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MỞ ĐẦU MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DOAN THI HUONG GIANG RESEARCH ON IMPROVING THE SUBMERGED TOLERANCE OF R[.]

AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

DOAN THI HUONG GIANG

RESEARCH ON IMPROVING THE SUBMERGED TOLERANCE

OF RICE VARIETY AS996 BY MOLECULAR MARKERS

Major: Genetics and Crop Breeding Code: 96.20.111

SUMMARY OF AGRICULTURE DOCTORAL THESIS

Hanoi - 2022

The thesis was completed at:

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

Supervisors:

1 Prof Dr Le Huy Ham

2 Assoc Prof Dr Luu Minh Cuc

Reviewer 1: ……… Reviewer 2: ……… Reviewer 3: ……… ……

The thesis will be defeneded in the Institute Committee of PhD

Dissertation Examination at Vietnam Academy of Agricultural Sciences

On /2022

The thesis can be found at:

- National Library of Vietnam

- Library of Vietnam Academy of Agricultural Sciences

INTRODUCTION

1 The necessary of the research

Rice (Oryza sativa L.) is the most improtant food crop in Vietnam With a

geographical location near the sea, having a dense system of rivers and fertile alluvial soil, wet-rice agriculture is the main job, bringing outstanding economic efficiency Growing rice plays an important role in the structure of agricultural production and plays a key role in ensuring food security, making Vietnam one of the world’s largest rice exporters

Recently, due to the impact of climate change, it has caused a change in some biotic and abiotic factors in an unfavorable direction, affecting the growth and development of rice, reduce rice production in the world in general and Vietnam in particular One of the most influential factors is that waterlogging occurs in almost all rice growing areas, but the most affected area is the Mekong River Delta, where

having an area and largest rice production in Vietnam (Wassmann et al., 2004; Hoanh et al., 2004) Therefore, the breeding research and development of rice sources that have improved flood tolerance and high yield are essential and meaningful in ensuring food safety and increasing farmers' incomes

The study discovered that the Sub1 gene locus is the main QTL (Quantitative trait loci) controlling the quantitative trait related to the flood tolerance mechanism in rice, which has been detail mapped by Xu and Mackill, 1996 Up to now, the method of molecular marker selection and backcrossing (MABC) has been successfully applied in bringing together gene/QTL loci into new varieties AS996 rice variety is one of the high-quality rice varieties that has been popularly grown in the Mekong Delta and Southeast provinces from 2003 to present However, AS996 variety is very sensitive to waterlogged conditions, so in recent times, yield has been greatly affected when flooding conditions have increased in the field Therefore, improving the flood tolerance of rice variety AS996 is one of the urgent problems posed for the Mekong Delta

Stemming from the requirements of production practice, we carried out the project:

"Research on improving the submerged tolerance of rice variety AS996 by molecular markers", to create a new rice variety AS996-Sub1 capable of submerged tolerance adaptive for the Mekong Delta region

2 Research objective

Improvement of rice variety AS996 using the method of markers assisted crossing (MABC), to create new rice varieties AS996-Sub1 with submerged tolerance and adaptability for the Mekong Delta

back-3 Scientific and practical significances

3.1 Scientific significances

- The results of the thesis project will provide scientific data in gathering

QTL/target gene Sub1 by MABC method applied to breeding submerged tolerance

rice varieties

- The research results of the thesis are also a reference for teaching, learning and scientific research, contributing to supplementing and perfecting the method of accurately assessing the presence of target genes on the expression of flood tolerance for a new rice variety with improved submerged tolerance

3.2 Practical significances

- Successfully selecting and creating submerged tolerance rice variety OM351 by MABC method has opened up the possibility of wide application in the improvement of rice varieties to improve the resistance to biotic and abiotic stresses

of the cultivars which are in mass production

- The OM351 rice variety has agro-biological characteristics similar to AS996 but has a higher yield than the AS996 variety, especially with submerged tolerance, which will contribute to the diversity of rice varieties to be cultivated for low-lying and flooded areas in the Mekong Delta provinces

4 Research materials and scope

4.1 Research materials

Rice variety AS996 which is popular cultivated in the South provinces Rice

variety IR64-Sub1 having Sub1 locus gene, this variery is imported from

International Rice Research Institute (IRRI)

