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Doctoral thesis summary of agriculture: Research and application of molecule markers in breeding salttolerant Bacthom 7 rice variety MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DONG THI KIM CUC “Research and application of molecule markers in breeding salttolerant Bacthom 7 rice variety” Major: Genetics and Breeding Code: 62.62.01.11 DOCTORAL THESIS SUMMARY OF AGRICULTURE HaNoi 2014
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES DONG THI KIM CUC “Research and application of molecule markers in breeding salt-tolerant Bacthom 7 rice variety” Major: Genetics and Breeding Code: 62.62.01.11 DOCTORAL THESIS SUMMARY OF AGRICULTURE HaNoi - 2014 The Doctoral thesis was completed in: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisos: 1. Assoc.Prof. Le Huy ham 2. Dr. Le Hung Linh Objection1: Objection2: Objection3: The Doctoral thesis is defelded at Institute Committee of PhD Dissertation Examination: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES At……day…….month ………… 2014 PhD thesis can be found at: - Nationa Library of VietNam. - Library of VietNam Academy of Agricultural Sciences 1 INTRODUCTION 1. Imperativeness of the thesis Rice production and yield are significantly losses due to diseases and pests infestation and the environmental impacts. Of these, the noticeable factor is salt-influenced to rice. The cultivated lands have been affected from the salinity by 380 million ha, accounted for 1/3 total worldwide cultivated areas. Salt – affected lands is the main factor which has curbed to develop productivity of rice, and also caused influence to food security in general. Hence, to reduce the salinity affect to the rice plant has paid much attention to research [124]. To meet this demand, to generate the salinity tolerance rice variety is necessary work. It is needed to explore the natural plant resource against salinity tolerance by either directly selecting or by genetic selection, or marker assisted selection. Application of molecular markers may help to identify the present of salinity tolerance gene(s), which is very useful for the breeder to select the effective crossed combination. Hence, It would be accelerated to breed rice salinity tolerance, shortened the selection time, and expenses and labour. As aforementioned, the topic entitled “Application the molecular markers to improve salinity tolerance of Bac Thom 7” 2. Objectives 2.1. General objectives Study on evaluating and developing some salinity tolerance of rice which derived from the the IRRI and India, and some grown rice varieties in Vietnam were used in this thesis. Applying marker assisted backcrossing to improve salinity tolerance of rice which are adapted for the Red River Delta. 2.2. Specific Objectives Identifying the salinity tolerance and phenotype of the rice lines which carrying the Saltol locus (donor plant), imported from International Rice Research Institute, as well as selecting the polularly grown rice cultivar were used as the receipient plant Applying marker assisted backcrossing (MABC) to pyramid salinity tolerance Saltol locus into Bac Thom 7, in order to generate the high quality rice variety with salinity tolerance for growing the coastal areas in Red River Delta 3. Scientific and Practical Significance 3.1. Scientific Significance 2 Based on the successful archivements of application of marker assisted backcrossing to transfer salinity tolerance QTL into other rice varieties will be widely applied for rice breeding to cope with climate change in the foreseeable future. Application of molecular breeding to combine with the traditional breeding to accelerate and identify the salinity tolerance rice materials, to pyramid it into Bac Thom 7 which could help to overcome the constrains of traditional breeding, especially with the heterzygote salinity tolerance QTL, reducing the cost of experiments, shortening the time and rapidly applying in practice. 3.2. Practical Significance The success of Saltol transfering into Bac Thom 7 based on molecular breeding will be widely applied for rice molecular breeding. The improved Saltol salinity tolerance of Bac Thom 7 lines would be selected and grown in a larger scale, especially for the coastal areas in the north of Viet Nam, where the most adversed influence from the climate change are. The most other significance of the current thesis was to simultaneously develop the salinity tolerance line/variety with the highest genetic background of the Bac Thom 7 and carried the Saltol QTL. The newly improved lines would grow well in the salt areas. 