of high-quality rice. Amylose and amylopectin contents and their structures determine the cooking, processing and culinary characteristics of the rice grain. Generally, rice grains contain 16%–30% of amylose content in the storage starch. Starch properties of rice grains are determined by AAC, GC and GT (Juliano, 1998; Bergmann et al, 2004). Amylose content is primarily controlled by waxy gene (Wx) located on chromosome 6. Wx encodes the enzyme granule-bound starch synthase, which plays a key role in amylose synthesis in rice (Tan et al, 1999; Fan et al, 2005). GT of the rice flour is controlled by alkali degeneration locus (alk) encoding a soluble starch synthase IIa (SSIIa) isoform (Fan et al, 2005; Bao et al, 2006). GC is reportedly associated with Wx gene (Tan et al, 1999; Fan et al, 2005; Fitzgerald et al, 2009a). Fragrance is very important in high-quality rice because it plays a considerable role in determining the price and market. Fragrance of rice grains depends on the combination of many biochemical compounds (Petrov et al, 1996). The most important compound is 2-acetyl-1-pyrroline (2AP), whose production is controlled by a single recessive gene located on chromosome 8. BADH2 (Niu et al, 2008) and Os2AP (Vanavichit et al, 2008) have been identified as genes responsible for the synthesis of 2AP (Bradbury et al, 2005).

Paw San Hmwe (PSM) from Myanmar, Basmati 370 from India, Jasmine rice (KDML105) from Thailand and Koshihikari from Japan are the best rated among the high-quality rice varieties. These varieties are priced higher because of their flavor and some other quality characteristics. These varieties constitute important genetic resources for improving cooking properties of rice in several rice breeding programs. PSM is one of Paw San rice varieties, grown in the rain-fed lowland areas of Ayeyarwady Division in Myanmar. It is well adapted to the particular local environmental conditions especially in delta region of Myanmar, and known as Pearl Paw San in the international market because of its strong aroma, good cooking quality and substantial grain elongation during cooking (Thein, 2011). With such exceptionally good qualities, 100% of this variety is exported in super-vacuum packages under the brand ‘Longevity’. In 2011, PSM was awarded as the ‘World Best Rice 2011’ and is considered to be the top-grade rice in Europe. The culinary traits and genetic and phenotypic characteristics of many varieties have been extensively studied. However, the molecular marker-based analysis of these properties in the Paw San rice has not been undertaken. The objective of this study was to investigate the genetic characterization of PSM

accessions obtained from the Myanmar germplasm bank, based on the alleles of known genes associated with grain quality and their corresponding phenotypes. Our results may encourage the utilization of PSM rice as a genetic resource in rice breeding programs.

MATERIALS AND METHODS

Rice materials

We obtained 31 PSM accessions from the gene bank of Department of Agricultural Research (DAR), Myanmar. Of these 31 accessions, 15 were collected from Aeyawady division, 7 from Pago division, 4 from Yangon division, and 1 each from Chin, Kayin, Mon, Rakhine and Sagaing divisions (Table 1). Three rice varieties, Basmati 370, KDML105 and IR57514, were used as standard controls to compare the allele forms of the specific gene markers in this study. The plants were grown 25 cm apart with 25 cm between rows. One plant in the inner portion of the plot was selected for leaf and grain samples for DNA and grain quality analysis.

Table 1. Information of Paw San Hmwe (PSM) accessions used in this study.

