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PART III - PROGRESS IN RICE GENETIC IMPROVEMENT FOR FOOD SECURITY


Biotechnology for rice breeding: progress and impact - G.S. Khusha and D.S. Brarb

a Former Programme Leader and b Plant Breeder
Plant Breeding, Genetics and Biochemistry Division, IRRI, Philippines

Rice is the most important food crop and the staple food for 40 percent of the world population. More than 90 percent of rice is produced and consumed in Asia. It is grown under a wide range of agroclimatic conditions. There have been major advances in increasing rice production worldwide thanks to the large-scale adoption of modern high-yielding rice varieties and improved cultural practices. World rice production more than doubled from 257 million tonnes (Mt) in 1966 to 599 Mt in 2000. This was mainly achieved through the application of the principles of classical Mendelian genetics and conventional plant breeding methods. The current world population of 6.1 billion is expected to reach 8.0 billion by 2030 and rice production must increase by 50 percent in order to meet the growing demand. If this goal is to be met, it is necessary to use rice varieties with higher yield potential, durable resistance to diseases and insects and tolerance to abiotic stresses.

Plant breeding comprises two phases:

Recent advances in cellular and molecular biology and rice biotechnology have produced new tools to increase the efficiency of both phases (Khush and Brar, 1998).

EVOLUTIONARY PHASE: BROADENING THE GENE POOL OF RICE CULTIVARS

Genetic variability for agronomic traits is the key component of breeding programmes for broadening the gene pool of both rice and other crops. A number of rice cultivars and elite breeding lines characterized by resistance to major diseases and pests, tolerance to abiotic stresses and improved quality characteristics have been developed through conventional plant breeding approaches. In such breeding programmes, it is mainly the genetic variability available in O. sativa germplasm which has been exploited. However, the genetic variability for many traits, such as tolerance to stem borer, tungro, sheath blight and salt stress, is limited in cultivated germplasm. Breeders therefore search for genetic variability in other gene pools involving wild relatives of Oryza and new techniques are applied for the creation and transfer of variability through somaclonal variation and genetic engineering.

Wide hybridization

Wide hybridization involving hybridization between rice and related wild species is adopted to broaden the gene pool of rice. The genus Oryza comprises 24 species representing AA, BB, CC, BBCC, CCDD, EE, FF, GG, HHJJ and HHKK genomes. Of these species, O. sativa (2n=24 AA) is cultivated worldwide, whereas O. glaberrima (2n=24 AA) is limited to certain areas in West Africa. Wild species are an important reservoir of useful genes for resistance to major diseases and pests, tolerance to abiotic stresses and cytoplasmic male sterility. However, several problems occur when transferring useful genes from wild species to cultivated rice (Brar and Khush, 1997). The barrier most commonly encountered is lack of crossability resulting from chromosomal and genic differences. Biotechnology tools, such as embryo rescue and protoplast fusion, have been employed to overcome this difficulty, resulting in the production of several interspecific hybrids. More recently, molecular techniques have been employed for precise monitoring of alien gene introgression.

TABLE 1
Introgression of genes from wild Oryza species into cultivated rice

Trait

Donor Oryza species

Wild species

Genome

Accession number

A. Transferred to Oryza sativa

Grassy stunt resistance

O. nivara

AA

101508

Bacterial blight resistance

O. longistaminata

AA

-

O. officinalis

CC

100896

O. minuta

BBCC

101141

O. latifolia

CCDD

100914

O. australiensis

EE

100882

O. brachyantha

FF

101232

Blast resistance

O. minuta

BBCC

101141

Brown planthopper resistance

O. officinalis

CC

100896

O. minuta

BBCC

101141

O. latifolia

CCDD

100914

O. australiensis

EE

100882

Whitebacked planthopper resistance

O. officinalis

CC

100896

Cytoplasmic male sterility

O. sativaf. spontanea

AA

-

O. perennis

AA

104823

O. glumaepatula

AA

100969

Tungro tolerance

O. rufipogon

AA

105908

O. rufipogon

AA

105909

B. Progenies under evaluation for introgression

Yellow stem borer

O. longistaminata

AA

-

Sheath blight resistance

O. minuta

BBCC

101141

Increased stem elongation ability

O. rufipogon

AA

CB751

Tolerance to acidity, iron and aluminium toxicity

O. glaberrima

AA

many

Weed competitiveness

O. glaberrima

AA

many

Source: Brar and Khush, 2002 (modified).

Gene transfer from wild species to rice

Hybrids between cultivated rice and AA genome wild species can be produced through normal procedures. Hybrids between rice and distantly related wild species, on the other hand, are usually difficult to produce; low crossability and abortion of hybrid embryos are common features in such crosses. Hybrids have been produced through embryo rescue between elite breeding lines or varieties and several accessions of wild species representing BBCC, CC, CCDD, EE, FF, GG, HHJJ and HHKK genomes. A number of useful genes for resistance to brown planthopper (BPH), whitebacked planthopper (WBPH), bacterial blight (BB), blast and tungro disease have been transferred from wild species to rice (Jena and Khush, 1990; Khush et al., 1990; Brar and Khush, 1997) (Table 1). The first example of transfer of a useful gene from wild species is the introgression of a gene for grassy stunt virus resistance from O. nivara to cultivated rice (Khush, 1977). Grassy-stunt-resistant varieties (IR 28, IR 29 and IR 30) were released for cultivation in 1974. Other grassy-stunt-resistant varieties (IR 32, IR 34 and IR 36) were later released. The grassy stunt resistance gene has now been incorporated into numerous varieties developed at the International Rice Research Institute (IRRI) or by national rice improvement programmes.

