In this article we will discuss about:- 1. Introduction to Application of Biotechnology in Rice Production 2. Phases for Biotechnology of Rice 3. Molecular Maps for Rice Genetics 4. Gene Tagging 5. QTL Mapping 6. Pyramiding Genes for Bacterial Blight (BB) Resistance 7. Cytoplasmic Diversification.
Contents:
- Introduction to Application of Biotechnology in Rice Production
- Phases for Biotechnology of Rice Production
- Molecular Maps for Rice Genetics
- Gene Tagging in Rice Production
- QTL Mapping for Production of Rice
- Pyramiding Genes for Bacterial Blight (BB) Resistance
- Cytoplasmic Diversification of Rice
1. Introduction to Application of Biotechnology in Rice Production:
Major advances have been made in increasing rice production as a result of large-scale adoption of modern high-yielding varieties and improved production technologies. The world rice production has more than doubled from 257 million tonnes in 1966, to 560 million tonnes in 1996. High-yielding varieties were mainly developed through the application of classical Mendelian principles and conventional plant breeding methods.
To meet the need of growing human populations, we will have to produce 70% more rice by the year 2025. To meet this challenge, we need varieties with higher yield potential. Moreover, several biotic and abiotic stresses continue to limit rice productivity. To overcome these constraints to rice production, varieties must have multiple resistances to diseases and insects, and tolerance of abiotic stresses.
Plant breeding consists of two phases – the evolutionary phase, where variable populations are created; and the evolutionary phase, where superior genotypes are selected on the basis of evaluation. Recent advances in cellular and molecular biology have provided scientific tools to increase the efficiency of both phases.
Some of the exciting developments in biotechnology include:
(i) Successful plant regeneration from protoplasts of several japonica and indica cultivars;
(ii) Production of somatic hybrids and cybrids through protoplast fusion;
(iii) Production of transgenic plants carrying agronomically important genes;
(iv) Development of a comprehensive molecular map consisting of more than 2000 DNA markers;
(v) Tagging, via linkage with molecular markers, several genes governing resistance to major diseases and insects;
(vi) Mapping of quantitative trait loci (QTL) governing several traits of low heritability;
(vii) Development of polymerase chain reaction (PCR)-based markers;
(viii) Development of protocols for marker-aided selection;
(ix) Determination of synteny relationships between genomes of rice and other cereals;
(x) Construction of bacterial artificial chromosome (BAC) libraries for physical mapping of the rice genome and their utilization in map-based cloning of genes; and
(xi) Transfer of useful genes from wild into cultivated rice across crossability barriers.
2. Phases for Biotechnology of Rice Production:
i. Evaluation Phase:
As early as 1968, Niizeki and Oono reported the production of haploids from anther cultures of rice. Since then, the anther culture technique has been greatly refined. The effect of the genotype, the physiological state of the donor plant, the stage of pollen development, the pretreatment of anthers, and the media composition for callus induction and efficiency of plant regeneration from anther cultures have been extensively investigated. As a result, it is now possible to produce haploids from the anther cultures of many japonica and indica rices, although the frequency of regenerated plants is relatively lower in indicas.
Another culture is important in developing true breeding lines in the immediate generation from any segregating population, thereby shortening the breeding cycle of new varieties. In conventional breeding, after a cross is made between desired parents, the segregating populations from F2 to F7 are grown to ultimately develop true breeding homozygous lines; anther cultures-derived lines lack heterozygosity and can be multiplied and evaluated in the immediate generation. The selection efficiency with doubled haploid (DH) lines is higher, especially when dominance variation is significant.
Early generation lines (F3-F5) show phenotypic differences because of additive and dominance variance. In contrast, there is no dominance variance among DH lines and heritability of the trait is high. DH lines are also useful for developing mapping populations for molecular analysis.