4.2 Research scope

- The thesis focuses on crossbreeding and using molecular markers to identify

individuals carrying the Sub1 locus gene and the genetic background of the AS996

variety in breeding generations

- Evaluation of submerged tolerance, agro-biological characteristics, yield potential and adaptability and development of new varieties in some representative provinces of southern ecological regions

The study period was from 2010 to 2017

5 New contributions of the thesis

- Analysis of polymorphism between AS996 and IR64-Sub1 varieties identified

71 polymorphic markers Among them, 69 markers used to screen genetic

background of the ASS996, the two markers ART5 and SC3 are located in the Sub1 locus gene to select individuals carrying the Sub1 locus in the backcross

populations

- Applying the MABC method has successfully improved, selected and created the submergence rice variety AS996-Sub1 (newly named OM351) The OM351 vaeiety has agro-biological characteristics, quality and resistance to pathogen and diseases similar to AS996, higher yield than AS996, especially with 14-days submerged tolerance ability

6 Thesis structure

The thesis consists of 154 pages including 6 parts: Introduction (4 pages); Chapter 1 Literature Review (36 pages); Chapter 2: Materials, Contents and Research Methds (20 pages); Chapter 3: Research Results and Discussion (72 pages); Conclusions

and Recommendations (2 pages); List of published works related to the thesis (1 page); References (19 pages) included 25 Vietnamese documents, 121 English documents and 5 websites The thesis has 41 data tables, 32 figures, 3 appendices and 3 published works

Chapter 1 LITERATURE REVIEW 1.1 Impact of climate change to agricultural production

According to the assessment of the Intergovernmental Panel on Climate Change (IPCC), Vietnam is one of the five countries most affected by climate change In the climate change scenario to the year 2100: 1,8 million hectares of rice land are affected by climate change; accounting for 91,8% of the total area of rice land in the whole region; 1,8 times higher than the climate change scenario in 2030; increased 2,2 times compared to 2020 Accordingly, the rice cultivation area lost 2,2 million hectares and the damaged rice output corresponds to 55,6% of the total rice production of the region The most affected rice cultivation areas are the key rice-growing provinces in the Dong Thap Muoi region, the Long Xuyen Quadrangle such as Kien Giang, Long An, An Giang, and Dong Thap The provinces that lost the most rice production were Kien Giang, Soc Trang, and Tra Vinh (Huong and

Pathirana, 2013)

1.2 Study on the mechanism of submerged tolerance of rice

Under flooded conditions, the morphological changes in the roots and shoots were affected, the factors involved in the plant growth were affected, such as number of

branches, plant height, relative growth rate, internode length, chlorophyll a, b

When flooding happened, the oxygen content of the water falls below the level suitable for plant growth, respiration changes from aerobic to anaerobic causing an energy crisis It is the anaerobic fermentation that leads to the accumulation of cytotoxic compounds In the condition of complete flooding, the plant's organs suddenly reduce the rate of gas diffusion, limiting the introgression of oxygen and thus photosynthesis is also reduced, the process of respiration increases rapidly, taking up all the stored carbohydrates, leading to plant death (Das et al., 2009) The higher photosynthetic capacity in flood-tolerant cultivars is due to the protection of photosynthetic organs, such as higher chlorophyll content, better stomatal conductivity after flooding (Panda and Sarkar, 2017) The good flood tolerance mechanism is also related to the accumulation of unstructured carbohydrates

The unstructured carbohydrate composition is necessary for the survival of the plant under adverse conditions Carbohydrate concentration before flooding is considered

a key trait for survival under flooded conditions (Panda and Sarkar, 2014) The carbohydrate stored in the plant is used to provide the energy needed to sustain the plant while submerged Research groups have shown that flooded varieties contain 30%-50% of carbohydrate reserves (Afrin et al., 2018) Plant hormone components such as ethylene gas, gibberellic acid, and abscisic acid are considered to play important roles through synergistic and antagonistic actions for plant survival under