Cytoplasmic diversification

Wild species with AA genome have been an important source of cytoplasmic male sterility (CMS) - the major tool for breeding commercial rice hybrids. Lin and Yuan (1980) reported the development of a male sterile line with the cytoplasm of wild species (O. sativa L. f. spontanea) and the nuclear genome of rice. This cytoplasmic source has been designated as wild abortive (WA), i.e. a male sterile wild rice plant with abortive pollen. About 95 percent of the male sterile lines used in commercial rice hybrids grown in China and other countries have WA cytoplasm. A new CMS source from O. perennis was transferred into indica rice (Dalmacio et al., 1995). A newly developed CMS line has been designated as IR 66707A; it has the cytoplasm of O. perennis and the nuclear genome of cultivated rice IR 64. Genetic studies show that the male sterility source of IR 66707A is different from that of WA. Another CMS line (IR 69700A) with the cytoplasm of O. glumaepatula and the nuclear genome of IR 64 has been developed. Both IR 66707A and IR 69700A are completely stable for male sterility. Restorers of these CMS sources are currently being sought.

Tagging alien genes with molecular markers

Alien genes for resistance to BPH, BB and blast have been tagged with molecular markers. RFLP (restriction fragment length polymorphism) 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 (Jena et al., 1992). Most introgressed segments were detected by single RFLP markers and the flanking markers were negative for introgression. RFLP analysis was carried out on the introgression line IR 65482-4-136-2-2 with resistance to three BPH biotypes derived from O. sativa x O. australiensis. Of the 14 probes previously mapped to chromosome 12, only RG457 detected introgression from O. australiensis. Co-segregation for BPH resistance and RG457 showed that the gene for resistance to BPH is linked to the molecular marker RG457, with a crossover value of 3.68 + 1.29% (Ishii et al., 1994). Such tight linkage should facilitate selection for BPH resistance during the transfer of this resistance to other elite breeding lines of rice.

TABLE 2
Some examples of transgenic rice plants with agronomically important genes

Transgene

Transfer method

Useful trait

Reference

bar

Microprojectile bombardment

Tolerance to herbicide

Cao et al., 1992

bar

PEG-mediated

Tolerance to herbicide

Datta et al., 1992

Coat protein

Electroporation

Tolerance to stripe virus

Hayakawa et al., 1992

Coat protein

Particle bombardment

Tolerance to rice tungro spherical virus

Sivamani et al., 1999

Chitinase

PEG-mediated

Sheath blight resistance

Lin et al., 1995; Datta et al., 2001

cryIA(b)

Electroporation

Tolerance to striped stem borer

Fujimoto et al., 1993

cryIA(b)

Particle bombardment

Tolerance to yellow stem borer and striped stem borer

Wunn et al., 1996; Ghareyazie et al., 1997

cryIA(c)

Particle bombardment

Tolerance to yellow stem borer

Nayak et al., 1997

cry1A(b), cryIA(c)

Agrobacterium-mediated

Tolerance to striped stem borer and yellow stem borer

Cheng et al., 1998

cry1A(b), cryIA(c)

Particle bombardment

Tolerance to yellow stem borer

Tu et al., 2000

cry1A(c), cry2A, gna

Particle bombardment

Tolerance to stem borer, leaf folder and brown planthopper

Maqbool et al., 1998, 2001

CpTi

PEG-mediated

Tolerance to striped stem borer and pink stem borer

Xu et al., 1996

gna

Particle bombardment

Insecticidal activity for brown planthopper

Rao et al., 1998

Corn cystatin (CC)

Electroporation

Insecticidal activity for Sitophilus zeamais

Irie et al., 1996

Xa21

Particle bombardment

Resistance to bacterial blight

Tu et al., 1998; Zhang et al., 1998

codA

Electroporation

Increased tolerance to salt

Sakamoto and Murata, 1998

Soybean ferritin

Agrobacterium-mediated

Increased iron content in seed

Goto et al., 1999; Lucca et al., 2001

psy

Particle bombardment

Phytoene accumulation in rice endosperm

Burkhardt et al., 1997

psy, crt1, lcy

Agrobacterium-mediated

Provitamin A

Ye et al., 2000

Source: Khush and Brar, 2002 (modified).

Somaclonal variation

Somaclonal variation refers to the variation arising through tissue culture in regenerated plants and their progenies. Somaclonal variation is reported in various plant species and occurs for a series of agronomic traits, such as disease resistance, plant height, tiller number and maturity, and for various biochemical traits. The technique consists of growing callus or cell suspension cultures for several cycles and regenerating plants from these 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 sterility, have been isolated. Heszky and Simon-Kiss (1992) tested several somaclonal variants of anther culture origin, one of which was released as the variety, Dama: it is resistant to Pyricularia and has good cooking qualities.

