Abstract: Amylose content was determined in 41 traditional glutinous rice varieties of Assam classed as Birain, Bora and Chokuwa group during 2004-06. Average apparent amylose content in 6 accessions of Chokuwa (9.368%) was higher than 20 accessions of Bora group (0.502%) and 15 accessions of Birain (0.191%) genotypes. Mahsuri, a non-glutinous rice variety contained intermediate amylose content (21.2%). Eight SSR markers were used to assess genetic variability. The size of amplified fragments ranged from 100 to 500 bp. Among all genotypes, average Polymorphism Information Content (PIC) was 0.923. The average genetic similarity within the Birain accessions ranged from 0.119 to 0.571. Within Bora-Chokuwa accessions, similarity value ranged from 0.047 to 0.667. The average similarity was 0.228, which reflected that the Bora group could be more diverse than the Birain group. Amylose content is said to be highly influenced by environmental conditions. Since, Birain accessions were from the same Barak valley agro-climatic condition and Bora as well as Chokuwa were from the Brahmaputra valley, an analysis was made with corresponding pair-wise relative rate of increase (%) in apparent amylose contents as well as corresponding values of pair-wise Jaccard’s co-efficient of similarity among the accessions of Birain, Bora and Chokuwa groups of glutinous rice. It showed the existence of a matching relation between the increased values of respective apparent amylose content and the genetic similarity. It seems that apparent amylose content though cannot play a solid indicator for genetic variability in glutinous rice germplasm. However it may help to gauge biochemical bases towards genetic variability under same environmental condition.
INTRODUCTION
Glutinous rice is also called sticky rice, sweet rice, Waxy rice, Botan rice, Mochi rice and pearl rice. It is a type of short-grained rice that is especially sticky when cooked and is grown in Japan, Korea, China, Philippines, Thailand, Indonesia, India and Vietnam. This class of rice was introduced to Assam from Thailand or Burma a considerable time ago (Sharma et al., 1971). The Waxy rice of Assam has been classified in two groups, Bora (glutinous) and Chokuwa (semi-glutinous) based on apparent amylose content. Birain or Beruin rice is also another class of glutinous rice popular in Barak valley in the southern region of Assam, India and adjoining areas of Bangladesh. It is called glutinous in the sense of being glue-like or sticky and not in the sense of containing gluten. Its sticky properties should not be confused with the other varieties of rice that become sticky to one degree or another when cooked. Waxy rice of Assam has significance in social and religious ceremonies and forms a popular daily breakfast diet in rural Assam. Milled rice is also used in the preparation of snacks, flat rice, puffed rice, bamboo rice, sweet rice beer and other dishes. The parboiled polished Chokuwa rice swells on soaking and becomes soft and is used by the people of Assam as a fast food with curd. These multiplicities of uses make the glutinous rice very popular among farmers, in spite of the advent of modern high yielding rice varieties. What distinguishes glutinous rice from other types of rice is having no (or negligible amounts of) amylose content. Amylose, a linear polymer of glucose is one of two components of starch, the other being amylopectin. The apparent amylose content of Chokuwa varieties varies from 15 to 20%, while that of Bora varieties was found in trace quantities (Dutta and Barua, 1978).
To date more than 2000 SSR markers of cultivated rice are available, which provides a powerful tool for studying Oryza genus as a whole, as SSR markers have good cross-species amplification (McCouch et al., 2002). Waxy locus has been evaluated with SSR markers. Ayres et al. (1997) determined the relation between the Wx microsatellite polymorphism and the amylose content in the 92 US rice strains. They demonstrated that the Wx microsatellite is polymorphic enough to distinguish most rice strains in different amylose classes and the inheritance of the CT repeats can be traced through a cross-section of the US rice pedigree. Bergman et al. (2001) demonstrated that the CT repeats classes of Wx gene with n = 10 or 11, 14, 20 and 17 and 18 were categorized as high amylose, intermediate amylose and low-amylose types, respectively. Bao (2002) reported the usefulness of (CT)n microsatellitic markers of Wx gene in rice starch quality improvement. Prathepha (2003) characterized 68 rice strains belonging to two species of Oryza by using Wx microsatellite, (CT)n repeats, that are closely linked to the rice waxy gene. Prathepha (2003) observed the predominance of (CT)17 repeats in the Wx locus, in addition to identification of a unique microsatellite class,(CT)18 repeats, in the strains tested. While evaluating amylose content or identifying Waxy locus, no study has been reported to link amylose content with indices of genetic relationship among the glutinous germplasm for appropriate characterization. The present investigation was carried out to study variation for apparent amylose content in context of polymorphism information content along with indices of genetic relationship derived through SSR markers in Birain, Bora and Chokuwa groups of traditional glutinous rice of Assam.