flooded conditions (Huang et al., 2019) ) The activities of ethylene and gibberellic acid stimulate prolonged plant growth under flooded conditions (Voesenek et al., 2013; Goswami et al., 2017) Ethylene increases the response of growth tissues to gibberellic acid by reducing abscisic acid, a potent antagonist of gibberellic acid, which enhances the plant's tolerance to flooding (Du et al., 2014; Locke et al., 2018 ) Experimental inhibition of gibberellin biosynthesis before flooding increased the survival rate of plants that were flooded many times Plant hydrogen peroxide (H2O2) plays a key role in flooding, as a signaling molecule that regulates various physiological processes (Fukao et al., 2019) This molecule reduces inhibitory levels in low light, and can alter expression levels of hundreds of genes in plants (Yun et al., 2010) Peroxidase activity in rice leaf sheaths increased from pre-inundation to post-flood in all tested varieties This increase was stronger in tolerant varieties

The molecular mechanism of "submergence" is controlled by the Sub1A gene,

formed in response to elevated ethylene levels, which accumulate during flooding,

to inhibit both ethylene production and gibberellic responsiveness acid (GA), leading to a long-lasting inhibition during submersion The decreased GA responsiveness is due to the accumulation of Della proteins such as slender rice 1

(SLR1), which inhibits GA responses (Fukao and Bailey-Serres, 2008) The

Sub1A-1 gene increases brassinosteroid (BR) content after flooding by downregulating the

transcription of the BR fusion gene, and this causes GA repression by metabolism

of the GA-inactivating enzyme OsGA2ox7 (Schmitz et al., 2013) Furthermore,

recent work has shown that Sub1A suppresses carbohydrate and nitrogen

metabolism and controls responses to leaf growth-regulating hormones under flood stress (Alpuerto et al., 2022) Tolerance to full submergence is a genetic trait that helps rice plants to recover from full submersion (10-14 days)

1.3 The method of selection by molecular markers combines backcrossing and its application in plant breeding

DNA molecular markers are markers that are polymorphic in nature In this study, the SSR (Simple Sequence Repeats) indicator is used, which amplifies the simple repeats or microsatellites Microsatellites are sequences of DNA that repeat in an orderly fashion, consisting of repeating units from 2 to 6 nucleotides, in a short repeating pattern and several dozen times The advantage of the SSR directive is that it is easy to implement and less expensive SSR is a co-dominant marker capable of detecting very high polymorphisms and is species-specific SSR markers are accurate and effective in studying genetic diversity, in analyzing organism genomes, building genetic maps of gene linkages, and selecting for resistance/tolerance to some biological and abiotic traits, learning to apply in breeding

The method of selective breeding by molecular markers combined backcross (MABC) has been widely used in recent years and has brought many successes in plant breeding The aim of MABC is to transfer one or several genes/QTLs of interest from a genetic material source into elite lines/varieties to improve the target

trait (Neeraja et al., 2007), MABC applied based on DNA markers that have a relationship or association with the gene/QTL of interest instead of just phenotyping the target trait like traditional backcross methods Recipient plant genomes can rapidly combine target gene selection and combined genetic background selection using traditional backcross methods (Hasan et al., 2015) The MABC breeding method is very important, supporting traditional selection and determining the success of the breeding process (Collard et al., 2005) There have been many researches on applying the method of using gene-linked molecular markers in plant breeding, and achieved success in the world and in Vietnam

1.4 Studies on the application of molecular markers in the selection and breeding of submerged tolerant rice varieties

The International Rice Research Institute mapped the QTL associated with the flood tolerance trait (Sub1) located on chromosome 9, and successfully introduced 6 rice varieties for flooded areas by MABC breeding The research team at IRRI has selected and released several QTL/Sub1 gene carriers in 6 countries including 4 varieties in Indonesia, 2 varieties in Nepal, 1 variety in Burma, 2 varieties in India and 2 varieties in Bangladesh and 2 varieties in the Philippines In Indonesia, in two years, Sub1 gene was successfully transferred into two rice varieties Ceherang and PSB RC18 by MABC method (Septiningsih et al., 2015)