4. Plant materials and the Scope study of thesis 4.1. Plant materials to study The inbred rice varieties carrying the salinity tolerance (Saltol) which were imported from the IRRI, and the inbred rice varieties are popularly grown in Vietnam, as well as using the related molecular markers in the current study. 4.2. Places and time to conduct experiments The experiments were carried out at the Molecular Biology Division, Agricultural Genetics Institute, (Tu Liem, Hanoi); and the Center of Technological Exchange and Extension (Thanh Tri, Hanoi), and Giao Thuy, Nam Dinh Province. Time period: From 2010 to 2013 5. Significant Contribution Application of molecular assisted backcrossing (MABC) is one of the initial research to improve Bac Thom 7 with salinity tolerance for growing in the coastal areas of Red River Delta. Applying MABC method which can be transferred the target gen/QTL in the other variety via 2-3 generations, while, traditional backcrossing has needed about 8 breeding generations 3 Application of molecular assisted backcrossing could pyramid the saltol into Bac thom 7 which also has carried the enough desire traits of Bac Thom 7, but can be grown in the salinity affected areas upto 6 ‰ 6. Structure of the Thesis The current thesis was presented by 159 pages, of which included 25 Tables and 31 Fingures, and separated into 4 chapters: Chapter I: An overview (50 pages), Chapter II: Materials and Methods (15 pages); Chapter III: Results and Discussion (88 pages); Chapter IV: Conclusion and Suggestions (2 pages). One hundred and ten literature references were used to cite for this thesis, in which there are 23 Vietnamese references and 89 English reference and 16 link webpage were also used. 4 CHAPTER I OVERVIEW AND SCIENTIFIC BACKGROUND 1.1. The adversed impacts from climate change to worldwide agricultural production and Vietnam 1.1.1. Adversed effects from climate change to worldwide agricultural production According to the report of FAO (2010), over 800 million ha of cultivated areas have been affected by salinity and 20%, approximately 45 million ha, have been also affected due to salinity penetration at different levels [38]. In Asia, if sea level rise will be at 1m, approximately 10.000 km2 cultivated and fishery areas will be influenced and become the salinity swarmp. 1.1.2. The effects of climate change to agricultural production in Vietnam Vietnam is among the most influenced by sea level rise. The scientists reported that when the sea level rise, some cultivated areas in Cuulong delta and Red River delta and some other coastal delta will be inundated by sea water, and the sea level rise will be more increase, the most effected areas will be Red River delta and Cuulong delta. 1.2. Salt affected land and salt affected areas in Vietnam 1.2.1. Salt affected land The land consists of 50-60% ratio of argillaceous. The land shows high tight level and poor absorbent level, tought patter, and chapped and difficult to do tillage. Because the salt land is composing of much Na+ under the NaCl dissovel, thus, the pressure of Na 2 SO4 endosmosis is so high that can be influenced to water and nutrition absobtion of the plant. Also, neutral and alkali in salt land are causing low activation of the microoganism 1.2.2. General introduction of the rice field affected salinity in Vietnam Accoring to Hoang Kim and Bien and Howeler (2003), in Vietnam, there are two larger rice field affected salt is: Red River Delta included some sub-area such as Thai Binh, Hai Phong, Nam Dinh, Ninh Binh, while approximately 1.8 to 2.1 millio ha of land have been affected by salinity where are located in Ca Mau, Bac Lieu, Ben Tre, Kien Gian, Tien Giang, Tra Vinh and Soc Trang Provinces. Most of cultivated land areas have been affected salinity and alum and flooding [4]. 1.3. Genetic reseaches on salinity tolerance of rice 1.3.1. Mechanism of salinity tolerance of rice • Phenomenom of salt prevention • Phenomenom of leaf to leaf patition • Phenomenom of re-absorbance • Tolerance ability by the tissues 5 • Moving from root to bud •Dillution influence 1.3.2. Genetics of Salinity tolerance 1.3.2.1. Reseach on genetics of quantitative traits of salinity tolerance According to Mishra et al (1998), the trait of salinity tolerance in plant is polygenic trait, negligible causing effect from the parental plant (recipient plant) because these genes are not located in the cytoplasm [72]. During development stage of rice, the plant height, and rice production under salt affection are controlled by the additative genes (Mishra et al 1990)[73]. 