Code Accession No. Local name Region Type

PSM1 807 Paw San Bay Kyar Aeyawady Indica PSM2 870 Mee Don Aeyawady Japonica PSM3 923 NgaKywe Yangon Japonica PSM4 930 Paw San Hmwe Pago Indica PSM5 962 Paw San Hmwe Pago Indica PSM6 998 Mee Don Saba Kayin Indica PSM7 1 045 ZawGyiPyan (Mee don) Aeyawady Indica PSM8 1 105 NgaKywe (U To) Aeyawady Japonica PSM9 1 139 Paw San Hmwe Pago Indica PSM10 1 207 Paw San Hmwe Aeyawady Japonica PSM11 1 495 Paw San Hmwe Yangon Indica PSM12 1 627 Paw San Hmwe Mon Japonica PSM13 1 641 Paw San Hmwe Aeyawady Japonica PSM14 1 981 NgaKywe Yangon Indica PSM15 2 117 Mee Don Hmwe Aeyawady Indica PSM16 2 176 Hnan War Mee Don Rakhine Indica PSM17 2 447 NgaKywe Pago Indica PSM18 2 460 Paw San Bay Kyar Aeyawady Indica PSM19 2 499 Paw San Hmwe Pago Indica PSM20 2 500 Paw San Hmwe Pago Japonica PSM21 2 501 Paw San Hmwe Yangon Japonica PSM22 2 502 Paw San Hmwe Aeyawady Japonica PSM23 2 522 Paw San Hmwe Aeyawady Japonica PSM24 2 578 Paw San Shwe War Aeyawady Indica PSM25 2 620 Paw San Hmwe Aeyawady Indica PSM26 2 878 Hnan War Mee Don Sagaing Indica PSM27 2 946 Paw San Hmwe Pago Indica PSM28 3 225 Mee Don Yoe Sein Aeyawady Indica PSM29 3 530 Bay Kyar Lay Aeyawady Japonica PSM30 5 802 Paw San Hmwe Chin Japonica PSM31 11 800 Paw San Hmwe Aeyawady Indica

Kyaw Swar OO, et al. Grain Quality Characterization of Paw San Hmwe Accessions 55

DNA markers

Seven specific gene markers for SSIIa, Wx and Os2AP loci were used to genotype all plant materials (Table 2).

DNA markers SNP2340-41 and SNP2209, located within SSIIa gene on the short arm of chromosome 6 (Lanceras et al, 2000; Bao et al, 2006) and considered as the most informative single nucleotide polymorphisms (SNPs), were used to distinguish between high and low GT. These markers were developed by the Rice Gene Discovery Unit (Katengam et al, 2008).

Waxy (CT)n and Waxy (G-T) markers were used to characterize the alleles of the Wx gene. Waxy (CT)n, a microsatellite marker, is used to amplify the fragment containing a (CT)n repeat in the 5′ untranslated region of Waxy exon 1 (Bligh et al, 1995) and putative 5′ splice site of the leader intron in the Wx gene (Bligh et al, 1995; Ayres et al, 1997). When the fragment is cut by AccI restriction enzyme, an SNP in the putative 5′ splice site of the leader intron, located on the short arm of chromosome 6 (Ayres et al, 1997; Chen et al, 2008), can be detected. It can differentiate between high (Wxa) and low (Wxb) amylose content.

Three DNA markers, Aromarker (Vanavichit et al, 2008), FMbadh2-E2A (Shi et al, 2008) and 3In2AP (Myint et al, 2012a), were used to characterize Os2AP alleles. Aromarker has been developed for the detection of the 8-bp deletion (creating a stop codon in fragrant rice) in the seventh exon of Os2AP (Vanavichit et al, 2008), and FMbadh2-E2A for a region with 7-bp deletion in the second exon of the Os2AP gene on chromosome 8, which is discovered in Wuxiangjing 9 (japonica) (Shi et al, 2008). The 3In2AP marker has been developed using the region with a 3-bp insertion in the thirteenth exon of Os2AP in Myanmar rice Yangon Saba (Myint et al, 2012a). These markers can distinguish between the alleles associated with aromatic and non-aromatic rice.

DNA extraction and PCR amplification

Young leaf samples were collected from each accession,

placed in plastic bags, and stored on ice for DNA extraction. Total genomic DNA was isolated from 0.5 g of leaf tissue (from PSM accessions and controls) using the DNA Trap® kit developed in the DNA Technology Laboratory (http://dnatec.kps.ku.ac.th), Kasetsart University, Kamphaeng Saen Campus, Thailand.

PCR reactions for polyacrylamide gel electrophoresis (PAGE) analysis contained 50 ng of DNA template, 1 × PCR buffer, 2 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.2 µmol/L each of forward and reverse primers, and 1 U of Taq DNA polymerase. The volume was adjusted to 10 µL with distilled water. PCR reaction was initiated by denaturation at 94 ºC for 3 min, followed by 35 cycles of 94 ºC for 30 s, 55 ºC for 30 s, and 72 ºC for 2 min. Final 7 min incubation at 72 ºC completed the primer extension.

