Therefore, breeding of rice varieties is both able to adapt well to saline conditions and high yield, good quality low amylose content and rich nutrition.. In 2011, CuuLong Delta Rice In
Trang 1MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
SUMMARY OF DOCTORAL THESIS
Specialization: Biotechnology
Code: 62 42 20 01
NGUYEN THI MY DUYEN
BREEDING OF SALINITY TOLERANT RICE WITH HIGH IRON AND LOW AMYLOSE
CONTENT
Can Tho, 2019
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THIS STUDY WAS COMPLETED AT
CAN THO UNIVERSITY
Scientific supervisor: Assoc Prof Doctor TRAN THI CUC HOA NGUYEN THI LANG
The dissertation defended at the university examination committee
At The Room of Thesis Defend (Room 3), The Second Floor, Operation Building, Area 2,CanTho University
At … hour …, ondate … month … year …
Referee 1: Prof.Dotor.Nguyen Hoang Loc
Referee 2: Doctor Truong Thi Bich Van
Confirmation of the Chairman
of the board
Prof.Dotor.Le Van Hoa
The dissertation is available at Libraries:
1 Central library of Can Tho University
2 National library of Vietnam
Trang 3PUBLISHED PAPERS
1 Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa,
2018 Evaluation of genetic material for salt tolerance rice with high iron content and low amylose Science and Technology
Journal of Agiculture and Rural Development, 1:24-28 (in
Vietnamese)
2 Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa,
2018 Breeding salt – tolerant rice lines by backcrossing to the salt – tolerant rice variety Pokkali and using molecular marker
selection Science and Technology Journal of Agiculture and
Rural Development, 11:11-16 (in Vietnamese)
3 Nguyen Thi My Duyen, Vu Anh Phap, Tran Thi Bich Xuan and
Tran Thi Cuc Hoa, 2018 Improved quality of
OM5451/Pokkali by backcross breeding Can Tho University Journal of Science 45(7B): 6-12 (in Vietnamese)
4 Nguyen Thi My Duyen, Vu Anh Phap and Tran Thi Cuc Hoa
Hydridization and selection salinity tolerant rice lines from the
hybrid and backcross OM238/Pokkali Can Tho University Journal of Science (Accepted, in Vietnamese)
Trang 43
Chapter 1: INTRODUCTION
1 Necessity of the dissertation
Rice (Oryza satiava L.) is the staple food of more than half of
the world's population, especially countries in Asia, Africa and Latin America countries In Vietnam, rice is the main agricultural crop In 2015, Vietnam ranked the third in the world in terms of rice exports after India and Thailand In particular, the CuuLong River Delta (CLRD) is the largest rice export region According to statistics of the General Statistics Office, the total area of 2017 rice cultivation in Vietnam was estimated at 7.72 million hectares (down by 26.1 thousand hectares compared to 2016), the estimated yield was 5.55 tons/ha (down by 0.02 ton/ha compared to 2016), the production was estimated at 42.8 million tons (down by 318.3 thousand tons compared to 2016) (Vietnam Business Monitor, 2017)
Agricultural production has been facing climate change, in which salinity is the second most important factor after drought About a fifth of the world's irrigated arable land has been affected and over 800 million hectares have been damaged by salinity In CLRD region, in the period of 2015-2016, the extent of the influence of the "4 g/l" saltwater boundary (salinity cannot be drained or produced) affected 10/13 provinces of this region, with a total area rice damage was nearly 139,000 ha Among them, 86,000
ha of rice damaged over 70% and 43,000 ha of rice damaged 70% of productivity (vtv.vn, 2016) According to the General Department of Irrigation, the Ministry of Agriculture and Rural Development (MARD), the flow of the Mekong River to CLRD had been changing complicatedly, sharply decreasing compared to the beginning of the dry season, the possibility of increasing invasion Saline intrusion was higher than the same period in 2017 (Bich Hong, 2018) Thus, with the influence of global climate change, the area of rice cultivation in the CLRD has been
Trang 530-increasing, leading to a serious reduction in Vietnam's rice production
Coping with this problem, the selection of salinity-tolerant rice varieties has been studied by scientists This was also one of the solutions given in MARD's proposal in 2015 (TTO, 2015) In addition to some salinity tolerant varieties, it has been selected to create and cultivate effectively in some countries around the world Many sources of local rice varieties such as Nona Bokra, Bura Rata were well tolerated with saline conditions, equivalent to the Pokkali varieties that have been identified In the late 20th century, breeders used genetic changes to create rice varieties with high yield potential, good rice quality, resistance to some major pests, and tolerance to the disadvantage conditions such as drought, flooding, salinity
In CLRD, rice varieties grown in saline areas have limitations such as high-yielding rice varieties that are low or medium tolerant