In Vietnam, Dao Van Khoi has applied a molecular marker to breed flood-tolerant SHPT2 with Sub1 gene from Khang Dan 18 rice variety, which has been recognized and put into production in Vietnam (Dao Van Khoi, 2019) Most of the other results have only been recorded at the initial level of success in crossing over and checking the presence of the Sub1 gene locus in lines, creating breeding materials or promising lines (Ta Hong Linh, 2012; Cuc et al 2012, Luu Thi Ngoc Huyen et al., 2014)

In summary: MABC breeding method is currently one of the most effective methods in improving the breed with the desired trait through the integration of target genes into the genome The development of broad-adaptive rice varieties that respond to changes in biotic and abiotic stresses increased by climate change is needed Researches on improving rice varieties in the world as well as in the country are always aimed at that In fact, in recent years, flooding is one of the leading and most serious challenges facing of the rice production industry in Vietnam Therefore, improving and enhancing the submerged tolerance of popular rice varieties outside of production in the southern provinces is one of the issues that need to be prioritized today

Chapter 2 RESEARCH MATERIAL, CONTENTS AND METHODS

2.1 Research Materials

The rice variety AS996, receiving the tolerance gene, is widely grown in production

in the southern provinces The rice variety IR64-Sub1, carrying the Sub1 -

submergence locus gene, was imported from the International Rice Research

Institute (IRRI) The use of 400 SSR molecular markers, as well as agricultural materials and specialized materials in molecular biology

Content 3 Evaluation of submergence tolerance, agro-biological characteristics and yield potential of some promising rice lines/varieties in selective generations Content 4 Production test and author’s test of submerged tolerance rice variety AS996-Sub1 (OM351) in some southern provinces

2.3 Research Methods

2.3.1 Evaluation of starting materials for the improvement of submergent tolerant rice varieties

2.3.1.1 Material source evaluation

* The experiment to evaluate the materials was conducted with 8 rice varieties

a Experimental design: The experiment was arranged in a randomized complete block (RCB) with 3 replications Area of each experimental plot: (5 m x 2 m) = 10

m2 Total experimental area: 10m2/plot x number of test cells x 3 replicates (excluding protection band) Implementation period: Fall-Winter 2010

b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Yield components (number of branchs/clump, number of firm seeds/branch, weight of 1000 seeds, percentage of empty seeds, actual yield)

* The experiment to evaluate the tolerance of materials was carried out with 8 rice varieties according to the method of Pamplona et al., 2007

a Experimental design: a completely randomized one-factor experiment with three replicates, 24 seeds/replication Conduct the assessment when the plant has developed a new leaf after 15-21 days of submergence, count the number of live plants

b Calculating submergent tolerance according to IRRI's scale (2013)

The rice varieties participating in the experiment were evaluated according to the following formula:

CS%= (S1%/S2%)*100

In which: CS% – survival rate; S1% – survival rate of the variety to be evaluated;

S2% – survival rate of the control variety carrying the Sub1 gene

Scale for assessing submerged tolerance based on CS%: score 1 (CS% = 100 or more); point 3 (CS% = 95-99%); score 5 (CS% = 75-94%); score 7 (CS% = 50-74%); score 9 (CS% = 0-49%)

2.3.1.2 Evaluation of broodstock in the selection and breeding of submergent tolerant rice varieties

a Experimental design: The experiment was arranged in a randomized complete block (RCB) with 3 replications Area of each experimental plot: (5 m x 2 m) = 10 m2 Total experimental area: 10m2/plot x number of test cells x 3 replicates (excluding protection band) Implementation period: Winter-Spring 2010-2011

b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Yield components (number of branchs/m2, number of firm seeds/branch, weight of 1000 seeds, actual yield)

2.3.1.3 Determination of molecular marker on 12 chromosomes for polymorphisms between IR64-Sub1 and AS996

a DNA extraction and purification techniques

Rice DNA was extracted and purified by CTAB method (Laboratory of Genetics, University of Ghent, Belgium)

b Survey of polymorphism between the two parent breeds

A total of 400 SSR markers on 12 chromosomes were selected

*PCR with SSR primers were performed in 0.2 ml eppendorf or 96-position PCR plate and amlified on an Eppendorf Master Cycle Pro S machine

* Electrophoresis analysis of PCR results on polyacrylamide gel:

* Recording results: for each SSR marker, if the DNA band size is different between the 2 surveyed varieties, it will be recorded as having polymorphism and will be used for screening for resistance genes or genetic background in the folowing steps

2.3.2 Crossbreeding and selection of crossbred individuals carrying both the Sub1 submerged tolerance locus gene and the AS996 genetic background by molecular markers and backcrossing (MABC)

2.3.2.1 Method of sexual hybridization, back-crossing

Conduct crossbreeding between variety AS996 (mother) with variety having

Sub1 tolerance locus IR64-Sub1 (father) Using hybrid generations F1, BC1F1,

BC2F1 backcrossed with AS996 to create hybrid population BC1F1, BC2F1, BC3F1 respectively

2.3.2.2 Selection of crossbred individuals carrying the Sub1 locus gene and the genetic background of AS996 using SSR molecular markers

Sowing F1 hybrid seeds of generations BC1F1, BC2F1, BC3F1 The F1 hybrid seeds were grown until 20 days old, then DNA extraction was carried out, the

presence of Sub1 locus gene was checked, and the genetic background of AS996

received by polymorphic molecular markers that were found in previous step

Select individuals having Sub1 locus gene and largest genetic background of AS996

in each generation aimed to create BC1F1, BC2F1, BC3F1 populations

Experimental steps were carried out as in section 2.3.1.3 Data were recorded and processed on Graphical genotypes 2 software (GGT2.0) and other statistical analysis methods The regulation was that, the DNA band of IR64-Sub1 variety was

A, the DNA band of AS996 variety was B, carrying both bands in the heterozygous state was H

2.3.3 Evaluation of submergence tolerance, agro-biological characteristics and yield potential of some promising rice lines/varieties in selective generations

2.3.3.1 Evaluation of submerged tolerance of some rice lines/varieties in BC 3 F 3

breeding generation

- Using the method of Pamplona et al., 2007 (mentioned in section 2.3.1.1) to evaluate When the plants get 1 more leaf during the 15 day recovery period, on the 21st day, evaluate the results by counting the number of live plants and calculate the survival rate according to the formula as in section 2.3.1.1

2.3.3.2 Evaluation of some agro-biological characteristics and yield potential of some promising rice lines/varieties from BC 3 F 3 to BC 3 F 5 generations

a Experimental design: The experiment was arranged in a randomized complete block (RCB) with 3 replications Area of each experimental plot: (5 m x 2 m) = 10

m2 Total experimental area: 10 m2/plot x number of test plots x 3 replicates (excluding guard area) Implementation period: Fall-Winter 2013; Winter Spring 2013-2014 and Summer Fall 2014

b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length); Level of infection with pathogens and diseases; Yield components (number of branchs/clump, number of firm seeds/branch, weight of

1000 seeds, percentage of empty seeds, actual yield)

c Cultivation technique:

- The soil is carefully made by machine, picking up weeds, leveling, ensuring there

is no stagnant water, creating trenches around to drain

- Sowing technique: planting density of 40 clumps/ m2

- Fertilizer method: + Primer: 100% organic fertilizer + 100% phosphorus + 25% urea + Promote: Phase 1: 7-10 days after sowing: apply 40% urea Phase 2: After phase one 10 days: apply 20% urea + 40% potassium Phase 3: When the rice is 1-2

cm long, apply 15% urea + 60% potassium

- Pest control: applied according to the scale of technical regulation 55:2011/BNNPTNT (National regulation on testing the value of cultivation and use

QCVN01-of rice varieties)

d Method of traits evaluation: according to "National technical regulation on testing the value of cultivation and use of rice varieties" (QCVN 01-55:2011/BNNPTNT)"

2.3.3.3 Evaluation of submerged tolerance, comparison of rice varieties under normal and submerged conditions

* Under normal conditions:

a Experimental design: The experiment was arranged in a randomized complete block (RCB) with 3 replications Area of each experimental plot: (5 m x 2 m) = 10

m2 Total experimental area: 10 m2/plot x number of test cells x 3 replicates (excluding protection area) Implementation period: Winter-Spring 2014-2015; Summer Fall 2015 and Winter Spring 2015-2016

b Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length, arista neck release, plant stiffness, leaf canopy); Level of

infection with pathogens and diseases; Yield components (number of arista/cluster, number of seeds/arista, number of firm seeds, ratio of empty seeds, weight of 1000 seeds, theoretical yield, actual yield)