To identify the waxy allele (Wxa/Wxb), PCR products obtained using Waxy markers were digested with AccI restriction enzyme in 15 µL solution containing 10 µL of PCR products with 1 × NE buffer and 1 U of restriction enzyme AccI (New England Bio Labs) for 1 h at 37 ºC.

PCR reactions for agarose-based primers SNP2209 and SNP2340-41 were performed in a total volume of 20 µL containing 50 ng of DNA template, 1 × PCR buffer, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.25 µmol/L each of primers P and Q, 0.125 µmol/L each of primers A/G and GG/TT, and 1 U of Taq polymerase. PCR reaction was initiated by denaturation at 95 ºC for 3 min, followed by 35 cycles of 95 ºC for 30 s, 62 ºC for 30 s and 72 ºC for 30 s. Final 5-min incubation at 72 ºC was used for the completion of primer extension.

PCR product electrophoresis

The amplified PCR products of Aromarker, FMbadh2-E2B, 3In2AP and Waxy (CT)n were loaded onto 4.5% polyacrylamide gels and separated by electrophoresis in 1 × TBE buffer at 50 ºC and 60 W. The PCR products were visualized using silver standing. The amplified PCR markers Waxy (G-T), SNP2209 and SNP2340-41 were loaded onto 1.5% agarose gels and

Table 2. Gene specific markers for grain qualities used in clustering 31 Paw San Hmwe accessions.

Trait Gene Chromosome Marker Reference

Amylose content Wx 6 Waxy (G-T) Ayres et al, 1997 6 Waxy (CT)n Bligh et al, 1995 Fragrance Os2AP 8 Aromarker Vanavichit et al, 2008

8 3In2AP Myint et al, 2012a 8 FMbadh2-E2A Shi et al, 2008

Gelatinization temperature SSIIa 6 SNP2340-41 Katengam et al, 2008 6 SNP2209 Katengam et al, 2008

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separated by electrophoresis in 0.5 × TBE buffer at 90 V for 60 min. The PCR products were visualized using ethidium bromide staining. Polymorphisms in the DNA profiles were scored using visual comparisons with standard controls and standard DNA gene ladder.

Evaluation of physical and chemical properties of rice grains

Seeds were collected from the plants whose leaves were used for DNA extraction. The harvested seeds were sun-dried until the moisture content was ≤ 14%, and stored for two months. Before analysis, the seeds were milled and whole grains were selected and oven-dried.

Brown rice length and width (BRL and BRW) Ten de-husked entire brown rice grains were measured using vernier calipers. Using the length/width ratio, the grains were classified as long slender (LS), short slender (SS), medium slender (MS), long bold (LB) and short bold (SB) (Dela Cruz and Khush, 2000; Anonymous, 2004).

Alkali spreading value (ASV) and clearing test Six milled rice grains were placed in Petri plates, 15 mL of 1.7% KOH was added, and the plates were kept at room temperature for 23 h. Then, the ASV was evaluated and scored. The values 1–3 were classified as high GT (more than 74 ºC), 4–5 as intermediate GT (70 ºC–74 ºC), and 6–7 as low GT (less than 70 ºC) (Perez and Juliano, 1978).

Apparent amylose content (AAC) AAC was measured using the procedure of Juliano (1971, 1985) with minor modification. Rice flour (100 mg) was mixed with 1 mL of 95% ethanol and 9 mL of 1.0 mol/L NaOH, and incubated at room temperature overnight to gelatinize the starch. The samples were diluted to 100 mL with distilled water. A 5 mL aliquot of this suspension was mixed with 1 mL acetic acid (1 mol/L) and 1.5 mL iodine solution (0.2% iodine and 2% potassium iodide), and the volume was adjusted to 100 mL with distilled water. The samples were incubated at room temperature for 20 min, and then the absorbance at 620 nm was measured. NaOH solution was used as a control. The AACs of different accessions were obtained using a standard graph (Perez and Juliano, 1978).