Genetic engineering

The introduction of alien genes from bacteria, viruses, fungi, animals and, of course, unrelated plants into crop species allows plant breeders to achieve breeding objectives which until just a decade ago were not considered possible. Several techniques are now available for the transformation of rice, e.g.: electroporation, polyethylene-glycol-induced uptake of DNA into protoplasts, microprojectile bombardment and, more recently, Agrobacterium-mediated transformation. Transgenic rices carrying agronomically important genes for resistance to stem borer and fungal pathogens, and tolerance to herbicide, have been produced in both japonica and indica rices (Table 2). Several laboratories have produced transgenic rices - mainly through protoplast-mediated DNA transformation, but also via microprojectile bombardment. Hiei et al. (1994) and Cheng et al. (1998) obtained a large number of fertile transgenic plants through Agrobacterium-mediated transformation, while Chen et al. (1998) produced transgenic rice with multiple transgenes. Transgenic plants were produced after co-bombarding embryogenic callus and cell suspensions with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two of the target genes and 17 percent more than nine. Plants containing multiple transgenes had normal morphology and 63 percent of the plants set viable seeds.

Transgenic rice for modifying yield potential

Starch biosynthesis plays an important role in plant metabolism. Several enzymatic steps are involved in starch biosynthesis in plants. ADP-glucose pyrophosphorylase (ADPGPP) is a critical enzyme for regulating starch biosynthesis in plant tissues. Starch levels and dry matter accumulation were enhanced in potato tubers of plants transformed with glgC16 gene from E. coli encoding ADPGPP (Stark et al., 1992). The glgC16 gene has been introduced into rice and the yield potential of these lines will soon be evaluated.

The transfer of C4 traits into C3 rice is being explored with the aim of improving photosynthetic efficiency; however, it is difficult to incorporate genes for C4 traits into C3 plants through traditional plant breeding methods. Ku et al. (1999) used Agrobacterium-mediated transformation and introduced from maize a gene for phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants. Most transgenic rice plants showed high level expression of the maize gene; the activities of PEPC in the leaves of some transgenic plants were two to three times higher than those in maize, and the enzyme accounted for up to 12 percent of the total leaf soluble protein. The level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. The transgenic rice plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. These findings demonstrate a successful strategy for introducing the key biochemical component of the C4 pathway of photosynthesis into rice.

Transgenic rice for insect resistance

As early as 1987, genes coding for toxins from Bacillus thuringiensis (Bt) were transferred to tomato, tobacco and potato, where they provided protection against lepidopteran insects. A major target for Bt deployment in transgenic rice is the yellow stem borer, Scirpophaga incertulas (Walker). The pest is widespread in Asia and is the cause of potentially substantial crop losses. Improved rice cultivars are either susceptible to the insect or have only moderate levels of resistance; transgenic rice with Bt is therefore very promising for the control of yellow stem borer.

Fujimoto et al. (1993) introduced a truncated d-endotoxin gene, cry1A(b), into rice. Transgenic plants in the R2 generation expressing the cry1A(b) protein showed increased resistance to striped stem borer and leaf folder. Wunn et al. (1996) introduced cry1A(b) gene into IR 58 through particle bombardment. The transgenic plants in R0, R1 and R2 generation showed resistance to several lepidopteran insect pests, with feeding studies revealing a mortality rate of up to 100 percent for yellow stem borer and striped stem borer larvae. Nayak et al. (1997) transformed IR 64 through particle bombardment using cry1A(c) gene and identified six independent transgenic lines with high expression of insecticidal crystal protein. The transferred synthetic cry1A(c) gene was stably expressed in the T2 of these lines and the transgenic rice plants proved highly toxic for yellow stem borer larvae. Cheng et al. (1998) produced over 2 600 transgenic rice plants in nine strains through Agrobacterium-mediated transformation. The plants were transformed with two synthetic - cry1A(b) and cry1A(c) - coding sequences from Bacillus thuringiensis. Bioassays in R1 indicated that the transgenic plants were highly toxic for striped stem borer and yellow stem borer. Maqbool et al. (1998) transformed Basmati 370 and M7 cultivars using the novel endotoxin cry2A gene.

Transgenic rice for disease resistance

Sources of resistance to some diseases (blast and bacterial blight) have been identified within cultivated rice germplasm, and improved germplasm with resistance has been developed. However, sources of resistance to sheath blight are not available and there are only a few known donors for resistance to tungro disease (caused by two types of virus). A highly successful strategy, known as coat protein (CP) mediated protection, has been employed against certain viral diseases, such as tobacco mosaic virus, in tobacco and tomato. A CP gene for rice stripe virus was introduced into two japonica varieties by electroporation of the protoplasts (Hayakawa et al., 1992). The resultant transgenic plants had high levels of CP (up to 0.5% of the total soluble proteins) and exhibited a significant level of resistance to virus infection. The resistance was inherited in the progenies.