* In the condition of artificial flooding

a Experiment layout:

Choose a site right next to the experiment under normal conditions The experiment was arranged in a randomized complete block (RCB) design with 3 replicates and was arranged the same as for the experiment under normal cultivation conditions Planting density is 40 clusters/m2, 2 plants/cluster The time for the varieties participating in the experiment to be flooded was 10 days at the post-implantation stage (growth stage) Varieties are completely submerged at a depth of 50 cm After

15 days of water withdrawal, the rice plants create branches and new leaves appear, then calculate the survival rate after flooding according to the formula as in the section 2.3.1.1 mentioned above

b Monitoring indicators: Growth time; The height of trees; Survival rate of trees; Recovery time after flooding; Yield components (number of arista/cluster, number

of seeds/arista, number of firm seeds/arista, ratio of empty seeds, weight of 1000 seeds, theoretical yield, actual yield)

c Methods to evaluate the criteria: Evaluation of agro-biological characteristics, morphological characteristics, degree of pathogen infestation, yield according to IRRI (2002);

- Evaluation of the flood tolerance of seed lines according to the method of Pamplona et al., 2007 (mentioned in section 2.3.1.1);

- Evaluation of the Flood tolerance index (FTI) (Pham Thi Xuan et al., 2020):

determined according to each monitoring indicator, which is the ratio between the value of the same indicator in the treatment formula inundation compared with itself in the control formula

2.3.4 Production test and author’s test of submerged tolerance rice variety AS996-Sub1 (OM351) in some southern provinces

2.3.4.1 Production testing in the national variety testing system for crop product

a Experimental arrangement: Test at 7 sites (Tien Giang, An Giang, Co Do, Thoi Lai, Kien Giang, Binh Thuan and Tay Ninh) The experiment was arranged according to the randomized complete block method (RCB) with 3 replicates, the experimental plot area was 10 m2 (Gomez & Gomez, 1984) Implementation period: Winter-Spring 2014-2015; Summer Fall 2015

b Monitoring indicators: Similar to the experiment in section 2.3.3.3

+ Evaluation of rice and rice quality: Analysis of the percentage of flipped rice, head rice ratio, and grain size according to TCVN1643:2008; Analysis of gelatinization temperature according to TCVN5715:1993; Determination of amylose content according to TCVN5715-2:2008 and ISO 6647-2007; Determination of protein content according to TCVN 8133-2: 2011 and Iso/TS16634-2: 2009; Evaluation of rice quality according to TCVN8373:2010

2.3.4.2 Experiment of rice variety OM351

a Location: 06 provinces in the Mekong Delta (Dong Thap, An Giang, Long An, Tra Vinh, Bac Lieu, , Ben Tre)

b Implementation period: Summer-Autumn 2016; Winter-Spring 2016-2017 and Summer-Fall 2017

c Experimental arrangement: The experiment was arranged in both normal and flooded conditions Flood tolerant rice variety OM351 was planted next to control variety AS996, with an area of 1500 m2 The density of transplanting is 40 clusters/m2, transplanting 2 plants/hole

- Fertilizer for 1 hectare including 8 tons of organic fertilizer + 110kg N + 90 kg P2O5 + 90kg K2O Apply all organic fertilizers and phosphate fertilizers with 50%

N, 30% K2O Apply the second time when the rice takes root and turns green with 40% N + 40% K2O The remaining amount is applied in the pre-flowering stage about 20 days

- Prevention of pests and diseases and use of pesticides according to the guidelines

of the plant protection guidance in each locality where the trial is carried out

d Monitoring indicators: Growth time; Agro-biological characteristics (plant height, arista length, arista neck escape, tree stiffness, leaf canopy); Level of pest and disease infection; Yield components (number of arista/cluster, number of seeds/arista, number of firm seeds/arista, ratio of empty seeds, weight of 1000 seeds, theoretical yield, actual yield)

- Evaluation of agro-biological characteristics, resistance to pests and diseases in the field, yield components according to the standard system for evaluating rice genetic resources (IRRI, 2002)