1.3.2.2. Research on salinity tolerance at molecular level Based on the QTL mapping, salinity tolerance is controlled by multi-genes. Some markers such as AFLP and STS have been used, the major gene has been identified and located on the chromosome 1 and named as Saltol. The QTL (quantitative trait loci) mapping has been applied in case of the target gen has been controlled by many genes (example as salinity tolerance trait) 1.3.3. Expression of salinity tolerance of gene Based on the morphological, physiological and biochemical characteristics of the experiment, we have observed the present of Saltol-rice varieties and the sensitive rice varieties (without Saltol) 1.4. Molecular markers and their application 1.4.1. Molecular marker Molecular markers (or DNA markers) are polymorphic markers. They are included the molecular flow of DNA or the sequence information which are available and transiting in the database or internet (for example, sequence of primers SSR, STS, RAPD, AFLP ). 1.4.2. Some popular markers-use RFLP marker ((Restriction Fragment Length Polymorphism) RAPD, AFLP, STS marker SSR (Simple sequenced repeat) 1.4.3. Some applications of molecular markers 1.4.3.1. Study on genetic diversity Research on genetic diversity is very important to help to evaluate the plant germplasm, animals and use them more efficiency. Especially, it is possible to estimate the hererosis between the parents (the pairs of parents carrying higher the genetic distance which could obtain more the heterosis properly. 1.4.3.2. Studying on genetic mapping Application of molecular markers in QTL linkage mapping, has been applied the statistical analysis which could identify the linkage between the markers and gen loci (Quantitative trait loci). QTL mapping included 6 the architecture of genome mapping which could be useful to search for relationship between the traits and the polymorphic markers, and provide the close distance between QTL and markers, respectively. 1.4.3.3. Studying on plant breeding Along with the advanced development of molecular breeding technologies, the breeders have paid more attention to the issue as Marker assisted selection (MAS), has implied to use the markers that have linkage with the interested QTL/genes in plant breeding programe 1.4.4. Application of Marker assisted backcrossing (MABC) MABCis an a practical and efficient technique in transferring the interested QTL/gene into the elite rice variety to generate the improved rice variety in a short time carrying the desire QTL/gene and attain approximately 100% genetic background of the elite rice variety: The breeding programe may only implement at the BC3 or even thought in BC2 generation, respectively. 1.5. Some archievements in improving rice salinity tolerance 1.5.1. Some results and archivements in research on rice salinity tolerance in the world During the year 1977 to 1980, International Rice Research Institute (IRRI), was successfully selected the good rice salinity tolerance such as IR42, IR4432-28-5, IR4595-4-1, IR463-22-2, and IR9884-54-3 with the yield at 3,6 tones/ha. Gregorio et al (2002)[45] developed TCCP226-2-49-B-B- 3 rice cultivar with high salinity tolerance ability. Some local rice varieties which were derived in the East Asia have often been high salinity tolerance such as Nona Bokra (India), Pokkali (Sri Lanka), Getu (India), SR26B, Damodar, Cheriviruppu, Pat and Solla (India), Ketumbar (Indonesia), Khao Seetha (Thailand). Some rice varieties were in the template (subtropical countries) such as Harra (Spanish), Agami (Egypt), and Daeyabyeo (Korea). Several Japonica rice varieties such as Moroberekan have high salinity tolerance, which were origined in the affected salt areas. This variety has been researched and used as the donor plant (salinity tolerance) and population mapping (Kim et al, 2009)[55]. The rice varieties were Oryza glaberrima, which are mostly grown in the West African show lower salinity tolerance ability to compare with the rice varieties (Oryza sativa) (Awala và cs, 2010)[2]. Recently, in 2013, researchers in IRRI have successfully developed the high-super salt tolerance that could be very useful for the farmers to grow this rice cultivar in the affected salt areas such as the coastal areas. 