Zhang et al. (1998) introduced Xa21 using projectile bombardment of cell suspensions of elite indica rice varieties: IR 64, IR 72, Minghui 63, and BG90-2. Six of the 55 R0 lines carrying Xa21 showed a high level of resistance to BB in subsequent generations. Sheath blight in rice is caused by the fungus, Rhizoctonia solani, which has a wide host range. About six chitinase genes have been identified in rice and are being manipulated to increase the level of resistance to fungal diseases (Zhu and Lamb, 1991). Lin et al. (1995) introduced 1.1 kilobase rice genomic DNA fragment containing a chitinase gene through PEG-mediated transformation.

Transgenic rice for abiotic stress tolerance

A series of abiotic stresses, such as drought, excess water, mineral toxicities/deficiencies in soil and unfavourable temperature, affect rice productivity. Genetic engineering approaches hold great promise for the development of rice cultivars with higher levels of tolerance to abiotic stresses. Sakamoto and Murata (1998) introduced the codA gene for choline oxidase from Arthrobacter globiformis. The codA gene was inherited into the second generation of transgenic rice and its expression was stably maintained at levels of the mRNA, the protein and enzyme activity. Levels of glycinebetaine were estimated to be as high as 1 and 5 m mol/g of fresh leaves in two types of transgenic plants (Chl COD and Cyt COD plants) in which choline oxidase was targeted to the chloroplasts and cytosol, respectively. Preliminary results showed that the transgenic plants grew better than the control plants during recovery from treatment with 0.15 M NaCl for 7 days. Further analysis of transgenic plants demonstrated their ability to synthesize betaine and confer enhanced tolerance to salt and the cold.

Transgenic rice for improved nutritional quality

Rice contains neither b-carotene (provitamin A) nor C40 carotenoid precursors in its endosperm. Rice in its milled form (as it is usually consumed) is therefore entirely without vitamin A and its carotenoid precursors. Millions of rice consumers who depend on rice for a large proportion of their calories suffer from vitamin A deficiency. Ye et al. (2000) produced transgenic rice (Golden Rice) with the provitamin A(b-carotene) biosynthetic pathway engineered into its endosperm. Agrobacterium-mediated transformation was applied to introduce three genes: phytoene synthase (psy), phytoene desaturase (crt1) and lycopene cyclase (lcy). HPLC (high performance liquid chromatography) analysis revealed the presence of b-carotene in transgenic seeds. The transformed rice Taipei 309 is no longer cultivated. Efforts are now underway at IRRI to transfer the genes for b-carotene into widely grown varieties, such as IR 64, through conventional backcrossing and transformation.

Goto et al. (1999) introduced the entire coding sequence of the soybean ferritin gene into kita-ake, a rice cultivar via Agrobacterium-mediated transformation. The introduced ferritin gene was regulated by the rice seed storage protein glutelin promoter, GluB-1, and terminated by the Nos polyadenylation signal. Synthesis of soybean ferritin protein was confirmed in each of the transformed rice seeds by western blot analysis, and specific accumulation in endosperm was determined by immunological tissue printing. The iron content of T1 seeds was up to three times higher than in untransformed seeds.

EVALUATION PHASE: INCREASING SELECTION EFFICIENCY

Anther culture

As early as 1968, Niizeki and Oono reported the production of haploids from anther culture of rice. Since then, the anther culture technique has been refined greatly; it is now possible to produce haploids from the anther culture of many japonica and indica rices, although the frequency of plant regeneration is lower in indica varieties. Anther culture is important for the development of true breeding lines in the immediate generation from any segregating population, producing a shorter breeding cycle in new varieties. Selection efficiency in doubled haploid (DH) lines is higher, especially when dominance variation is significant. 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 culture of IR 64 x Azucena (indica/japonica cross) and CT9993 x IR 62266 are being used in the molecular mapping of genes with quantitative trait loci (QTL).

A number of varieties and improved breeding lines have been developed through anther culture in China, the Republic of Korea, Japan and the United States. Most of the anther-culture-derived varieties are japonica. Indica rices are generally regarded as recalcitrant for anther culture. At IRRI, the anther culture technique is being employed to obtain DH lines from many crosses for different rice breeding objectives. In the Philippines, one of the anther-culture-derived lines, IR 51500-AC-11-1, has been named as variety PSBRc50. Many DH lines produced through anther culture are now being used as parents in breeding programmes. DH lines are being produced to overcome sterility in the progenies of O. sativa x O. glaberrima crosses (Enriquez et al., 2000).

Molecular marker technology

One of the most exciting developments in rice biotechnology is the advent of molecular markers. A series of molecular markers, e.g. random fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites, have become available. These markers offer new opportunities for various studies in genetic and breeding research, particularly in the construction of saturated molecular maps, gene tagging, QTL mapping, marker-assisted breeding, gene pyramiding, physical mapping of genome, map-based gene cloning, alien introgression and DNA finger-printing of pathogen populations (Tanskley et al., 1989; Paterson et al., 1991; Mohan et al., 1997).

