Agronomic traits associated with drought tolerance of tropical - Derived germplasm for hybrid maize breeding

MINISTRY OF EDUCATION AND TRAININ MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES --------------------------------- DO VAN DUNG AGRONOMIC TRAITS ASSOCIATED WITH DROUGHT TOLERANCE OF TROPICAL-DERIVED GERMPLASM FOR HYBRID MAIZE BREEDING Specialized: Genetics and Plant Breeding Code: 9.62.01.11 SUMMARY OF AGRICULTURAL DISSERTATION HANOI - 2018 The work was completed in: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Science advisor: 1. Ph.D. Le Quy Kha, Institute Of Agricultural Science for Southern Vietnam 2. Ph.D. Pervez Haider Zaidi, International Maize and Wheat Improvement Center (CIMMYT) in India. The dissertation would be defended on Foundation level examination board Vietnam Academy of Agricultural Sciences August, 2018 Dissertation can find out at: 1. Vietnam Academy of Agricultural Sciences 2. Maize Research Institute 1 GENERAL INTRODUCTION In Vietnam, drought is one of major constraints for maize production. Consequently, damage caused by drought was estimated more than 30%, up to 70- 80% or no harvest at all. Vietnam is one of the most affected countries by climate change and drought has been more and more regular and severe. Therefore, it is annually about 0.6 and 0.7 million ha maize area facing more stresses especially drought. Maize area, grain yield and production in Vietnam in 2017 was 4.6 tons.ha-1 respectively that it is lower than the global average (5.5 tons.ha-1). Meanwhile, the demand becomes more and more, leading to exceeding the supply Therefore, breeding drought tolerant maize varieties is the most important orientation for rainfed maize areas. Therefore, it is essential for the thesis “Agronomic traits associated with drought tolerance of tropical-derived germplasm for hybrid maize breeding” for increasing an efficiency in breeding gain may be crucial. RESEARCH OBJECTIVES Studies on agronomic traits associated with drought tolerant ability of some materials were carried out for selection of inbred lines and development of promising maize hybrids for production. SCIENTIFIC AND PRACTICAL MEANINGS OF THE THESIS - The meanings of science Providing more and more scientific data on phenotyping and evaluating combining ability of F2:3 BP progenies populations under managed drought and optimal condition, in combination with identifying genome regions associated with quantitative trait locus (QTLs) controlling drought tolerance at the early generations during improvement and development of drought tolerant maize varieties. - The meanings of practice + Based on the results of evaluating drought tolerance of 8 BP progenies at F2:3 generations [8 BP populations × testers] for selection and development of 9 inbred lines with good combing ability and drought tolerance, high yield under drought stress, higher efficiency in drought tolerant maize breeding. + Having identified 2 significant gene clusters, including the first on chromosomes 1 (bin 1.05-1.07), 7 (bin 7.01-7.03) and the other on chromosome 8 (bin 8.02-8.03), controlling anthesis- ilking interval, leaf senescence and grain yield relating to drought tolerance. 2 + Having developed 9 inbred lines (RA1, RA2, RA3 ... RA9) used as drought tolerant materials for breeding maize toward rainfed condition. + Having developed 2 promising hybrid combinations (LVN72, ÐH17-1) suitable for rainfed condition in Vietnam. NEW CONTRIBUTIONS OF THE THESIS Giving more data on phenotypes and quantitative trait loci (QTLs) on genome regions of chromosomes 1, 4, 6 7 and 8 associated with drought tolerance for development of germplasm and maize breeding for rainfed condition. Having developed 9 inbred lines with good combining ability, drought tolerance, high grain yield and initially introduced 2 promising hybrid combinations (LVN72, ÐH17-1) for production. OBJECTS AND SCOPE OF RESEARCH Researches on 8 F2:3 populations (790 families), crossed from 10 CIMMYT derived tropical inbred lines and test-crosses (2 testers called CML451, CLO2450), through which 9 best families were selected to develop into 9 inbred lines, crossed into 36 crosses by diallel. Local checks: in India these were PAC754, 30V92, HTMH5401 and 900MG; while using LVN10, VN8960, LVN61, NK67, C919, DK9901 in Vietnam. Experiments were carried out under managed drought and optimal conditions at International in Hyderabad, India and Ninh Thuan Province, Vietnam; Testing hybrid combinations were done in the northern region of Vietnam. ORGANIZATION OF THE THESIS The thesis contains 138 pages, 32 tables, 15 pictures and graphs with 5 sections general introduction (4 pages), chapter 1: Literature review (44 pages), chapter 2: Materials and methods (13 pages), chapter 3: Results and discussion (77 pages), and Conclusion and suggestion (2 pages); References of 201 documents with 29 Vietnamese ones and 169 others in English and 3 cited from websites; in which 2 research works published on Journal of Vietnam Agricultural Science and Technology and the other at the 12th Asian Maize conference in Bangkok, Thailand. Chapter 1. LITERATURE REVIEW 1.1. The global maize production situations and in Vietnam 1.1.1. World maize production Maize (Zea mays L.) have been of profound changes. Maize area, grain yield and production in 2010 higher increase than in 2005 by 9.8% area, 7.8% 3 productivity and 18.3% production. And there was, in 2015, an increase of 9.5% area, 5.27% yield and 15.3% production. In 2017/2018, the forecast of maize production will be 1,046 million tons, 42 million tons lower than in 2016/2017 (1,088 million ton). These data show that an increase in maize production has been slower for recent years but in the future, more and more will demands on maize be, mainly for feed and by 2050 doubled with 1,178 million tons and 194 million hectares. In fact, world maize areas, grain yield and production develop drastically but mainly in developing countries where maize cultivation still depends on rainfed conditions. Therefore, it is necessary to continuously improve drought tolerance of maize varieties for an increase in grain yield and production. 1.1.2. Maize production in Vietnam Maize production were obtained from 1995 to 2004, with annual increase in areas (5.3%), yield (4.8%) and production (10.7%) respectively. During 2005- 2015, domestic maize production still went on the same trend but more slowly that it was annually only 2.2% in grain yield, 2.0% in area và 5.0% in production. On the other hand, for more than 10 last years, the livestock gets an average increase of 8 - 12% per year, so maize must be imported for domestic demand and it was about 8,8 million tons in 2017 and as predicted, 10.5 million tons in 2018. Therefore, it is necessary to continuously improve grain yield, quality and drought tolerance of maize varieties. 1.2. Impacts of drought on maize production in the world and Vietnam 1.2.1. Impacts on global maize production More and more severely and unpredictably has climate change been taking place worldwide, in which drought can be considered as one of main factors. Annually, the loss of maize production due to drought is around 8%. By 2025, drought will have got more severe and regions will have become drier in all over the world but mainly in Africa and Asia. Impacts due to climate change, at present and in the future, can globally affect nearly 160 million hectares and reduce maize production by 6 - 23%. Therefore, in fact, it is essential to develop drought tolerant maize varieties remain stability and increase in production, satisfactory to more and more demands. 1.2.2. Impacts on maize production in Vietnam In Vietnam, drought is a major constraint for maize production, there is about 4 0.3 million ha facing the shortage of water and the loss of 0.5-0.7 million tons. Though, drought takes place in all 8 maize regions, in which the most severe drought stress in North Central Region, South Central Coast Region and Central Highland Region; intermediate stress in Mountainous Region, South East Region and the Mekong River Delta Region; moderate stress in the Red River Delta Region. Otherwise, global total surface water in 2025 will be about 96% of in 2010, by 2030 total surface water inflows at the upstream, deltas and the Red river valleys will have been reduced by 2.4%; 2.9% and 1.9%, respectively and water shortage will become serious in 50 years. Hence, breeding maize with high yield, drought tolerance and stability is really crucial for enhancement of maize production. 1.3. Research and usage of drought tolerant maize varieties 1.3.1. Research and usage of drought tolerant maize in the world Over the past 38 years, breeders have been selecting and improving drought tolerance in maize. The results of having developed drought tolerant maize since 2008 were summarized: breeding gain with traditional methods is 50 kg.ha-1 (equivalent to 1.4% per year) while with marker assisted selection- MAS and gene transfer methods are 20 kg.ha-1 (equivalent to 0.6%) and 30 50 kg.ha-1 (equivalent to 0.7%) respectively. Studying characteristics of active genes or molecular markers closely related to genes controlling drought tolerance is an important step in applying the selection of genotypes in improving drought tolerance in maize. 