GC In 13 mm × 150 mm test tubes, 200 µL xylene solution was added to each 100 mg rice powder sample. The tubes were vortexed, 2.0 mL of 0.2 mol/L KOH was added and the tubes were vortexed again for 1 min.

The tubes were covered with glass marbles and placed in a boiling water bath for 8 min, then removed from the bath and kept at room temperature for 5 min. The tubes were cooled in ice-cold water for 15 min. Finally, the tubes were laid down horizontally over a graphing paper with mm-scale. The gel was classified as soft for more than 60 mm spread, medium for 40–60 mm, and hard for less than 40 mm (Bhattacharya, 1979).

Aroma Five seeds of the brown rice from each accession were added to 200 µL distilled water. The tubes were incubated at 65 ºC for 3 h and then cooled. The lids were opened one by one, and samples were sniffed by three panelists and scored as strongly scented (2), mildly scented (1) and non-scented (0) (Wanchana et al, 2003).

Statistical analysis

Analysis of variance All grain quality traits were subjected to statistical analysis using CropStat Version 7.2. Least significant difference (LSD) test was used to compare the means of grain quality traits in the different accessions. Simple linear regression and analysis of variance were used to analyze the genetic associations.

Analysis of genetic dissimilarity The data were scored using the bands of the quality markers for 31 PSM accessions and controls (KDML105, Basmati 370 and IR57514). Nineteen simple sequence repeat (SSR) markers (RM1, RM5, RM237 and RM431 on chromosome 1, RM154 on chromosome 2, RM338 on chromosome 3, RM124 on chromosome 4, RM538 on chromosome 5, RM510 on chromosome 6, RM11 on chromosome 7, RM25, RM44 and RM447 on chromosome 8, RM215 and RM316 on chromosome 9, RM271 and RM474 on chromosome 10, RM287 on chromosome 11 and RM1227 on chromosome 12) (Myint et al, 2012b) were also used to genotype the PSM accessions and controls, which were used in grouping the accessions. Khin Myo Win, who initially established the grouping of several Myanmar accessions, kindly supplied the genotypic data of IR8, IR64, Basmati 217, Aus 143, Baran Boro, Indane, Azucena, Nipponbare, Leikkalay Meedon, Paw San Taung Pyan Hmwe and Nga Bya Yin Kauk Hnyin Net rice (Myint et al, 2012b). We used them as controls to help in the group assignments of the PSM accessions. To evaluate the genetic dissimilarities between the genotypes, the polymorphic bands were used to construct a binary matrix. The gene-specific markers were obtained from

Kyaw Swar OO, et al. Grain Quality Characterization of Paw San Hmwe Accessions 57

the dissimilarity matrix computed by a shared allele index using DARwin software (Perrier and Jacquemoud-Collet, 2006). An unweighted neighbor-joining (NJ) tree was built using this dissimilarity matrix.

RESULTS

Grain quality and agronomic characteristics

PSM rice had an average aroma sensory score of 0.9 (Table 3). There were nine accessions with aroma score ≥1.0 (2.4 was the highest score), which were clustered together. In terms of cooking properties, the average ASV, GC and AAC of PSM were 5.4, 30.8 mm and 23.6%, respectively. We also found that most PSM accessions were non-aromatic, with low-to-intermediate ASV, hard GC and intermediate-to-high AAC. Our results showed that almost all of the aromatic PSM accessions had low ASV. There were some variants with intermediate ASV, important for maintaining the desired characteristics of PSM rice (Table 3).

Most of the PSM accessions had high AAC. Most accessions had GC less than 40 mm (hard consistency) except for PSM17, which belonged to the medium consistency group. We also examined physical grain

properties such as the length and width of paddy and brown rice grains and obtained the length/width ratios. The average paddy length, width, and length/width ratio were 7.8 mm, 3.4 mm, and 2.3, respectively; the same parameters for the average brown rice grains were 5.5 mm, 3.0 mm, and 1.8 (Table 3 and Fig. 1). Brown rice seeds of most PSM accessions were short or medium length, with a previously reported characteristic of PSM (Calingacion et al, 2014).