Seeds of such populations can be distributed to workers in other laboratories, and populations can be grown repeatedly in many different environments, greatly facilitating the additional mapping of both DNA markers and genetic loci controlling traits of agronomic importance. In rice, mapping populations (DH lines) produced through anther cultures of IR64 X Azucena, an indica/japonica cross, and CT9993 X IR62266 are being used in the molecular mapping of genes including quantitative trait loci.
Use of anther cultures in the development of rice varieties has been reported in several countries, including China, Japan, the Republic of Korea and the US. Most of the anther culture-derived varieties are japonicas. Indica rices are planted on 90% of land under rice cultivation, but they are generally regarded as recalcitrant, not only to anther cultures but also to other tissue culture techniques.
At the International Rice Research Institute (IRRI), the anther culture technique is being employed to obtain DH lines from many crosses for different rice-breeding objectives. To date, about 8000 DH lines have been regenerated. On average of about 20-50 DH lines are produced from one cross.
This efficiency is being further increased to reach the target of 100 lines per cross. Several superior DH lines have been selected from crosses for salinity tolerance. One of the anther culture derived lines, IR51500-AC-11-1 has been named as a variety (PSBRC50) in The Philippines. Many DH lines produced through anther cultures are now being used as parents in breeding programmes.
ii. Evolutionary Phase:
The genus Oryza, to which cultivated rice belongs, has 22 wild species. Diploid O. sativa (2n = 24) has the AA genome and is the predominantly cultivated species, while O. glaberrima, also with the AA genome, is cultivated in a limited area in West Africa. The wild species of rice have either 2n = 24 or 48 chromosomes with AA, BB, BBCC, CC, CCDD, EE, FF, GG and HHJJ genomes. Genetic variability for some traits, such as resistance to tungro, sheath blight and yellow stem borer, and tolerance of abiotic stresses, is limited in the cultivated germplasm. Wild species offer useful sources of genes for rice improvement.
Several barriers are encountered in transferring useful genes from wild species to cultivated rice. The barrier most commonly encountered is lack of crossability because of chromosomal and genie differences. Biotechnology tools such as embryo rescue and protoplast fusion have become available to overcome the crossability barriers, and several interspecific hybrids have been produced. Molecular techniques have been employed in the precise monitoring of alien gene introgression and in the transfer of useful genes to cultivated crop plants.
Production of interspecific hybrids through embryo rescue Abortion of embryos at different stages of development is a characteristic feature of wide crosses. Hybrids have been produced through embryo rescue between elite breeding lines or varieties of rice and several accessions of 11 wild species representing BBCC, CC, CCDD, EE, FF, GG and HHJJ genomes. These hybrids have been produced to transfer useful genes for resistance to brown plant hopper (BPH), bacterial blight, blast, sheath blight, tungro virus, and yellow stem borer.
Additional hybrids involving rice and AA genome wild species have been produced for the diversification of cytoplasmic male sterility and for the transfer of tolerance to acid sulphate conditions. In most interspecific crosses, the F1 hybrids are completely sterile.
Progenies are advanced through embryo rescue in subsequent backcrosses to the respective recurrent rice parents until plants with 2n = 24 and 25 chromosomes become available. Monosomic alien addition lines (MAALs) have been produced involving alien chromosomes of five wild species. These MAALs serve as an additional source of alien genetic variation.
3. Molecular Maps for Rice Genetics:
The availability of a comprehensive molecular genetic map for rice comprising more than 2000 DNA markers has been a major advance in rice genetics. A molecular genetic map of rice, based on restriction fragment length polymorphisms, was developed at Cornell University, Ithaca, USA, in collaboration with IRRI.
This map was based on an indica X japonica F2 population and mapped sequences were cloned from a genomic library derived from the indica variety IR36. A second RFLP map was based on a different indica X japonica cross. Causse developed the map, comprising 726 markers.