Molecular maps

The availability of a comprehensive molecular genetic map in rice comprising more than 2 300 DNA markers represents a major advance in rice genetics. A molecular genetic map of rice based on RFLPs was developed at Cornell University, Ithaca, USA, in collaboration with IRRI (McCouch et al., 1988). This map was based on F2 population derived from an indica x japonica cross, and mapped sequences were cloned from a genomic library derived from the indica variety IR 36. Many new markers were later added to the map. A comprehensive molecular genetic map consisting of 2 275 DNA markers was developed at the Rice Genome Research Program in Japan (Harushima et al., 1998). Singh et al. (1996) mapped centromeres on the molecular genetic map of rice and determined the correct orientation of linkage groups.

Molecular marker-aided selection (MAS)

Numerous genes of economic importance, such as those for disease and insect resistance, are repeatedly transferred from one varietal background to another by plant breeders. Most genes behave in a dominant or recessive manner and transfer is a time-consuming process. Screening procedures are sometimes cumbersome and expensive and require a large amount of field space. Tagging such genes by tight linkage with DNA or isozyme markers means that time and money can be saved in transferring them from one varietal background to another. A molecular marker very closely linked to the target gene can act as a “tag” which can be used for indirect selection of the gene(s) in the breeding programme.

Gene tagging in rice

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). The development of varieties with durable resistance to these diseases is the focus of a joint coordinated effort by IRRI and National Agricultural Research Systems (NARS) using molecular marker technology. Several genes for bacterial blight and blast resistance have been tagged with molecular markers (Table 3).

The incorporation of resistance to blast has long posed a serious challenge in rice improvement. Resistance based on single major genes has generally been overcome in a matter of years in blast-prone environments. Resistance in some cultivars, however, has proven to be longer-lasting. In several cases, durable resistance to blast is believed to be associated with quantitative or polygenic inheritance. Under these conditions, there is little or no gain in fitness for a pathogen variant to overcome only a fraction of the polygenes. Many rice improvement programmes now aim to incorporate quantitative or polygenic resistance into rice varieties. Wang et al. (1994) identified gene Pi5, which confers complete resistance, including nine regions of the genome with quantitative effects on blast resistance. The latter were considered to be putative QTL for blast resistance. Three of the QTL found to be associated with partial resistance in this study had previously been identified as being linked to genes for complete resistance. Analysis of major resistance genes has also been carried out using near isogenic lines (NILs). The NILs have been used for mapping genes Pi2 and Pi4.

TABLE 3
Some examples of rice genes of agronomic importance mapped with molecular markers

