Darshan S. Brar

Alien introgression in rice.

Rice (Oryza sativa L.) productivity is affected by several biotic and abiotic stresses. The genetic variability for some of these stresses is limited in the cultivated rice germplasm. Moreover, changes in insect biotypes and disease races are a continuing threat to increased rice production. There is thus an urgent need to broaden the rice gene pool by introgressing genes for such traits from diverse sources. The wild species of Oryza representing AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ genomes are an important reservoir of useful genes. However, low crossability and limited recombination between chromosomes of cultivated and wild species limit the transfer of such genes. At IRRI, a series of hybrids and monosomic alien addition lines have been produced through embryo rescue following hybridization between rice and several distantly related species. Cytoplasmic male sterility and genes for resistance to grassy stunt virus and bacterial blight have been transferred from A genome wild species into rice. Similarly, genes for resistance to brown planthopper, bacterial blight and blast have also been introgressed across crossability barriers from distanly related species into rice. Some of the introgressed genes have been mapped via linkage to molecular markers. One of the genes Xa-21 introgressed from O. longistaminata has been cloned and physically mapped on chromosome 11 of rice using BAC library and flourescence in-situ hybridization. RFLP analysis revealed introgression from 11 of the 12 chromosomes of C genome species into rice. Introgression has also been obtained from other distant genomes (EE, FF, GG) into rice and in majority of the cases one or two RFLP markers were introgressed. Reciprocal replacement of RFLP alleles of wild species with the alleles of O. sativa indicates alien gene transfer through crossing over. The rapid recovery of recurrent phenotypes in BC2 and BC3 generations from wide crosses is an indication of limited recombination. Further cytogenetic and molecular investigations are required to determine precisely the mechanism of introgression of small chromosome segments from distant genomes in the face of limited homoeologous chromosome pairing. Future research should focus on enhancing recombination between homoeologous chromosomes. Introgression of QTL from wild species should be attempted to increase the yield potential of rice.

Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106

Abstract Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21.

Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza

Abstract Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in Oryza. Forty two genotypes including 17 wild species, representing AA,BB,CC,EE,FF,GG,BBCC,CCDD, and HHJJgenomes, two cultivated species, Oryza sativa (AA) and Oryza glaberrima (AA), and three related genera, Porteresia coarctata, Leersia and Rhynchoryza subulata, were used in ISSR analysis. A total of 30 ISSR primers were screened representing di-, tri-, tetra- and penta-nucleotide repeats, of which 11 polymorphic and informative patterns were selected to determine the genetic diversity. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered 42 genotypes according to their respective genomes. ISSR analysis suggests that the genus Oryza may have evolved following a polyphyletic pathway; Oryza brachyantha (FF genome) is the most divergent species in Oryza and Oryza australiensis (EE genome) does not fall under the Officinalis complex. DNA profiles based on ISSR markers have revealed potential diagnostic fingerprints for various species and genomes, and also for individual accessions/cultivars. Additionally ISSR revealed 87 putative genome/species-specific molecular markers for eight of the nine genomes of Oryza. The ISSR markers are thus useful in the fingerprinting of cultivated and wild species germplasm, and in understanding the evolutionary relationships of Oryza.

Phylogenetic relationships among Oryza species revealed by AFLP markers

Abstract The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms.

Genomic paleontology provides evidence for two distinct origins of Asian rice (Oryza sativa L.)

The origin of rice domestication has been the subject of debate for several decades. We have compared the transpositional history of 110 LTR retrotransposons in the genomes of two rice varieties, Nipponbare (Japonica type) and 93-11 (Indica type) whose complete sequences have recently been released. Using a genomic paleontology approach, we estimate that these two genomes diverged from one another at least 200,000 years ago, i.e., at a time which is clearly older than the date of domestication of the crop (10,000 years ago, during the late Neolithic). In addition, we complement and confirm this first in silico analysis with a survey of insertion polymorphisms in a wide range of traditional rice varieties of both Indica and Japonica types. These experimental data provide additional evidence for the proposal that Indica and Japonica rice arose from two independent domestication events in Asia.