1.3.2. Researches of drought tolerant maize in Vietnam Since 1990s, there has been of full researches on drought tolerance at the stages of maize (from the seedling to flowering); During 1988-1998, studies were focused on high plant density, anthesis-silking interval (ASI), leaf senescence, grain yield and ect; Recently, applying biotechnology in maize breeding, in which, mainly focusing on two fields: tissue culture and recombinant DNA technology for an improvement in grain yield has been developed. Identifying molecular markers relating to drought tolerance such as Dhn gene helped select drought tolerant germplasm exactly. Hence, it may be concluded that combining conventional and modern approaches is a basis for breeding maize varieties tolerant to drought in Vietnam. 1.4. Scientific basis of drought and drought tolerance in maize 1.4.1. Definitions of drought Drought is a harsh condition and the consequence of the shortage of rainfall or no rain for prolong more than crop season or no enough water. Drought is classified as 5 following: In low land tropics a marginal rainfed maize environment may be defined as having seasonal precipitation below 500mm; in highlands seasonal precipitation below 350 mm; During bracketing- flowering stage, less than 100mm rainfall is considered as drought, between 100-200 mm as being marginal for maize production. 1.4.2. Maize under drought environment Though, every crop stage of maize has some susceptibility to drought, however, three stages of early growth stage (when plant stand are established), flowering and mid-to-late grain filling stage, are considered critical stages to drought, especially at flowering. Drought leads to reducing the growth of leaf, silk, stem, root, grain yield. 1.5. Genetics of drought tolerance in maize Drought tolerance in maize is controlled by multiple genes (multigene) and their environment. Maize can express drought tolerant ability in many ways such as drought avoidance and tolerance during growth and development in order to reduce grain yield loss. The heritability, the correlation between parents and generations, helps predict heterosis based on the correlation of secondary traits and grain yield. The importance of secondary traits for breeding maize for drought tolerance is their correlation with grain yield, in which, it should be more focus on traits of anthesis-silking interval, ear per plant, leaf senescence and ect. 1.6. Useful traits for breeding maize tolerant to drought Some secondary traits are used for maize breeding for drought tolerance: 1) Leaf rolling at crop stage of seedling to pre-flowering; 2) root system; 3) Ears per plant; 4) Anthesis-silking interval (ASI); 5) Leaf senescence (SEN); 6) stay- green; 7) Shelling percentage; 8) efficient ear length; 9) Grain yield. 1.7. Application of molecular assisted selection 1.7.1. Application of molecular assisted selection for maize breeding Since the beginning of the 20th century, marker assisted selection (MAS) has been a useful tool in maize development and improvement. This approach enables breeding based on genotypes, so markers associated with one or many genes controlling interested traits through which it is able to identify germplasm with stress tolerant genes. Now, applying quantitative trait loci (QTLs) mapping on genome regions of chromosomes helps exactly identify anticipated materials and save time. 1.7.2. Single nucleotide polymorphism (SNP) Single nucleotide polymorphism (SNP) called as "snips", is the most popular type of genetic variation. Each SNP represents a variation in a single nucleotide 6 that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g.> 1%). 1.7.3. QTL Mapping and genetics of quantitative traits A quantitative trait locus (QTL) is a region of DNA which is associated with a particular phenotypic trait, which varies in degree and which can be attributed to polygenic effects, i.e., the product of two or more genes, and their environment. To define genes controlling traits should be based on the combination of genotypic and phenotypic analysis at segregating populations while mapping QTLs based on mathematical models. 1.7.4. Improvement by conventional approaches and QTL mapping By effectively using genetic diversity, developing various elite lines, particularly through recurrent selection for interested genotypes in heterozygous and recombinant populations (F2, F2:3). The combination of traditional methods, phenotypic assessments under different environments and assistance of advanced biotechnology tools (such as SNPs) showed, through each selection cycle, grain breeding 7% under optimal conditions and 1% under drought stress and also an increase in the frequency of useful alleles, from 0.51 (at C0 cycle) to 0.52 (at C2 cycle). 