Gene

Trait

Chromosome

Link marker

Reference

Pi1

Blast resistance

11

Npb181

Yu, 1991

Pi2

Blast resistance

6

RG64

Yu et al., 1991; Hittalmani et al., 1995

Pi4

Blast resistance

12

RG869

Yu et al., 1991

Pita

Blast resistance

12

RZ397

Yu et al., 1991

Pi5

Blast resistance

4

RG498, RG788

Wang et al., 1994

Pi6

Blast resistance

12

RG869

Yu, 1991

Pi7

Blast resistance

11

RG103

Wang et al., 1994

Pi9

Blast resistance

6

RG16

R. Nelson (pers. comm.)

Pi10

Blast resistance

5

RRF6, RRH18

Naqvi et al., 1995

Pi11

Blast resistance

8

BP127

Zhu et al., 1992

Pi12

Blast resistance

12

RZ869

Zheng et al., 1996

Pib

Blast resistance

2

RZ123

Miyamoto et al., 1996

Pi20

Blast resistance

12

XNbp88

Imbe et al., 1997

Pikm

Blast resistance

11

R1506

Kaji and Ogawa, 1996

Pb1

Blast resistance

11

S723

Fujii et al., 2000

Xa1

Bacterial blight resistance

4

Npb235

Yoshimura et al., 1992

Xa4

Bacterial blight resistance

11

Npb181

Yoshimura et al., 1992, 1995

xa5

Bacterial blight resistance

5

RG556

McCouch et al., 1991

Xa10

Bacterial blight resistance

11

OP07

Yoshimura et al., 1995

xa13

Bacterial blight resistance

8

RZ390, RG136

Yoshimura et al., 1995; Zhang et al., 1996

Xa21

Bacterial blight resistance

11

RG103

Ronald et al., 1992

Xa22

Bacterial blight resistance

1

RZ536

Lin et al., 1996

Stvb1

Rice stripe virus resistance

12

Npb220


RTSV

Rice tungro spherical virus resistance

4

RZ262

Sebastian et al., 1996

RYMV

Rice yellow mottle virus resistance

12

RG341

Ghesquiere et al., 1997

Bph1

Brown planthopper resistance

12

XNpb 248

Hirabayashi and Ogawa, 1995

bph2

Brown planthopper resistance

12

G402

Murai et al., 2001

Bph9

Brown planthopper resistance

12

S11679


Bph10

Brown planthopper resistance

12

RG457

Ishii et al., 1994

Bph

Brown planthopper resistance

-

MRF0P16938

Jena et al., 1998

Qbph1

Brown planthopper resistance

3

R1925

Huang et al., 2001

Qbph2

Brown planthopper resistance

4

C820

Huang et al., 2001

ef

Early flowering

10

CDO98

Ishii et al., 1994

fgr

Fragrance

8

RG28, RM223

Ahn et al., 1992

Wph1

Whitebacked planthopper resistance

7

-

McCouch, 1990

WBPH

Whitebacked planthopper resistance

11

RG103

Kadirvel et al., 1999

Gm1

Gall midge resistance

-

OPK7

Katiyar and Bennett, 2001

Gm2

Gall midge resistance

4

RG329, RG214

Mohan et al., 1994

gm3

Gall midge resistance

-

OPQ12

Nair et al., 1995

Gm4

Gall midge resistance

8

OPM12

Mohan et al., 1997

Gm5

Gall midge resistance

12

OPB14

Katiyar and Bennett, 2001

Gm6

Gall midge resistance

4

RG214

Katiyar et al., 2002

Rf1

Fertility restoration

10

OSRRF

Akagi et al., 1996

Rf2

Fertility restoration

1

CDO686/RZ58

Zhang et al., 1997

Rf3

Fertility restoration

1

RG532

Zhang et al., 1997

Rf5

Fertility restoration

1

RG374

Shen et al., 1998

S5

Wide compatibility

6

RG213

Yanagihara et al., 1995

Se1

Photoperiod sensitivity

6

RG64

Mackill et al., 1993

Se3

Photoperiod sensitivity

6

A19

Maheswaran, 1995

sdg

Semi-dwarf

5

RZ182

Liang et al., 1994

sd1

Semi-dwarf

1

RG109

Cho et al., 1994

tms1

Thermosensitive male sterility

8

-

Wang et al., 1995b

tms3

Thermosensitive male sterility

6

OPAC3640

Subudhi et al., 1997

tms4

Thermosensitive male sterility

2

RM27


rtms1

Thermosensitive reverse male sterility

10

RM239

Jia et al., 2001

pms1

Photoperiod sensitive male sterility

7

RG477

Zhang et al., 1993

pms2

Photoperiod sensitive male sterility

3

RG191

Zhang et al., 1993

pms3

Photoperiod sensitive male sterility

12

C751/RZ261

Li et al., 2001

Sub1

Submergence tolerance

9

RZ698

Xu and Mackill, 1996; Nandi et al., 1997

Salt

Salt tolerance

7

RG64

Zhang et al., 1995

OSA3

Salt tolerance

12

RG457

Zhang et al., 1999

Source: Khush and Brar, 1998 (modified).

Efforts to detect markers closely linked to bacterial blight resistance genes have taken advantage of the availability of a set of ten NILs. These lines each contain a single gene for resistance and, in the case of Xa21, the gene has been introgressed from the wild species of rice, O. longistaminata (Khush et al., 1990). Segregating populations have been used to confirm cosegregation between the RFLP markers and Xa1, Xa2, Xa3, Xa4, xa5, Xa10, xa13 and Xa21.

Genes for aroma, wide compatibility, fertility restoration and thermosensitive genetic male sterility, BPH resistance and tolerance to tungro virus and submergence have been tagged with molecular markers (Table 3). PCR (polymerase chain reaction)-based markers have been developed for some of these traits to facilitate selection. Nair et al. (1995, 1996) developed PCR-based markers for gall midge resistance genes, Gm2 and Gm4 - useful in MAS for developing gall-midge-resistant varieties. Ichikawa et al. (1997) developed PCR-based markers for Rf1, a fertility restorer gene. The emphasis is now 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.

QTL mapping

Although a number of important characters are determined by loci which have major effects on phenotype, most economically important traits, such as yield, quality and tolerance to 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. Such loci are termed “quantitative trait loci” (QTL). The genes governing such traits - known as polygenes or minor genes - also follow Mendelian inheritance but are greatly influenced by the environment.

The advent of molecular markers has made it possible to map QTL with a large genotypic effect on phenotype. These molecular markers allow the transformation of QTL into Mendelian or quasi-Mendelian entities that can be manipulated in classical breeding programmes. Through linkage analysis of large segregating populations (polymorphic for molecular markers and QTL for yield and yield components), locations of the QTL can be determined. Several QTL for traits of economic importance, such as blast resistance (Wang et al., 1994), root traits (Redoña and Mackill, 1996; Yadav et al., 1997; Zheng et al., 2000), submergence tolerance (Nandi et al. 1997) and yield components (Xiao et al., 1996, 1998), have been mapped with molecular markers.

Wang et al. (1994) mapped Pi5 and Pi7 for blast resistance on chromosomes 4 and 11, respectively. Nine QTL with quantitative resistance to isolate PO6-6 of blast were also identified. Li et al. (1995) identified six QTL contributing to resistance to Rhizoctonia solani. These QTL are located on six of the 12 rice chromosomes and accounted for 60 percent of the genotypic variation in the cross Lemont x Tequing. Nandi et al. (1997) used AFLP markers and identified four QTL for submergence tolerance on chromosomes 6, 7, 11 and 12 of rice. In addition, a major gene (Sub1) for submergence tolerance was localized on chromosome 9. Xiao et al. (1996, 1998) 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 an 18 and 7 percent increase in grain yield per plant. A total of 68 significant QTL were identified and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Moncada et al. (2001) used advanced backcross QTL analysis on O. sativa x O. rufipogon derivatives and found that certain regions of rice genome harbour genes which are useful in a range of environments. 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 mapping orthologous QTL. Future research should focus on:

Pyramiding major genes

The development of saturated molecular maps and 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. Protocols for PCR-based MAS in rice have been developed (Zheng et al., 1995). 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 the early seedling stage and in various environments. MAS helps overcome interference from interactions between different alleles of a locus or of different loci. MAS increases the efficiency and accuracy of selection, especially for traits which are difficult to phenotype. MAS was applied for pyramiding four genes for BB resistance (i.e. Xa4, xa5, xa13 and Xa21). Breeding lines with two or three genes were also developed.