The Oryza map alignment project: The golden path to unlocking the genetic potential of wild rice species

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the ‘Oryza Map Alignment Project’ (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project’s finished reference genome – O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara – thought to be the progenitor of modern cultivated rice.

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F2 population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F3 families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.

Genetics Of Resistance To Insects In Crop Plants

Publisher Summary This chapter reviews the genetics of resistance to insects in crop plants. These genes have been utilized in developing insect-resistant cultivars. Insect-resistant cultivars of major food crops—such as rice, wheat, corn, sorghum, and barley— are grown on millions of hectares annually. Farmers growing these save billions of dollars in insecticide costs and supply lower cost food to the consumer. Insect-resistant cultivars have greater yield stability and thus are responsible for greater food security. The chapter presents several cases, such as rice variety IR36, which is resistant to brown planthopper, green leafhopper, yellow stem borer, striped stem borer, and gall midge was planted in over 10 million ha of riceland of the world annually (IRRI, 1982). Its cultivation alone yielded an additional income of one billion dollars annually to rice growers and processors. Numerous rice cultivars with multiple resistances to insects have been developed and are widely grown. However, for the continued success of host resistance, one must have dynamic programs for gene identification. New genes for resistance must be continuously utilized in breeding programs to ensure the availability of resistant cultivars when old ones become susceptible because of the development of new biotypes.

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F1 hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F1 hybrids were completely male sterile. We did not obtain any BC1 progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F1 hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC1 plants varied from 28 to 31, and all were male sterile. BC2 plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC2 F2 progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC2 progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC2 F4 progenies 4 were resistant to BPH and 1 to race 6 of BB. F3 segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.

Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv. to rice, Oryza sativa L.

Oryza latifolia, a tetraploid wild relative of cultivated rice is an important source of resistance to bacterial blight (BB), the brown planthopper (BPH) and the whitebacked planthopper (WBPH). Interspecific hybrids were obtained between an elite breeding line (IR31917-45-3-2) of Oryza sativa (2n = 24 AA) and O. latifolia Acc. No. 100914 (2n = 48 CCDD). The crossability in F1 was 7.58% and it ranged from 0.11 to 0.62 in backcross generations. The F1 hybrid showed 2–6 II, 0–2 III, 0–1 IV and 22–32 I; the mean being 3.92 II + 0.11 III + 0.02 IV + 27.30 I per cell at diakinesis. Monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. latifolia were characterized based on morphology and isozyme banding pattern. The MAALs were designated as MAAL-1, MAAL-2, MAAL-4, MAAL-5, MAAL-6, MAAL-7, MAAL-8, MAAL-9, MAAL-10, MAAL-11 and MAAL-12. The female transmission rates of the alien chromosome varied from 4.4 to 35.5%, whereas 8 of the 11 MAALs transmitted the alien chromosome through the male gamete, the range being 1.7% (MAAL 10) to 11.9% (MAAL 12). Disomic progenies in BC3 and BC4 generations had complete resemblance to the O. sativa parent. Of the 2,295 disomic BC3F3 progenies, 309 showed introgression for resistance to BPH and 188 each for WBPH and BB resistance. Four plant progenies which were resistant to both BPH and WBPH were also resistant to BB race 2 of the Philippines. Nine of the 34 BC3F1 plants showed introgression for ten allozymes of O. latifolia, such as Est5, Amp1, Pgi1, Mdh3, Pgi2, Amp3, Pgd2, Est9, Amp2 and Sdh1, located on 8 of the 12 chromosomes. Alien introgression was also detected for morphological traits such as long awns, earliness, black hull, purple stigma and apiculus. Abnormal plants with many wild-species traits suddenly appeared in normal disomic progenies. These plants showing instability and abnormal segregation behaviour are being investigated for the activation of transposons.