1.5.2. Application of molecular markers in improving salinity tolerance of rice The fine mapping of Saltol QTL was made on the chromosome 1 by the researcher groups (Gregorio 1997; Bonille et al 2002; and Niones 2004) which explained about 40-65% salinity 7 tolerance in rice [44][28][85]. Mohammadi – Nejad et al (2010) used 33 SSR polymorphic markers on the chromosome 1 “Saltol QTL” in order to identify the linkage and the utinity of the markers for rice breeding [76]. 1.5.3. Some results and archivements of research on salinity tolerance of rice in Vietnam 1.5.3.1. Use of the SSR markers which have tightly linked to Saltol QTL in rice breeding Application of molecular markers and anther culture to improve salinity tolerance of rice were conducted, total 72 rice lines were generated by anther culture (Lang et al, 2008). Also, Buu et al (2000) used 30 SSR markers to map the salinity tolerance trait in the F3 generation that including 257 segregation population from the crossed between IR28/Đoc Phung. 1.5.3.2. Improving the rice salinity tolerance Đo Huu At (2005) made mutation by Coban (Co 60 ) to generate CM1, CM5, [1]. Also, Dang Minh Tam et al (2003), reported that 10 rice line were developed from the local and high yield rice cultivars, shown a medium salt tolerance (3-5 point). Moreover, they showed high regenerative percentage in the NaCl culture at 1,0 and 1,5% [35]. Ngo Dinh Thuc (2006) applied the anther culture technique to create 8 soma variation liné from the OM576, IR64, Basmati and VD20 which could withstand salinity tolerace at 5 level at the test of rice seeding with EC = 12 dS/m [19]. 1.5.3.3. Screening salinity tolerance of rice From 1992 – 1995, Institute of Southeast Agriculture and Science reported that 14 potential rice cultivars involving salinity tolerance were selected as the following: Nep ao Gia; Trang Diep; Mong Chim; Mong Chim Roi; and Nep Bo Rieng [8]. Also, Cuulong Rice Research Institute has reported to attain 30 rice lines with promising in salinity tolerance as from 2000 to present. The Field Crops Research documented that M6 is a salinity tolerance which obtained from the crossed Bau Hai Phong/1548 during the year of 2001-2005. 8 CHAPTER III MATERIALS, CONTENTS AND METHODS 2.1. Materials The rice materials included: Total 14 rice lines/varieties carrying Saltol QTL salinity tolerance were imported from IRRI and some popularly grown rice varieties in Red River Delta Chemical argents and research facilities: SSR markers used: 447 markers Research instruments: Experimental tools of Agricultural Genetics Institute 2.2. Areas to conduct experiments The Laboratory of Molecular Biology Division-Agricultural Genetics Insititute. Tuliem-Hanoi The Net house and paddy fields conducted for experiment at the Center of Technology Exchange and Extension, Vinh Quynh, Thanh Tri, Hanoi Experiments for evaluation of growth and development of the imported rice lines/varieties were conducted in two provinces: Nam Dinh and Hanoi Perious to implement: From 2010 to 2013 2.3. Contents 2.3.1. Content 1: Research, evaluation of the salinity tolerance and agronomical traits, rice yield and some sub-traits involving in rice yield of the Salton-lines/varieties imported from IRRI and some popularly grown in the Red River Delta. It has been an important for further research on rice salinity tolerance for the coastal areas in North Vietnam 2.3.2. Content 2: Application of marker assisted backcrossing to transfer the Saltol QTL into Bacthom 7, an elite rice cultivar 2.3.3. Content 3: Evaluating some main agronomical traits and several components involving in rice yield traits, salinity tolerance level, rice quality of the lines carrying Saltol QTL in the net house and the paddy field test 2.4. Methods * MABC (Marker Assisted Backcrossing) to improve salinity tolerance of rice 2.4.1. Methods to conduct field test 2.4.2. Methods for evaluation of salinity tolerance of rice: Screening in the artificial conditions. 2.4.3. Methods to implement experiments in the laboratory . Research and application of molecule markers in breeding salt-tolerant Bacthom 7 rice variety Major: Genetics and Breeding Code: 62.62.01.11 DOCTORAL THESIS SUMMARY OF AGRICULTURE . polymorphic markers on the chromosome 1 “Saltol QTL” in order to identify the linkage and the utinity of the markers for rice breeding [76 ]. 1.5.3. Some results and archivements of research on salinity. tolerance of rice in Vietnam 1.5.3.1. Use of the SSR markers which have tightly linked to Saltol QTL in rice breeding Application of molecular markers and anther culture to improve salinity tolerance