The mapping population was derived from the backcross between the cultivated rice Oryza sativa and a wild species, O. longistaminata. More recently, a comprehensive molecular genetic map consisting of 1383 DNA markers has been developed under the Rice Genome Research Program in Japan. The markers, distributed along 1575 cM on 12 linkage groups, comprise 883 cDNAs, 265 genomic DNAs, 147 random amplified polymorphic DNAs (RAPDs) and 88 other DNAs. Recently, Singh mapped centromeres on the molecular genetic map of rice and determined the correct orientation of linkage groups.
Tagging Alien Genes with RFLP Markers:
Alien genes introgressed for resistance to BPH, bacterial blight and blast have been tagged with molecular markers. RFLP analysis was carried out on the introgression line IR65482-4-136-2-2 with resistance to three BPH biotypes derived from O. sativa X O. australiensis.
We surveyed the recurrent parent, the wild species (donor) and the introgressed line for RFLP polymorphism. Fourteen probes, previously mapped to chromosome 12, were found to be polymorphic between the recurrent parent and the wild species. Thirteen probes did not detect any introgression. Only RG457 detected introgression from O. australiensis.
Co-segregation for BPH reaction and RG457 was studied in the F2 population. The results showed that the gene for resistance to BPH is linked to molecular marker RG457, with a crossover value of 3.68 + 1.29%. Such tight linkage should facilitate selection for BPH resistance during the transfer of this resistance to other elite breeding lines of rice. Analysis of this introgressed line indicates that the mechanism of alien gene transfer is genetic recombination rather than substitution of whole or large segments of the chromosome of wild species.
4. Gene Tagging in Rice Production:
Two of the most serious and widespread diseases in rice production are rice blast caused by the fungus Pyricularia oryzae, and bacterial blight caused by Xanthomonas oryzae pv. oryzae. Development of varieties with durable resistance to these diseases is the focus of a coordinated effort at IRRI using molecular marker technology. Early gene mapping efforts have been directed at tagging several single genes conferring resistance to these pathogens. As a result, several genes for bacterial blight and for blast resistance have been tagged with molecular markers.
Resistance to blast has long posed a serious challenge in rice improvement. Resistance, particularly when based on single major genes, has generally been overcome in one or a few years in blast prone environments. Some cultivars, however, have shown longer lasting or durable resistance. In several cases, durable resistance to blast is believed to be associated with quantitative or polygenic inheritance. Under these conditions, there would be little or no gain in fitness for a pathogen variant that could overcome only a fraction of the polygenes. Many rice improvement programmes now aim at incorporating quantitative or partial resistance into rice varieties.
An RFLP mapping study was conducted to understand the genetics of blast resistance in Moroberekan, a traditional West African upland cultivar considered to have durable resistance. A population of F7 recombinant inbred lines was used for this analysis. One gene conferring complete resistance was identified on chromosome four and was designated Pi-5(t). Both interval analysis and linear regression have identified nine regions of the genome with quantitative effects on blast resistance. These were considered to be putative QTL for blast resistance.
It was interesting that three of the loci found to be associated with partial resistance in this study had been previously identified as being linked to genes for complete resistance. This raises the possibility that the same loci conditioning complete resistance under some circumstances may act as QTL under others. Analyses of single resistance genes have also been carried out using near isogenic lines (NILs). The NILs have been used for mapping of the genes Pi-2(t) and Pi-4(t).
Efforts to detect markers closely linked to bacterial blight resistance genes have similarly taken advantage of the availability often sets of NILs. These lines each contain a single gene for resistance and, in the case of Xa-21 the gene has been introgressed from the wild relative of rice, O. longistaminata. Segregating populations have been used to confirm co-segregation between the RFLP markers and Xa-1, Xa-2, Xa-3, Xa-4, xa-5, Xa-10, xa-13 and Xa-21.
To test the utility of markers as selection tools and to study the effect of gene combinations in a particular genetic background, pairs of genes were combined by marker assisted selection (MAS) and confirmed when possible by phenotypic selection. These markers provide immediate information on plants which carry a target gene and whether the gene exists in a homozygous or heterozygous state.