Agronomic characteristics such as average days to flowering (143.0 d), plant height (144.5 cm), panicle length (26.6 cm), yield (4.7 t/hm2), grain weight per hill (27.5 g), grain weight per panicle (3.8 g), effective tiller number per hill (9.9), filled grain number per panicle (122.5), seed-setting rate (82.3%), and 100-grain weight (3.3 g) are presented in Table 3.

Allele analysis using grain quality markers

Amylose content G/T in the putative 5′ splice site of the leader intron of the Wx gene can be used to distinguish between high and low amylose content alleles. PCR product of amplification was digested with AccI and yielded 233 bp and 77 bp for G allele while 310 bp for T allele. Twenty-six PSM accessions had G allele suggesting high amylose content similar to the control varieties

PSM17

PSM6 PSM16

PSM15

PSM26

PSM18

PSM14 PSM19 PSM31

PSM12

PSM11

PSM24

PSM3 PSM5 PSM9 PSM22

Fig. 1. Morphology of paddy and milled rice grains of some selected Paw San Hmwe (PSM) accessions.

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Kyaw Swar OO, et al. Grain Quality Characterization of Paw San Hmwe Accessions 59

Basmati 370 and IR57514 (Table 4). Two PSM accessions showed T allele suggesting low amylose content, like KDML105. Three accessions were heterozygous for these alleles.

Waxy (CT)n microsatellite was used to screen the PSM germplasm. We detected five alleles of (CT)n (Table 4): the first allele of 330 bp, the second of 326 bp, the third of 310 bp and two heterozygous alleles (330/310 bp and 326/310b bp). The 330 bp allele was detected in almost all accessions. There were some exceptions. PSM6 had the third allele (310 bp), PSM3, PSM19 and PSM21 had the heterozygous allele (330/310 bp), PSM14 and PSM26 had the heterozygous allele (326/310 bp), and PSM17 had the second allele. KDML105 and Basmati 370 also had the second allele, indicative of low amylose content due to 17 CT repeats (Prathepha, 2003), while IR57514 had 11 CT repeats.

GT We used SNP2340-41 marker to determine the distribution of GC and TT alleles. All PSM accessions used had the GC allele associated with high GT (ASV = 1–3) except for PSM14, which was heterozygous (GC/TT). The control variety KDML105 with low GT had TT allele, while GC allele (high GT) was found in Basmati 370 and IR75714 (Table 4). SNP2209 were used to determine the distribution of G and A alleles, and 23 accessions had G allele associated with high GT, while the remaining accessions were heterozygous (G/A). The control rice variety KDML105 showed G allele associated with high GT (Table 4). Considering the combination of SNP2340-41 and SNP2209, the haplotype 2209G-2340-41GC was associated with optimal SSIIa activity and high GT, while the haplotype 2209G-2340-41TT and 2209A-2340-41GC

Table 4. Thirty one Paw San Hmwe (PSM) accessions and controls assayed with seven rice quality markers.

Code Waxy (G-T) Waxy (CT)n SNP2340-41 SNP2209 3In2AP FMbadh2-E2A Aromarker

PSM1 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM2 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM3 T 19/11 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM4 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM5 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM6 G 11 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM7 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM8 G 19 High GT High GT Non aroma (197/194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM9 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM10 G 19 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM11 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM12 G 19 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM13 G 19 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM14 G/T 17/11 High/low GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM15 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM16 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM17 G 17 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM18 G/T 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM19 G 19/11 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM20 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM21 T 19/11 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM22 G 19 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM23 G 19 High GT High GT Non aroma (197/194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM24 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM25 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM26 G 17/11 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM27 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM28 G 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM29 G/T 19 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) PSM30 G 19 High GT High GT Aroma (197 bp) Non aroma (207 bp) Non aroma (400 bp) PSM31 G 19 High GT High/low GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp) KDML105 T 17 Low GT High GT Non aroma (194 bp) Non aroma (207 bp) Aroma (392 bp) Basmati 370 G 17 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Aroma (392 bp) IR57514 G 11 High GT High GT Non aroma (194 bp) Non aroma (207 bp) Non aroma (400 bp)

G means 233/77 bp Wxa, high amylose content; T means 310 bp Wxb, low amylose content. Waxy (CT)n repeat was considered by comparison between allele and CT repeat in previous reports. The values in this column are the number of

CT repeats, and 19, 17 and 11 correspond to 330, 326 and 310 bp alleles, respectively.