MATERIALS AND METHODS
Seeds of 41 glutinous rice varieties (Table 1) were used in the present study during 2004-06. Among them, 15 (suffixing the word Birain) were collected from Regional Agricultural Research Station (RARS), Assam Agricultural University (AAU), Karimganj and the remaining 26 (suffixing the words Bora and Chakuwa) were from RARS, AAU, Titabor, representing distinct agro-climatic variation within the plains of the two river valleys, the former one from the Barak valley and rest two from the Brahmaputra valley of the northeast India. The traditional names as given by the farmers were retained in the germplasm accessions for field level identification. Karnel of Pusha Birain, Das Birain, Mow Birain, Kala Birain, Agirjal Birain, Akib Birain, Gela Bora, Memon Bora, Chokuwa Bora-2 and Parochokuwa Sali were red while rests were white. The accessions like Uba Birain, Gela Bora, Jaisungam Birain, Das Birain, Maju Chokuwa and Pakhiloga Bora could be classed as slender grain and rest were as either non-slender or bold.
Amylose content was determined by the method described by Sawbhagya
and Bhattacharya (1979). Defatted moisture-free powdered (100 mg) sample
was taken in a stoppered conical flask. One milliliter of distilled alcohol
was added to wet the powder. Ten milliliter of 1 N NaOH was then gently added
by the side and kept for overnight and heated next day in a boiling water bath
for 2-3 min. After cooling, the volume was made up to 100 mL with distilled
water. Five milliliter of above dispersion was transferred into a 100 mL volumetric
flask. Fifty milliliter of water was added to it, followed by the addition of
1 mL of acetic acid and 2 mL of iodine (0.2%) solution.
| Table 1: | Apparent amylose content in (AACP) and corresponding pair-wise relative rate of increase (%) in AACP as well as corresponding values of pair-wise Jaccard’s co-efficient of similarity within Birain, Bora and Chokuwa groups of glutinous rice accessions of Assam |
| ! Value corresponding to glutinous rice accession in the previous row | |
The volume was made up to the mark with distilled water; pH of this solution was maintained at 4.5. One milliliter of standard amylose solution was taken and treated using the same procedure. A reagent blank was also prepared as such except for adding amylose extract or the standard amylose solution. After 30 min of interval, the intensity of colour developed was measured in spectrophotometer (Systronics UV-VIS, Model-118) at 630 nm against the reagent blank. Amylose content was calculated using the following relationship:
Where:
| R | = | 630 nm reading for sample dispersion |
| A | = | 630 nm reading for standard amylose solution |
| a | = | Amount of the standard amylose taken |
| r | = | Amount of sample taken |
The estimation was done in triplicate and their mean was recorded as g of amylose per 100 g in moisture free sample.
For studying genetic variation through SSR techniques, the total genomic DNA from each of the genotypes was extracted following the protocol of Plaschke et al. (1995) with slight modification. Genetic relatedness among the genotypes was then computed by using the Jaccard’s coefficient of similarity using SIMQUAL module of NISYS-pc. The pair-wise genetic similarity index was calculated as per Jaccard’s coefficient of similarity (Jaccard, 1908) is given below:
where, F is similarity index; NAB1 is number of bands present (Scored 1) in both accessions A and B; NAB0 is number of bands present in all test entries but not present in accession A or B; NT is total number of bands scored in the study.
RESULTS AND DISCUSSION
Apparent Amylose Content
The Birain genotypes had the lowest amylose content (0.191%) followed
by bora genotypes (0.502%) and Chokuwa genotypes (9.368%), respectively.
Among the glutinous rice accessions, the maximum and minimum amylose containing
genotypes were Kalamdani Chokuwa (11.400%) and Jaisungam Birain
(0.136%), respectively. Among Birain genotypes, the maximum and minimum
amylose containing genotypes were Pusha Birain (0.250%) and Jaisungam
Birain (0.136%), respectively. Among Chokuwa genotypes, the maximum
amylose containing genotype was Kalamdani Chokuwa (11.400%) and the minimum
amylose containing genotype was Sam Chokuwa (5.360%). Considering Bora
genotypes alone, the maximum and minimum amylose containing genotypes were Ghew
Bora (0.708%) and Joha Bora (0.151%), respectively. Present investigation
revealed that the amylose content of indigenous glutinous rice ranged from 0.136
to 11.4%, indicating some of them as sticky or Waxy rice. In general, Birain
group (0.191%) has lower amylose content than Bora genotypes (0.502%).
Based on amylose content, Juliano et al. (1981)
classified rice as Waxy (0-2% amylose), very low (5-12%), low (12-20%), intermediate
((20-25%) or high (25-30%). Dutta and Baruah (1978) reported
amylose content in Bora genotypes as trace amount and that in Chokuwa
from 17.14 to 19%, which were more than that observed in the present study.