to salinity, or low quality rice Local rice varieties are saline tolerant such as Tep Hanh and Mot Do Do, but with long duration, low yield and hard grain Therefore, it is necessary to breed tolerant rice varieties at the average salinity threshold (EC 4 dS/m) and high salinity tolerance (EC above 6 dS/m) or more, and other interested characteristics such as short duration, high yield and good resistance to pests and diseases
Currently, consumer’s demands are getting higher and higher The quality of rice with low in amylose content and rich in micronutrients is being paid attention The creation of rice varieties with high iron content in grain to contribute to reducing the rate of diseases related to iron deficiency, especially poor farmers in remote areas is very necessary Therefore, breeding of rice varieties
is both able to adapt well to saline conditions and high yield, good quality (low amylose content) and rich nutrition Moreover, nowaday, the rapid development of biotechnology, especially molecular genetics, has effectively supported the new plant
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breeding, helping to select the main hybrid more accurate, shorten the time for breeding In particular, the SSR (Simple sequence repeats) was chosen by many scientists because of its homogeneous nature (codominan), a high degree of reliability (Nguyen Thi Lang and Bui Chi Buu, 2011)
In 2011, CuuLong Delta Rice Institute released the iron rich rice variety OM6976 with medium salinity tolerance, but high amylose content, and OM5451 variety with low amylose content (18%) but low salinity tolerance Currently, there is no rice variety that is not only saline tolerant but also high iron and low amylose
content in grain Therefore, the thesis "Breeding Of Salinity Tolerant Rice With High Iron And Low Amylose Content" was
carried out through backcrossing method and detection of salinity tolerance gene by molecular markers SSR, thereby, rice varieties with both high salinity tolerance and high quality grain (low amylose and iron-rich content) to meet regional production and market demand for rice
2 Objectives
2.1 General objective:
Breed and select rice lines that adapt and thrive in saline ecological regions, and have high iron content and low amylose content (≤ 20%) in grain by the combination of traditional backcrossing and molecular markers
2.2 Specific objective:
One to two rice lines were bred and selected by traditional methods combined with molecular markers These lines were capable of adapting in saline ecological areas (~ 4 ‰) and good in quality (amylose content ≤ 20% and high iron content (iron in brown grain > 14 mg/kg and iron in milled grain ≥ 6 mg/kg)
3 Research subjects and scope of the study
Improved rice varieties from Genebank of Department of Biotechnology, CuuLong Delta Rice Institute
Trang 74 Location and duration
The experiments were conducted at the CuuLong Delta Rice Institute, Can Tho; Kien Giang (Minh Luong at Summer-Fall crop and An Bien at Winter-Spring); and Soc Trang (Tran De)
Duration: from January 2014 to February 2018
5 New contributions of the dissertation
Genetic resources were effectively exploited through the
selection of materials for Saltol genes (well-known salt-tolerant
rice varieties was Pokkali) Pokkali was affected by the photoperiod, the height of plant was over 150 cm, the grain quality was very bad, and the grain yield was not high However, effective application of SSR molecular markers in selection in rice backcross populations, the elite rice lines that were both salininity tolerant and high iron and low amylose (≤ 20%) in grain were selected
In addition, these selected rice lines not only achieved the thesis objectives but also attached other interested traits, such as strong plant, long grain, less chalkiness, wide adaptation, high yield and insect and disease resistance
6 Outline of the dissertation
The thesis was 146 pages in length, including 5 parts, introduction, overview, materials and methods, results and discussions, conclusions and recommendations, references and appendix The thesis consited of 74 tables, 44 figures and 142 references
Trang 8- CuuLong Delta Rice Institute
Molecular markers: for saline gene Saltol on chromosome 1
1 Normal growth, only the old leaves
show white tips while no symptoms on young leaves
Highly tolerant
3 Near normal growth, but only leaf tips
burn, few older leaves become whitish partially
Tolerant
5 Growth severely retarded; most old
leaves severely injured, few young leaves elongating
Moderately tolerant
7 Complete cessation of growth; most
leaves dried; only few young leaves still green
Susceptible
9 Almost all plants dead or dying Highly susceptible
(Source: IRRI, 1997)
The artificial salinity screening in the tray with the solution
of Yoshida (1976) plus salt at seedling stage according to the
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to identify donors from the material set
DNA extraction by CTAB, PCR and electrophoresis based on