The pyramided lines showed a wider spectrum and a higher level of resistance than lines with only a single gene (Huang et al., 1997). Yoshimura et al. (1995) developed lines carrying Xa4 + xa5 and Xa4 + Xa10 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 Xa4 + xa5 were more resistant to isolates of race 4 than were either of the parental lines.

Pyramided lines with different gene combinations are useful for developing varieties with durable resistance. Sanchez et al. (2000) used sequence-tagged site (STS) markers to pyramid three genes for BB resistance in an elite breeding line of rice. The pyramided lines with three or four genes in combination showed an increased and wider spectrum of resistance to bacterial blight than those with a single resistance gene. Singh et al. (2001) also used MAS to pyramid genes for BB resistance into a high-yielding indica rice cultivar, PR106, that is susceptible to BB. MAS was also employed to pyramid genes for resistance to blast (Hittalmani et al., 2000) and gall midge (Katiyar and Bennett, 2001). MAS also provides new opportunities to transfer and combine QTLs into agronomically desirable genotypes.

Molecular marker analysis is used to identify pathogen populations with wide diversity, which are then employed for screening and for developing genotypes with durable resistance. Gene deployment strategies are being employed based on an understanding of pathogen population genetics and on the genetic basis of durable resistance (Leung et al., 1993).

Synteny relationships

The development of molecular genetic maps has been important for understanding the homoeologous relationships between the genomes of various crop plants. Kurata et al. (1994a) analysed synteny relationships between the genomes of rice and wheat and found that many wheat chromosomes contained homoeologous genes and genomic DNA fragments in the same order as they are found in rice. Comparative genome mapping in rice, maize, wheat, barley, sorghum, foxtail millet and sugar cane demonstrates that gene content and order are highly conserved at both map and megabase levels between different species within the grass family, but the amount and organization of repetitive sequences have diverged considerably (Devos and Gale, 1997).

On the basis of comparative mapping information, gene location in one species can be used to predict the presence and location of orthologous loci in other species. Comparative mapping is accelerating map-based cloning of orthologous genes. The synteny relationships among cereals have resulted in the discovery of common genes, such as dwarf phenotype in maize (D8) and wheat (Rht1) based on genomic information derived from rice.

Map-based cloning of genes

The high density molecular genetic map and the development of BAC and YAC libraries have been important developments leading to the isolation of rice genes such as Xa1, Xa21 and Pib. Wang et al. (1995a) used a BAC library to identify clones linked to the Xa21 gene for BB resistance. Song et al. (1995) isolated the Xa21 gene using positional cloning; the isolated gene has been introduced into rice cultivars through transformation. Similarly, Xa1 for BB resistance has been cloned through a map-based cloning strategy (Yoshimura et al., 1998). The expression of Xa1, unlike that of previously studied resistance genes, was induced by wound and pathogen infection.

At IRRI, a BAC library was constructed from IR 64 genomic DNA (Yang et al., 1997), and it consists of 18 432 clones. BAC clones carrying xa5 for BB resistance have been identified (Yang et al., 1998). Efforts to clone xa5 are underway and, once cloned, it will be transferred to elite rice varieties through transformation. Wang et al. (1999) cloned the blast resistance gene Pib. The expression of Pib is induced when there are changes in the environmental conditions, such as temperature and darkness. Sanchez et al. (1999) identified BAC contigs flanking the xa13 locus for BB resistance, while Hayano-Saito et al. (2000) identified overlapping BAC clones flanking Stvb1, the gene for stripe virus resistance.

Functional genomics

Rice has become a model system for genomics research. Factors contributing to this situation include: the relatively small size of the rice genome; the synteny of its genome with those of other cereals; the availability of densely populated molecular maps containing more than 2 300 DNA markers; well-characterized YAC and BAC libraries; large-scale analysis of expressed sequence tags (ESTs); the vast amount of genetic resources (mutants, genetic stocks, wild species, mapping populations, introgression lines); and the comparative ease of transformation.