Markers also allow accurate identification of genotypes in which two or more genes have been combined in one individual, but where the phenotype of the individual is indistinguishable from those carrying fewer genes. On the basis of this capability, the hypothesis is being tested that polygenic forms of resistance can be effectively developed by combining genes that were originally identified as conferring qualitative or single gene resistance. Unexpected interactions between genes that have been well characterized individually can also be evaluated using this approach.
Genes for aroma, wide compatibility, fertility restoration and thermo-sensitive genetic male sterility, brown plant hopper resistance, and tolerance of tungro and submergence have been tagged with molecular markers. PCR-based markers have been developed for some of these traits to facilitate selection.
Nair developed PCR-based markers for Gm-2 and Gm-4(t) which are useful in MAS for developing gall midge resistant varieties. The emphasis now is on tagging genes of economic importance by developing PCR-based markers. This will enhance the efficiency of MAS and pyramiding of useful genes for tolerance to various biotic and abiotic stresses.
5. QTL Mapping for Production of Rice:
Although a number of important characters are determined by loci having major effects on phenotype, most economically important traits such as yield, quality and tolerance of various abiotic stresses (drought, salinity, submergence, etc.) are of a quantitative nature. Genetic differences affecting such traits (within and between populations) are controlled by a relatively large number of loci, each of which can make a small positive or negative contribution to the final phenotypic value of the traits.
The genes governing such traits, called polygenes or minor genes, also follow Mendelian inheritance but are greatly influenced by the environment. Biometrical procedures involving special experimental designs and data analyses are used to study genetics of quantitative traits. The advent of molecular markers has made it possible to map the QTL which have large genotypic effects on phenotype. These molecular markers provide methods to transform QTL into Mendelian or quasi-Mendelian entities that can be manipulated in classical breeding programmes.
Wang probed 281 recombinant inbred lines (RIL) with 156 RFLP markers. The RIL were evaluated for qualitative resistance using five isolates of blast pathogen, and for quantitative resistance using a single isolate in polycyclic tests in which multiple infection cycles were allowed to proceed. Two genes, Pi-5(t) and Pi-7(t), were mapped on chromosomes 4 and 11 respectively. Nine QTLs having quantitative effect on blast resistance to isolate Po6-6 were identified.
Redona and Mackill identified two QTL controlling root length, and five each influencing coleoptile and mesocotyl length. Nandi used amplified fragment length polymorphism (AFLP) markers and identified four QTL for submergence tolerance on chromosomes 6, 7, 11 and 12 of rice. In addition, a major gene, Sub-1 (t), for submergence tolerance was localized on chromosome 9. Xiao analysed QTL governing various agronomic traits. Some of these traits were controlled by one to six QTL.
Xiao analysed BC2 test cross families from the interspecific cross (O. sativa X O. rufipogon) and found that O. rufipogon alleles at marker loci RM5 on chromosome 1, and RG256 on chromosome 2, were associated with enhanced yield potential. The phenotypic advantage of the lines carrying O. rufipogon alleles at these loci corresponded to 18% and 17% yield increase over the O. sativa parent. These results indicate that molecular markers can be used to identify QTL from wild species responsible for transgressive segregation. Comparative mapping among different cereals has also increased the efficiency of orthologous QTL mapping.
Future research should focus on:
(i) Identification of QTL which could be exploited in different environments;
(ii) Exploitation of complementary QTL to isolate transgressive segregantes, particularly from interspecific crosses;
(iii) Identification of orthologous QTL among different species, as the conservation of such QTL among species may provide new opportunities for manipulation of economic traits;
(iv) High resolution of QTL to help determine whether QTL are single genes or clusters of tightly linked genes and whether over-dominance plays a significant role in heterosis; and
(v) Cloning of QTL, based on high resolution mapping to usher in a new era in molecular quantitative genetics.