Kumar et al (2007)
2.2.2 Content 2: Creation of the backcrossing populations with desirable traits
* Objectives: Breeding offspring populations with both
salinity tolerance and good quaility
* Content: Combination of high salinity tolerance from
donor parent (Pokkali) and high iron and low amylose content from recurrent parent was in order to creat the F1 offspring populations
2.2.3 Content 3: Breeding and selecting of BC populations
by salinity screening and identifying gene related to salinity tolerance through SSR markers
* Ojectives: Aim to shorten the time and increase the effect
of selection of salinity tolerant lines, after each generation,
separated lines would be selected by molecular markers
* Content: Breeding to creat the offspring populations to BC3
generation (Hình 2.1) Individual selection in all generations based on:
- Salinity tolerance at seedling stage through screening in saline condition
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- individuals carried genes associated with salinity tolerance
(Saltol)
- Evaluation of agronomic traits and yield
- Analysis of iron content and amylose content in grain
Figure 2.1 Breeding process of backcross population with salinity tolerance and high quality (high iron and low amylose content)
(P1: Recurrent Parent; P2: Donor Parent)
2.2.4 Content 4 and 5: Field trial of elite rice lines tolerant
to salinity, high iron content and low amylose content at 2 different ecological regions at the 2017 Summer-Fall crop and the 2018 Winter-Spring crop
* Objectives: The elite rice lines of BC3F4 and BC3F5 were grown in 2 ecological regions (normal – CuuLong Delta Rice
Trang 11Institute, saline - Soc Trang and Kien Giang) in the 2017 Fall and 2018 Winter-Spring crop to evaluate and select of elite lines with good adaptability to salinity and good morphological, agronomic, and grain characteristics
- Evaluation of agronomic traits and yield
- Analysis of GxE belonged to the model of Finlay and Wikinson (1963), Eberhart and Russell (1996)
- Analysis of iron content and amylose content in grain
2.2.5 Statistic analysis
Data were collect, input and store by Microsoft Excel 2010 Statistic analysis of data by SAS 9.1.3 software
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Chapter 3: RESULTS AND DISCUSSIONS
3.1 Content 1: EVALUATION OF INITIAL MATERIALS FOR BREEDING OF SALINITY TOLERANT AND HIGH QUALITY RICE VARIETIES
3.1.1 Evaluation of salinity tolerance in parental materials
by screening in saline environment
The results of screening of 36 rice varieties/lines showed that
7 varieties/lines were salinity tolerance in group I (score 1.0 - 2.9) and similar with Pokkali, such as OM107, OM6976, OM109, OM9577, SH1317, SH333 -91, OM108-200 Besides, there were
19 varieties/lines in group II (score 3.0 - 4.9) for salinity tolerance consisted of SH62, OM11, SH200, SH1-12, OM5451, OM241-5, OM40, OM326, OM121, OM10636, OM232, OM9584, SH514, OM241-7, OM240, SH713, SH891, OM239, OM9581 The rest varieties/lines belong to group III (above score 5.0), in the same group with IR29 including OM259, OM238, OM231, OM193, SH893-4, OM236, SH921, OM178, OM238-2, OM238-3
3.1.2 Analysis of amylose content, gel consistency and gelatinization temperature by biochemical method
Results of amylose content’s analysis recorded 15/36 varieties/lines with low content (≤ 20%) including OM238, OM231, OM5451, OM241-5, OM326, OM121, OM193, OM232, SH514, OM241-7, OM240, SH713, SH333-91, OM238-2 and OM238-3 All of these lines/cultivars had a gel strength of 72.7 - 100.0 mm, belonging to the low group (soft rice), in the same group as KDML105 Meanwhile, 9 varieties/lines with high
gelatinization temperature (score 1-3) were OM238, OM231,
OM326, OM121, OM193, OM232, OM241-7, OM240 and
SH333-91 Five (5) varieties/lines with average gelatinization temperature
(score 4-5) were OM5451, OM241-5, SH514, SH713 and
Trang 13OM238-2 Only one line was OM238-3 with low gelatinization temperature
Thus, based on salinity tolerance, Pokkali has been selected
as the father (donor) for the Saltol gene The varieties OM238, OM231, OM5451 and OM121 were selected as mothers (recurrents) because of their low amylose content and high iron content
3.1.4 Application of SSR molecular markers associated with saline tolerance genes to detect genetic material that carried the desired gene
Evaluation of polymorphism in parents with Saltol gene with
7 molecular markers is RM140, RM310, RM1287, RM3412b, RM7075, RM490 and RM10694 In particular, two molecular markers RM1287 (150 - 175 bp) and RM10694 (250 - 350 bp)
were selected as indicators for gene Saltol in selection of salinity
tolerant rice lines
3.2 BACKCROSSING TO CREAT NEW RICE LINES WITH DESIRABLE TRAITS
In F1 generation, the OM231/Pokkali popualtion collected
52 seeds, the OM 238/Pokkali population was 60 seeds, the OM5451/Pokkali population was 80 seeds and the OM121/Pokkali population was 48 seeds