But Kandali et al. (1995) reported 0.95 to 1.25%
of amylose in Bora accessions of Assam, which are in close correspondence
with that observed in the present investigation. There is a possibility of duplication
in Assam rice collection by having the similarity in names. It has been observed
that the accessions with similar names (two Chokuwa Bora accessions and
two Ranga Bora accessions) had different level of amylose content (Table
1). Moreover, amylose content is highly influenced by environmental conditions
(Juliano and Pascual, 1980). So, based on amylose content
alone, the duplicate nature of the four accessions could not be ascertained,
warranting the use of better system for analyzing genetic variability and duplicate
identification. However, for Birain group of Barak valley, no published
report on amylose content is available. Glutinous rice lacks the starch amylose,
which constitutes up to 30% of the total starch in non-glutinous rice endosperm
(Oka, 1988). The glutinous phenotype arises through the
disrupted expression of the amylose biosynthesis gene, (Wx), which encodes a
granule-bound starch synthase (Sano, 1984). Glutinous
rice contains a G to T mutation at the 5´splice site of Wx intron 1 that leads
to incomplete post-transcriptional processing of Wx pre-mRNA (Wang
et al., 1995; Bligh et al., 1998; Cai
et al., 1998; Hirano et al., 1998;
Isshiki et al., 1998). Glutinous rice does not
have detectable levels of spliced mRNA as a result of this mutation (Wang
et al., 1995; Bligh et al., 1998).
But some degree of amylose synthesis is restored in varieties that carry the
mutation due to display of cryptic splice site activation (Cai
et al., 1998; Olsen and Puragganan, 2002).
This might be the reason for detecting very low level of amylose in the present
study and other similar studies in glutinous rice of Assam. Dipti
et al. (2003) reported that the amylose content of Beruin
rice of Bangladesh ranged from 7.9 to 10%, which was much higher than that obtained
in Birain rice of Assam (0.136 to 0.250%) from the present study. Such
differences in amylose content might be due to differences in genotypes under
investigation along with environmental variation. However, methods to estimate
amylose content might influence the results, which cannot be confirmed
from the present investigation. Mahsuri, a non-glutinous rice variety
contained intermediate amylose content (21.2%) remains soft and fluffy on cooking.
Vanaja and Babu (2006) reported that Mahsuri contains
23.64% amylose, which is slightly more than the present study.
Therefore, it seems that the apparent amylose content in Chokuwa (9.368%) was higher than Bora (0.502%) and Birain (0.191%) genotypes.
Polymorphism Information Content (PIC)
Eight SSR markers were used in the study to access genetic variability.
The size of amplified fragments ranged from 100 to 500 bp. Average Polymorphism
Information Content (PIC) was 0.923 in all the genotypes. The marker RM-11 showed
a maximum PIC (0.954) and RM-164 showed a minimum PIC (0.876) across all the
genotypes (Table 2). Within Bora-Chokuwa group, RM-11
generated maximum PIC (0.949) and RM-251 revealed a minimum PIC of 0.837. Within
Birain accessions, RM-251 revealed a maximum PIC (0.981) and RM-315 showed
a minimum PIC of 0.906 (Table 3). A lower PIC was observed
for Bora-Chokuwa genotypes (0.903) than that for Birain genotypes
(0.947).
Indices of Genetic Relationship
In all the genotypes, the genetic similarity ranged from 0.029 to 0.667
with an average similarity of 0.238. Maximum similarity (0.667) was observed
between Parochokuwa Sali and Til Bora, while the minimum similarity
(0.029) was exhibited by Tepra Birain and Joha Bora (Table
4).
Within the Birain accessions, similarity value ranged from 0.119 to
0.571 with an average of 0.306. The highest similarity (0.571) was exhibited
by Chefa Birain with Aki Birain and Kala Birain, whereas,
the lowest similarity value (0.119) was observed between Mow Birain and
Akib Birain. Within Bora-Chokuwa accessions, similarity value
ranged from 0.047 to 0.667. The accessions. Parochokuwa Sali
and Til Bora exhibited maximum similarity (0.667) and Joha Bora
and Maju Chokuwa showed minimum similarity (0.047). The average similarity
was 0.228, which reflected that Bora group is more diverse than the Birain
group.