6. Pyramiding Genes for Bacterial Blight (BB) Resistance:
The development of saturated molecular maps, the possibility of finding tight linkage of target genes with molecular markers such as RFLPs and conversion of these markers to PCR-based markers have provided new opportunities to use MAS in rice breeding.
Recently, we have developed protocols for PCR-based MAS in rice. In MAS, individuals carrying target genes are selected in a segregating population based on tightly linked markers rather than on their phenotypes. Thus, the populations can be screened at an early seedling stage and in various environments.
MAS can overcome interference from interactions between alleles of a locus or of different loci. MAS increase the efficiency and accuracy of selection, especially for traits which are difficult to phenotype. We have successfully used MAS for pyramiding genes (Xa-4, xa-5, xa-13, Xa- 21) for bacterial blight resistance. Breeding lines with two, three or four BB resistance genes have been developed. The pyramided lines showed a wider spectrum and higher level of resistance than lines with only a single gene. Pyramided lines carrying four BB resistance genes are being used in MAS to transfer these genes into elite breeding lines of a new rice plant type.
Yoshimura selected lines carrying Xa-4 + xa-5 and Xa-4 + Xa- 10 using RFLP and RAPD markers linked to the BB resistant genes.
These lines were evaluated for reaction to eight strains of BB, representing eight pathotypes and three genetic lineages. Lines carrying Xa-4 + xa-5 were more resistant to isolates of race 4 than was either of the parental lines. Such pyramided lines with different gene combinations are useful for developing varieties with durable resistance.
In order to improve the durability of resistance of future rice varieties, we are using molecular marker analysis to identify pathogen populations with wide diversity, which are then employed for screening and for developing resistant genotypes. We are also using deployment strategies based on understanding of pathogen population genetics and on the genetic basis of durable resistance.
The high-density molecular genetic map of rice is also of great value in understanding genome organization and in map-based cloning of agriculturally important genes. Rice serves as a model for genome research in monocots because of its small genome size, excellent germplasm collection, good genetic stocks and relatively well-developed molecular genetic maps. Development of molecular genetic maps has been of great value in understanding the homoeologous relationships between the genomes of various crop plants.
Ahn found extensive homoeologies in a number of regions of the genomes of wheat, rice and maize. Kurata analysed synteny between rice and wheat and found that many wheat chromosomes contained homoeologous genes and genomic DNA fragments in a similar order to that found in rice. Comparative genome mapping in rice, maize, wheat, barley and sorghum is proceeding rapidly, and the rice research community is in an excellent position to take advantage of the possibilities for exchanging materials and information on these crops as opportunities arise.
Based on comparative mapping, the species with smaller genome size, such as rice, may be used to accelerate map-based cloning of orthologous genes. The small genome size of rice, about 40 times smaller than that of wheat and only 2.5 times larger than that of Arabidopsis thaliana makes rice an excellent candidate for isolation of genes through chromosome walking and map-based cloning. The possibilities of cloning rice genes based on map position have been greatly enhanced with the development of bacterial artificial chromosome (BAC) libraries. Wang used BAC library and identified clones linked to the Xa-21 bacterial blight resistance gene in rice.
Song isolated the Xa-21 gene by positional cloning and used this gene in rice transformation. At IRRI, a BAC library has been constructed from IR64 genomic DNA. The library consists of 18,432 clones. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Positive clones were analysed to generate 29 contigs. These contigs are serving as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards map-based cloning of disease resistance genes.
7. Cytoplasmic Diversification of Rice:
Most of the commercial hybrids of indica rice are based on the wild abortive (WA) source of cytoplasmic male sterility (CMS). More than 95% of the rice hybrids grown in China have WA cytosterile cytoplasm. Such cytoplasmic uniformity increases the vulnerability of hybrid rice to diseases and insects.
To overcome this problem, diversification of the cytoplasmic male sterility source is essential. We crossed 45 accessions of O. perennis and four accessions of O. rufipogon as the female parents with the widely grown varieties IR54 and IR64. Both IR54 and IR64 can restore the fertility of CMS lines possessing WA cytoplasm.