1.8. Combining ability Combining ability in crosses, including general combining ability (GCA) and specific combining ability (SCA) is defined as the ability of parents to combine amongst each other during the process of fertilization so that favourable genes or characters are transmitted to their progenies. Evaluating combining ability by top- cross method is to determine GCA and playa a very important role at the early stage of selection when materials is too numerous. Diallel cross method is used for evaluation of GCA and SCA of parental lines through which elite lines with high combining ability and hybrids are selected. Besides, applying GGEBiplot for evaluating the interaction of genotype with environment and determining combining ability gives crucial indices: GCA, SCA effects of parents; Heterotic groups; The best hybrids with high combining ability; The best lines . Chapter 2. MATERIALS, ACTIVITIES AND METHODOLOGY 2.1. Materials for development of F2:3 BP populations From 10 CIMMYT derived tropical lines showed in Table 2.1. These lines were divided into 2 heterotic groups: Group A including elite lines P1, P2, P3, P4 7 (male parents) and line P9 (drought tolerance, the female); Group B: lines P5, P6, P7, P8 (as the male parent) and P10 (drought tolerance, the female). Lines P9, P10 were crossed with others in the same group into 8 F1 populations: P9×P1, P9×P2, P9×P3, P9×P4 và P10×P5, P10×P6, P10×P7, P10×P8. Selfing F1 plants to develop 8 F2 populations, randomly selecting each cob and 100 cobs per each population and establishing 790 families. Continue selfing these families into F2:3 generations with the total of 790 F2:3 families represented in Table 2.2. Table 2.1. Elite and tolerant lines Table 2.2. Information of 8 populations and 790 F2:3 families Notice: ǂǂPopulations developed from Bi-parents method 8 2.2. Materials 2.2.1. Materials evaluate agronomic traits and identify QTL associated with drought tolerance of 8 F2:3 BP populations under drought stress and optimal condition at India 8 F2:3 progenies populations were developed from each of Bi-parent cross between drought tolerant lines P9, P10 with the elite (P1, P2, P3, P4 of Group A; P5, P6, P7, P8 Group B) of CIMMYT (Table 2). Crossing with 2 testers CML451 (T1) and CMLO2450 (T2). Local checks: LVN10 (ĐC1), VN8960 (ĐC2), NK67 (ĐC3), C919 (ĐC4), LVN61 (ĐC5). 2.2.2. Materials for evaluating and testing hybrid combinations of 8 populations with 2 testers (CNL451, CLO2450) in Ninh Thuan province. Including 1,605 entries: crossing 8 BP populations (790 F2:3 families) × 2 testers (CML451, CLO2450) = 1.580 hybrid combinations; 20 hybrid combinations of 10 parental lines × 2 testers; 5 Local checks (LVN10, VN8960, NK67, C919, LVN61). 2.2.3. Materials for researching combining ability, heterosis and drought tolerance and grain yield of 9 elite maize lines under severe, moderate drought stresses and optimal condition at Hyderabad, India. Including 36 diallel hybrid combinations of 9 lines and 4 local checks (PAC745, 30V92, 900MG, HTMH5401). These lines were original from 9 F2:3 families selected based on the results of testing agronomic traits, crossed and developed into inbred lines and named RA1, RA2, RA3, RA4, RA5, RA6, RA7, RA8, RA9. Table 2.3. The list of 9 lines and 36 diallel hybrid combinations 9 2.2.4. Materials for testing maize varieties: Hybrid combinations RA2/RA8 named LVN72, RA4/RA7 named ÐH17-1. 2.3. Research contents - Evaluate agronomic traits and identify QTL associated with drought tolerance of 8 F2:3 BP populations under drought stress and optimal condition; - Evaluate combining ability of 8 F2:3 BP populations and select some elite lines and promising hybrid combinations; - Testing promising hybrid combinations. 2.4. Time and research locations 2.4.1. Research locations - At CIMMYT, India: trials of testing 8 populations (790 families F2:3), 10 parental lines carried out on farm under drought stress and well-watering condition; trials of 9 lines (RA1, RA2 ... RA9) were crossed with diallel method in the field under severe, moderate drought stresses and optimal condition. - In Vietnam: trials for testing 8 populations x testers in the field under managed drought stress and optimal condition; Testing promising hybrid combinations LVN72, ĐH17-1. 2.4.2. Timelines/Milestone During 2011- 2014 in India: testing 8 populations and parental lines in the field and evaluating diallel crosses; During 2012-2017 in Vietnam: evaluating 8 populations x testers and testing promising hybrids. 2.5. Research Methodology 2.5.1. Experimental designs Under the guidance of CIMMYT and Maize Research Institute of Vietnam. 2.5.2. Methodologies of experimental evaluation in the field - Experimental designs of evaluation in the field under drought stress and well- watering conditions implemented by irrigation management models: well- watering; drought; re-irrigation (as CIMMYT guidelines). -Testing varieties according to QCVN01-56: 2011/BNN&PTNT. - Evaluating agronomic traits under guidelines of CIMMYT and Maize Research Institute of Vietnam. 2.5.3. Methodologies for genotyping and QTL linkage mapping DNA extraction and linkage analysis and QTL mapping were carried out under the guidance of CIMMYT with interval mapping m