- Actual yield is calculated according to the method of reaping 5 points diagonally, each reaping point is 50 m2 Calculating fresh yield, sampling 3 kg fresh yield for drying, measuring moisture content reach to 13%, calculating the ratio, converting fresh yield to dry yield Statistical processing data by pairwise comparison method (TTest) in Excel

2.3.5 Data processing methods

The data were synthesized by Excel 2013 The selection of submerged tolerancelines was determined through the results of experiments to evaluate growth, development and yield by the Selection Index according to Nguyen Dinh Hien Data of other experiments were analyzed and processed by software IRRISTAT 5.0; Statistix 8.2; analysis of variance (ANOVA) Genotypic data of molecular markers with lines in the selected population were recorded, collected and analyzed using Graphical genotypes 2 software (GGT2.0)

2.4 Time and place of study

- Research period: from 2010 - 2017

- Research location:

+ The experiments to determine the Sub1 tolerance loci and to screen the genetic

background by molecular markers were performed at the Laboratory of the

Molecular Biology Department - Institute of Agricultural Genetics (Co Nhue 1, Bac

Tu Liem, Hanoi)

+ Evaluation of material sources, artificial submerged tolerance, agro-biological characteristics of submerged tolerance rice lines/varieties in green house system,

fields belong to the Mekong Delta Rice Institute (Tan Thanh, Thoi Lai, Can Tho)

+ Testing submerged tolerance rice varieties in the national testing system in the provinces representing 2 ecological regions: Mekong Delta (Tien Giang, An Giang,

Co Do, Thoi Lai, Kien Giang), Southeast region (Binh Thuan, Tay Ninh)

+ Authorization trial of submerged tolerance rice varieties AS996-Sub1 (OM351) tested in mass production in Dong Thap, An Giang, Tra Vinh, Bac Lieu, Long An, and Ben Tre provinces

Chapter 3 STUDY RESULTS AND DISCUSSION

3.1 Evaluation of starting materials for the improvement of submerged tolerance rice varieties

3.1.1 Evaluation of material resources, selection of parent varieties

3.1.1.1 Evaluation of material resources

The experiment was conducted on 8 varieties, they were INPARA3, IR5S713-2B-8-2B-1-2, IR64-Sub1, BR11-Sub1, PSB-Re68-Sub1, Samba Mahsuri-Sub1, AS996 and IR42 The experimental results shown that: the variety with short growing time, high yield in the imported variety group, with the highest submergence tolerant among the tested lines/varieties is IR64-Sub1 (yield 57,1 quintal/ha) Combined with the results of the assessment of submergence tolerant, the variety IR64-Sub1 has the highest submergence tolerant among the tested rice lines/varieties (point 1) with 100% survival rate after the experiment, play the standard tolerance role, and at 78,9% compared to itself The nature of IR64-Sub1

is an IR64 variety that carries the QTL/ submerged tolerance gene (Sub1) which is

currently being grown quite popularly in some South and Southeast Asian countries, including Vietnam IR64-Sub1 variety has good genetic background, wide adaptability and stable yield over the years, especially has the best submerged tolerance compared to the imported variety that has been tested in artificial

conditions It is the most suitable variety to use as the donor for Sub1 tolerance

gene The rice variety AS996 is not submerged tolerance, but is suitable in the Mekong Delta region, giving the highest yield (58.3 quintals/ha) of the surveyed

varieties, suitable for use as the recipient Sub1 tolerant gene in the study for

breeding rice varieties that are submerged tolerance

3.1.1.2 Results of evaluation of parental materials for breeding purpose

After determining the source of materials to use as donor variety (IR64-Sub1) and

recipient variety (AS996) for submerged tolerance gene Sub1, evaluate and survey

both varieties for agro-biological characteristics and productivity factor again The results of the survey and evaluation shown that: Regarding the growth time: the two parent varieties are in group A1 (short days), ranging from 102-110 days in the Autumn-Winter season and 98-103 days in the Winter-Spring season About tree height: variety IR64-Sub1 has a tree height (98.5cm) lower than that of AS996 (105.3cm) About arista length: variety IR64-Sub1 has a shorter arista length