| Table 2: | Level of polymorphism in few glutinous rice germplasm of Assam a s detected by SSR markers |
| Table 3: | Comparative analysis of level of polymorphism as detected by SSR markers in few glutinous rice germplasm of Assam |
| Table 4: | Pair-wise Jaccar’s coeffcient of smilarity based on SSR data in glutinous rise accessions under study |
| Table 4: | Continued |
| 1: Chefa_Birain, 2: Aki_Birain, 3: Kacha_Birain, 4: Pusha_Birain, 5: Das_Birain, 6: Mow_Birain, 7: Kala_Birain, 8: Tepra_Birain, 9: Pani_Birain, 10: Jhanki_Birain, 11: Agirjal_Birain, 12: Akib_Birain, 13: Jaisungam_Birain1, 14: Uba_Birain, 15: Garuchakhuki_Birain, 16: Bor_Bora, 17: Gela_Bora, 18: Memon_Bora, 19: Joha_Bora, 20: Jengoni_Bora, 21: Bora_2, 22: Chokuwa_Bora_2, 23: Ranga_Bora_3, 24: Ranga_Bora_1, 25: Boga_Bora, 26: Bor_Chokuwa, 27: Chokuwa_Bora_1, 28: Kalamdani_Chokuwa, 29: Boga_Chokuwa, 30: Chandra_Bora, 31: Maju_Chokuwa, 32: Rangali_Bora, 33: Kaun_Bora, 34: Rupohi_Bora, 35: Tangun_Bora, 36: Garuchokuwa_Bora, 37: Pakhiloga_Bora, 38: Parochokuwa_Sali, 39: Til_Bora, 40: Sam_Chokuwa, Cophenetic correlation, r = 0.663 | |
| Fig. 1: | Apparent amylose content (%) and corresponding pair-wise relative rate of increase (%) in apparent amylose contents as well as corresponding values of pair-wise Jaccard’s co-efficient of similarity in (a) Birain (b) Bora and © Chokuwa groups of glutinous rice accessions in Assam |
Apparent Amylose Content vis-à-vis Genetic Relationship
Amylose content is said to be highly influenced by environmental conditions.
As respective accessions of Birain, Bora and Chokuwa groups
were collected from the same environmental condition viz. the Barak sub-basin
and the Brahmaputra valley agro-climatic conditions, an analysis was therefore
made with corresponding pair-wise relative rate of increase (%) in AACP as well
as corresponding values of pair-wise Jaccard’s co-efficient of similarity
among the accessions of Birain, Bora and Chokuwa groups
of glutinous rice (Table 1, Fig. 1a-c).
It showed the existence of a matching relation between the increased values
of respective AACP and the genetic similarity. For instance, in two Birain
accessions viz., Akib Birain and Mow Birain, Das Birain and
Garuchakuki Birain, the AACP was 0.172 and 0.189, 0.200 and 0.246 percent,
respectively. Thus the AACP of Mow Birain, Garuchakuki Birain were
about 10 and 23% higher than Akib Birain and Das Birain, which in turn,
were about 4 and 2.6% higher in amylose content than that of Agirjal Birain
(0.165) and Kala Birain (0.195).
The Jaccard’s coefficient (measures similarity) was decreased between Akib Birain and Mow Birain (0.119) by more than 200% than that of Akib Birain and Agirjal Birain (0.400) or between Aki Birain and Garuchakuki Birain (0.222) by more than 39% than that of Kala Birain and Das Birain (0.308). Similarly, AACP in Bora group of accessions, viz., Ghew Bora (0.708) was 4% higher than Bora-2 (0.680) against its nearest (0.678) Bora accession Goruchokuwa Bora. The Jaccard’s coefficient of similarity between Goruchokuwa Bora and Bora-2 was 0.378. The genetic similarity between Ghew Bora and Bora-2 was decreased by about 61 percent. Thus relative increase in AACP might be a reflection of direct or inverse proportionality to the genetic similarity in Birain and Bora accessions, which had lower amylose contents. The same reflection was not visualized in Chokuwa accessions which had comparatively higher amylose content. Here Maju Chokuwa (7.840%) was 46 and 227% higher amylose content than Sam Chokuwa (5.360%) and Parochokuwa (2.400%), respectively. Genetic similarity between Sam Chokuwa and Maju Chokuwa (0.268) was however increased by 108% over Sam Chokuwa and Parochokuwa (0.129). Thus it appeared that the AACP though could not play a concrete indicator for genetic variability in glutinous rice germplasm, however it might help to gauge genetic similarity, especially under lower amylose content. Customarily of course, apparent amylose content may help to characterize geographical indication of Waxy rice germplasm under the existing international patent regime.
ACKNOWLEDGMENTS
The authors are grateful to the Department of Plant Breeding and Genetics, Department of Biochemistry, Regional Agricultural Research Stations of Karimganj and Titabor under the Assam Agricultural University, Jorhat for rendering necessary help towards the study. The study was conducted on the funding of the NATP, Govemment of India.