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had no SSIIa activity and was low GT (Katengam et al, 2008). Since the bases at the location 2340-41 of KDML105 are TT, GT of this variety is low although the allele at SNP2209 is G. For the PSM accessions, three sets of allele combinations were found for SNP2340-41 and SNP2209: GC-G/A (heterozygous at SNP2209), GC/TT-G (heterozygous at SNP2340-41) and GC-G. Most of the accessions had GC-G combination indicating high GT (Table 4).

Aroma Three functional DNA markers were used to distinguish fragrant from non-fragrant grain alleles of gene Os2AP on chromosome 8: Aromarker (Vanavichit et al, 2008), FMbadh2-E2A (Shi et al, 2008) and 3In2AP (Myint et al, 2012a).

We found that 25 accessions were non-aromatic with allele sizes of 194 bp or 197/194 bp when screeming with 3In2AP, while six accessions (PSM10, PSM13 and PSM22 from Aeyawady, PSM12 from Mon, PSM21 from Yangon and PSM30 from Chin) were aromatic with 197 bp allele. Aromatic rice Yangon Saba had 197 bp allele (Myint et al, 2012b), and aromatic rice KDML105 had 194 bp allele (Table 4).

The results of screening with FMbadh2-E2A

showed that all of the PSM accessions had non aroma allele similar to those of IR57514, Basmati 370 and KDML105 (Table 4).

No aromatic alleles of Os2AP were found in the PSM accessions using Aromarker. KDML105 and Basmati 370 rice had 392 bp segments indicating that these controls were from aromatic rice varieties, while a non-aromatic control IR57514 had 400 bp allele. These results suggest that the aroma rice of different origins may be controlled by different alleles of the Os2AP gene.

Cluster analysis and genetic relationships

Cluster analysis and binary matrix were obtained based on genetic dissimilarity among PSM accessions and reference controls. Those dissimilarities were clearly reflected in the alleles of grain quality markers and microsatellite markers [standard markers used in grouping rice accessions reported by Myint et al (2012b)]. Grouping of rice PSM accessions and controls is presented in Fig. 2. G1 included the indica group varieties with the indica controls such as KDML105 and IR57514. Surprisingly, two accessions PSM6 and PSM14 were included in this group. G2 included the Boro/Aus types, similar to the isozyme

Fig. 2. Dendrogram of 31 Paw San Hmwe (PSM) accessions (blue), reference checks (violet) and standard checks (red) generated by cluster analysis using unweighted neighbor-joining of DARwin software based on 7 rice quality markers and 19 SSR markers. G1, G2, G5A, G5B and G6 represent the cluster groups; SG1, SG2 and SG3 represent the sub-group in G5B based on the genotype and

phenotype of the PSM accessions.

Kyaw Swar OO, et al. Grain Quality Characterization of Paw San Hmwe Accessions 61

grouping. The Basmati group belonged to G5A, while the japonicas were in G6. Most PSM accessions belonged to G5B, as reported by Myint et al (2012b). Three sub-groups of PSM accessions could be determined in this group, based on the grain quality markers. It is clear that aromatic accessions (PSM10, PSM22, PSM30, PSM12 and PSM21) form SG1. Surprisingly, SG1 had low GT values and intermediate AAC. SG2 and SG3 contained the non-aromatic accessions except for PSM8, which was aromatic and heterozygous for 3In2AP marker. PSM8 belonged to SG3, which had low-to-intermediate GT and intermediate-to-high AAC. Gelatinization in this group cannot be well separated using the SNP markers for GT. SG2 had low-to-intermediate GT and intermediate AAC.

DISCUSSION

PSM rice can easily maintain its popularity due to its superior texture, taste and good milling recovery. Grains of PSM elongate up to three times during the cooking and have a strong aroma. In this study, we provide information on culinary and agronomic properties of PSM germplasm, which may be useful in rice breeding programs and would help to maintain the market position of PSM rice (Calingacion et al, 2014).