Mutants induced through transposable elements, retrotransposons, miniature inverted repeat transposable elements (MITEs) and T-DNA insertions have provided a wealth of genetic resources for functional genomics. Notable examples include: T-DNA-tagged insertional mutants with 30 000 lines carrying 42 000 T-DNA inserts (An et al., 2001); Tos17 retrotransposon insertional mutants with about 30 000 lines carrying more than 250 000 insertions (Hirochika et al., 2001); and deletion mutant populations produced by fast-neutron, gamma radiation and chemical mutagenesis (Leung et al., 2001). These plant materials have provided the necessary link between structural or sequence data and gene function through forward and reverse genetics. Furthermore, a large number of ESTs from various cDNA libraries have been produced for micro-array expression experiments. One major challenge is to determine the function of previously unknown rice genes revealed by sequencing projects. Another is to understand the functions of apparently redundant rice genes that may have different roles in different tissues or in response to different environments.

New high-throughput methods are being developed for expression analysis. Biochips are being used to follow changes in gene expression in response to abiotic stresses. Using gene chips or micro-arrays, the representative genes of rice can be analysed on a glass slide and used in RNA hybridization to reveal gene expression patterns and identify pathways by association. A combination of mRNA and proteomics will precisely reveal the function of rice genes involved in the phenotypic expression of different agronomic traits.

POTENTIAL IMPACT

Recent advances in cellular and molecular biology of rice offer new opportunities to enhance the efficiency of both the evolutionary and the evaluation phase of rice breeding. Biotechnology is becoming an important component of rice breeding (Tables 4 and 5). Anther culture has become an important technique for use by plant breeders to shorten the breeding cycle for the development of rice varieties, to fix recombinants and in overcoming sterility in distant crosses. Several rice varieties have been developed through anther culture. Doubled haploid populations are important for mapping genes governing agronomic traits including QTL.

TABLE 4
Overcoming some of the constraints of conventional rice breeding using biotechnology tools

Constraint

Biotechnology applications

Limited genetic variability for resistance to stem borer

Transgenic rice carrying Bt genes shows enhanced resistance to stem borer

Limited genetic variability for resistance to sheath blight

Transgenic rice carrying chitinase genes shows tolerance to sheath blight

Lack of genetic variability for b-carotene

Transgenic ‘golden’ rice carrying phytoene synthase (psy), phytoene desaturase (crt1) and lycopene cyclase (lcy) shows provitamin activity in rice seeds

Low selection efficiency to pyramid genes for durable resistance to pests

Marker assisted selection practised to pyramid genes for bacterial blight, blast and gall midge resistance

Longer breeding cycle of rice varieties

Varieties developed through anther culture in shorter period

Narrow gene pool for resistance to pests

Genes from wild species with broad spectrum of resistance to bacterial blight, brown planthopper and tungro incorporated into elite breeding lines of rice

Characterization of pathogen population difficult and laborious

DNA fingerprinting practised to characterize pathogen populations and for gene deployment for durable resistance

TABLE 5
Biotechnology products in rice

Biotechnology tools

Product

Anther culture

Several improved cultivars and elite breeding lines developed and released for commercial cultivation in China, Korea Rep. and Philippines.

Molecular marker assisted selection

Marker assisted selection practised. Pyramided lines with durable resistance carrying genes for resistance to bacterial blight, blast and gall midge developed in India, China, Philippines and Indonesia. Some of the pyramided lines have been field tested and are in the process of release for commercial cultivation.

Alien introgression

Elite breeding lines of rice carrying genes from wild species have been developed for resistance to bacterial blight, blast, brown planthopper, tungro and tolerance to acidic conditions. Varieties resistant to brown planthopper and tolerant to acidity have been released in Viet Nam.

Somaclonal variation

Early-maturing, high-yielding and blast-resistant varieties released in Hungary and Japan.

Transformation

Novel genes inserted into rice cultivars for tolerance to herbicide and resistance to stem borer, sheath blight and bacterial blight and provitamin A for improved nutritional quality. Field tests of transgenic rice carrying Bt gene for stem borer resistance have been made in China. However, no commercial release of transgenic rice has been made so far.

Molecular markers have led to the tagging of numerous genes for tolerance to major biotic and abiotic stresses. MAS has become an important tool in rice breeding: for moving genes from one varietal background to another; for pyramiding genes; and for the development of durable pest-resistant cultivars. Fine mapping of QTL should provide a means to pyramid QTLs for tolerance to major abiotic stresses. Map-based cloning has made it possible to isolate useful genes governing important agronomic traits and the incorporation of these genes into elite rice cultivars through transformation. Advances in tissue culture and molecular marker technology have resulted in broadening of the gene pool of rice and have enhanced the efficiency of introgression of useful genes from wild species across crossability barriers. Advances in genetic engineering have facilitated the introduction of cloned novel genes into rice through transformation. Transgenic rices with enhanced resistance to diseases and insects and improved nutritional quality have been produced and will have a great impact in terms of increasing rice production and improving the nutritional value of rice. Public and private efforts in sequencing the rice genome have added new dimensions for research in functional genomics to precisely reveal the function of rice genes. Identification of genes and their manipulation present another major breakthrough in rice genetics and breeding. As discussed above, many rice cultivars have been developed through the application of biotechnology tools. Many more will be forthcoming. Future food and nutritional security will depend upon the availability of rice cultivars with higher yield potential, durable resistance to diseases and insects, tolerance to abiotic stresses and higher levels of micronutrients in the grain. Conventional breeding methods and biotechnology tools will help meet these challenges.

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