Of all the backcross derivatives, one line with the cytoplasm of O. perennis and the nucleus of IR64 was found to be stable for complete pollen sterility. The newly developed CMS line has been designated IR66707A. Crosses of IR66707A with six restorers of WA cytoplasm also show almost complete pollen sterility, indicating that this source of CMS is different from that of WA cytoplasm.
Southern hybridization of IR66707A, O. perennis and IR66707B with eight mitochondrial DNA-specific probes was carried out. Of 40 combinations, 18 showed a monomorphic pattern, while in 22 polymorphic combinations the banding patterns of IR66707A and O. perennis were identical. The results indicated that IR66707A has the same mitochondrial genome as the donor O. perennis and that CMS may not be caused by any major rearrangement or modification of mitochondrial (mt)DNA.
Characterization of alien genetic variation through RFLP markers Both isoenzyme and RFLP markers detect extensive polymorphism between rice and wild species and have proved useful as genetic markers in the characterization of alien genetic variation. Introgression has been detected for isoenzyme loci from several wild species. RFLP analysis of the introgression lines derived from O. sativa X O. officinalis showed introgression of the chromosome segments in 11 of the 12 chromosomes of O. officinalis. Using molecular markers, introgression of small chromosome segments has been detected from chromosomes 10 and 12 of O. australiensis into rice.
We are now using RFLPs to determine introgression of the chromosome segments from distantly related genomes of wild species such as O. brachyantha and O. granulata into rice. Under an IRRI-Japan shuttle project, in situ hybridization techniques are being employed to precisely detect introgression of the chromosome segments from wild species into rice.
Somaclonal Variation of Rice:
Somaclonal variation refers to the variation arising through tissue culture in regenerated plants and their progenies. Somaclonal variation has been reported in various plant species and occurs for a series of agronomic traits such as disease resistance, plant height, tiller number and maturity as well as for various biochemical traits. The technique consists of growing callus or cell suspension cultures for several cycles and regenerating plants from such long-term cultures. The regenerated plants and their progenies are evaluated in order to identify individuals with a new phenotype.
Some useful somaclonal variants, including those for disease resistance and male sterile lines, have been isolated in rice. Heszky and Simon-Kiss tested several somaclonal variants of anther cultures origin. Of these, one was released as a variety named Dama.
This variety is resistant to Pyricularia and has good cooking quality. Similarly, Ogura and Shimamoto identified useful somaclonal variants from protoplast regenerated progenies of Koshihikari, and a new variety, Hatsuyume, was released. This variety is late by 1 week, shorter in height, lodging resistant and has a 10% higher grain yield than the mother variety, Koshihikari.
Somatic Hybridization of Rice:
Somatic hybridization involves the isolation, culture and fusion of protoplasts from different species and the regeneration of somatic hybrid plants. Since the first demonstration of plant regeneration from mesophyll protoplasts of tobacco, protoplasts have been successfully cultured and regenerated into plants from more than 300 plant species. Yamada were the first to successfully regenerate plants from rice protoplasts. Since then, many laboratories have regenerated plants from protoplasts of several japonica and indica cultivars. Hayashi produced somatic hybrids between rice and four wild species of Oryza.
One of the most important applications of somatic hybridization is the production of cytoplasmic hybrids (cybrids). In cybridization, the nuclear genome of one parent is combined with the organelles of the second parent in one step. For example, transfer of CMS to elite breeding lines requires five to seven backcrosses. The donor-recipient protoplast fusion method has made it possible to transfer CMS within several months.
Yang produced cybrid rice plants through asymmetric hybridization by electro-fusing the gamma-irradiated protoplasts of A-58 CMS and the iodoacetamine treated protoplasts of the fertile cultivar Fujiminori. The technique has been successfully used to transfer CMS from the indica rice Chinsurah Boro II into japonica cultivars.