Phenotypic and genotypic assessment of grain quality of PSM accessions

The aromatic PSM accessions have a 3-bp insertion in exon 13 of chromosome 8. No aromatic accession with a 8-bp deletion in exon 7 or a 7-bp deletion in exon 2 was found. The 3-bp insertion in the exon 13 of Os2AP is common in Myanmar aromatic accessions, particularly in the popular and widely grown aromatic varieties such as PSM and its relatives (Paw San Yin and Paw San Bay Kyar). Myint et al (2012b) suggests that the 3-bp insertion allele has originated in Myanmar region. However, a fragment with the 8-bp deletion in exon 7, another aromatic allele, can be found in most aromatic accessions grown in South and Southeast Asia, including the most popular aromatic rice accessions, KDML105 and Basmati 370. The 7-bp deletion in exon 2 is characteristic for Chinese accessions (Shi et al, 2008), while the 3-bp insertion is more restricted and mainly found in Myanmar. The localization of the 3-bp mutation in Myanmar reflects the confinement of the PSM accessions in the country. Myint et al (2012b) have reported the 8-bp deletion

found in some of the indica aromatic accessions from Myanmar. The authors believe that the presence of this deletion is the result of preference for indica varieties in the neighboring regions or preferences associated with changes in aroma caused by different mutations. However, the results of this study differed from those of Myint et al (2012a), in which only the 3-bp insertion was found in PSM accessions and the 8-bp deletion was not detected. Our study focused on relatively small number of PSM accessions that happened not to have the 8-bp deletion, with a low genetic diversity in the germplasm. Only eight accessions with 3-bp insertion were aromatic, indicating that PSM accessions from the gene bank of DAR, which should be aromatic, may have mutated or are contaminated and need cleaning up.

Intermediate-to-high AAC and GT are preferred in Myanmar (Calingacon et al, 2014). Thus, the PSM accessions are probably selected by the farmers to have such desired properties. GC, commonly determined by measuring the spread of cooled gel made from flour cooked in 0.2 mol/L KOH (Cagampang et al, 1973), is a measure of firmness of rice after cooking and is used to classify rice varieties with the same AAC, particularly in the high AAC class, into hard, medium and soft consistency types (Cagampang et al, 1973; Kohlwey, 1994). The gene coding for GC is located within the Wx locus (Tang et al, 1991; Tian et al, 2005). Quantitative trait loci of AAC have been also mapped onto this locus (Lanceras et al, 2000; He et al, 2006; Zheng et al, 2008), indicating a relationship between AAC and GC. Varieties with waxy and very low AAC tended to have soft GC (high GC values), while in the varieties from other AAC classes, GCs ranged from hard to soft. Thus, other factors apart from amylose content must be contributing to the strength of the cooling gel, and elucidating these mechanisms will help in breeding programs searching for the superior texture traits (Cuevas and Fitzgerald, 2012).

Among the markers developed to determine amylose content in rice, the G/T alleles explains 80%–90% of the variation observed in non-waxy rice accessions (Aryes et al, 1997; Bao et al, 2006). Consequently, there are rice accessions with T allele showing low or intermediate AAC, and some with G allele showing high or intermediate AAC. This observation suggests that there could be other positions in the Waxy gene that could be involved in the separation of AAC classes. According to Hoai et al (2014), the low AAC is associated with the C allele in the exon 10 of the

62 Rice Science, Vol. 22, No. 2, 2015

Waxy gene rather than the change from G to T at the splicing donor sites of the first intron. This allele change in the exon 10 may explain the intermediate AACs of PSM3 and PSM21, which had T allele at the splice site of the leader intron. The work of Biselli et al (2014) complements the findings of Hoai et al (2014) since accessions with AAC between 21% and 22% possess T alleles in the splice site of the leader intron. In our study, C allele at the exon 10 was not included in the analysis to predict the corresponding AAC. It is clear that the allele change from T to C in the exon 10 is associated with changes in AAC. Phenotypically, almost all PSM accessions showed intermediate-to-high AAC corresponding to the genotype. Accessions that were heterozygous for G/T alleles had high AAC, which could have been caused by the additive and dominant nature of the genes controlling AAC (Pooni et al, 1993; Kuo et al, 1997). The PSM accessions contained 11, 17 or 19 CT repeats, and most of them had 19 CT repeats. PSM3 and PSM21 had the T allele, which is normally associated with low AAC. However, in these two accessions, the results did not correspond to their AAC phenotypes. Chen et al (2008) have analyzed several rice accessions and shown that (CT)11 group has high AAC while (CT)17 and (CT)19 groups include both intermediate and low AAC. It might due to the combination of the G/T alleles, which corresponds to (CT)n repeat, was heterozygous allele 19/11 that caused the intermediate AAC observed in those PSM accessions, although this combination has not been detected in several haplotyping studies (Bao et al, 2006; Wan et al, 2007; Chen et al, 2008; Prathepha, 2008; Biselli et al, 2014).

For PSM accessions, three sets of allele combinations were determined for SNP2340-41 and SNP2209: GC-G/A (heterozygous at SNP2209), GC/TT-G (heterozygous at SNP2340-41) and GC-G. Although most PSM accessions had GC-G allele (Table 4) suggesting high GT, the GT phenotypes or ASV scores were mostly with intermediate GT (Table 3). It is possible that those SNPs do not correspond precisely to the intermediate GT phenotypes. Cuevas et al (2010) have suggested that intermediate GT is caused by the presence of DP24–35 amylopectin chains that can be synthesized by other enzymes. The genetic control of GT has not been fully elucidated. Nevertheless, the available GT markers can be used in varietal differentiation (with high and intermediate versus low GT) as an indicator of the cooking time of rice (Cuevas et al, 2010). It is an economically important

indicator of quality because shorter cooking time leads to significant savings in fuel costs (Fitzgerald et al, 2009b). Consequently, the intermediate GT of PSM accessions may be one of the features to be selected in rice breeding programs.

Cluster analysis and genetic relationships

Based on the dendrogram created for PSM accessions, standard controls and reference controls (Myint et al, 2012b), five groups were identified following the grouping reported by Myint et al (2012b). In the study of Myint, some of the Myanmar accessions, like NgaKywe and Meedon Yoyo, are grouped together with the indica accessions. NgaKywe and Mee Don Saba used in our study were also grouped with the indica varieties because of grain size and seed color characteristics (Fig. 1). However, a study by Thien et al (2012) has clearly separated Mee Don Saba and NgaKywe from an indica variety KDML105. These results show that the molecular characterization of these accessions can help in their grouping and demonstrating the possible contaminations in the gene bank. As mentioned by Glaszmann (1987), isozyme group V includes rice in the Indian subcontinent from Iran to Myanmar. The clustering of Myanmar PSM accessions in G5B corresponds with the study of Glaszmann (1987).

Our study presents a useful characterization and grouping of PSM accessions. The results are of great benefit to gene banks, such as the bank of the DAR in Myanmar. In some accessions, we found heterozygous allele for some markers, which may be due to contamination. Therefore, it has to be taken into account in maintaining the germplasm. We believe that the existing PSM accessions must be carefully checked and the contaminants should be removed to maintain the integrity of the PSM varieties in the international market. The rising popularity of these rice types in the world market can be maintained by distributing rice seed from controlled sources of national repute such as DAR.

ACKNOWLEDGEMENTS

This study was supported by the National Center for Genetic Engineering and Biotechnology, Thailand (Grant No. P1020028), and Generation Challenge Program, Mexico (Grant No. P0900835). The authors are grateful to the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office, Office

Kyaw Swar OO, et al. Grain Quality Characterization of Paw San Hmwe Accessions 63

of Higher Education Commission, Ministry of Education, Thailand for the advice in phenotyping of traits. This research was conducted at the Rice Gene Discovery Unit, Kasetsart University, Thailand, and the materials were provided by Department of Agricultural Research, Yezin, Nay Pyi Taw, Myanmar.

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Characterization of Myanmar Paw San Hmwe AccessionsUsing Functional Genetic Markers