Recurrent Parent Genome Recovery Research Articles

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC3F1 three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC3F2 generation.

Introgression of Shoot Fly (Atherigona soccata L. Moench) Resistance QTLs into Elite Post-rainy Season Sorghum Varieties Using Marker Assisted Backcrossing (MABC).

Shoot fly (Atherigona soccata L. Moench) is a serious pest in sorghum production. Management of shoot fly using insecticides is expensive and environmentally un-safe. Developing host–plant resistance is the best method to manage shoot fly infestation. Number of component traits contribute for imparting shoot fly resistance in sorghum and molecular markers have been reported which were closely linked to QTLs controlling these component traits. In this study, three QTLs associated with shoot fly resistance were introgressed into elite cultivars Parbhani Moti (= SPV1411) and ICSB29004 using marker assisted backcrossing (MABC). Crosses were made between recurrent parents and the QTL donors viz., J2658, J2614, and J2714. The F1s after confirmation for QTL presence were backcrossed to recurrent parents and the resultant lines after two backcrosses were selfed thrice for advancement. The foreground selection was carried out in F1 and BCnF1 generations with 22 polymorphic markers. Forty-three evenly distributed simple sequence repeat markers in the sorghum genome were used in background selection to identify plants with higher recurrent parent genome recovery. By using two backcrosses and four rounds of selfing, six BC2F4 progenies were selected for ICSB29004 × J2658, five BC2F4 progenies were selected for ICSB29004 × J2714 and six BC2F4 progenies were selected for Parbhani Moti × J2614 crosses. Phenotyping of these lines led to the identification of two resistant lines for each QTL region present on chromosome SBI-01, SBI-07 and SBI-10 in ICSB 29004 and Parbhani Moti. All the introgression lines (ILs) showed better shoot fly resistance than the recurrent parents and their agronomic performance was the same or better than the recurrent parents. Further, the ILs had medium plant height, desirable maturity with high yield potential which makes them better candidates for commercialization. In the present study, MABC has successfully improved the shoot fly resistance in sorghum without a yield penalty. This is the first report on the use of MABC for improving shoot fly resistance in post-rainy season sorghum.

Marker-assisted introgression of resistance to fusarium wilt race 2 in Pusa 256, an elite cultivar of desi chickpea.

Fusarium wilt caused by F. oxysporum f. sp. ciceris causes extensive damage to chickpea (Cicer arietinum L.) in many parts of the world. In the central part of India, pathogen race 2 (Foc 2) causes severe yield losses. We initiated molecular marker-assisted backcrossing (MABC) using desi cultivar, Vijay, as a donor to introgress resistance to this race (Foc2) in Pusa 256, another elite desi cultivar of chickpea. To confirm introgression of resistance for this race, foreground selection was undertaken using two SSR markers (TA 37 and TA110), with background selection to observe the recovery of recurrent parent genome using 45 SSRs accommodated in 8 multiplexes. F1 plants were confirmed with molecular markers and backcrossed with Pusa 256, followed by cycles of foreground and background selection at each stage to generate 161 plants in BC3F2 during the period 2009-2013. Similarly, 46 BC3F1 plants were also generated in another set during the same period. On the basis of foreground selection, 46 plants were found homozygotes in BC3F2. Among them, 17 plants recorded >91% background recovery with the highest recovery percentage of 96%. In BC3F1 also, 14 hybrid plants recorded a background recovery of >85% with the highest background recovery percentage of >94%. The identified plants were selfed to obtain 1341 BC3F3 and 2198 BC3F2 seeds which were screened phenotypically for resistance to fusarium wilt (race 2) besides doing marker analysis. Finally, 17 BC3F4 and 11 BC3F3 lines were obtained which led to identification of 5 highly resistant lines of Pusa 256 with Foc 2 gene introgressed in them. Development of these lines will help in horizontal as well as vertical expansion of chickpea in central part of India.

Introgression of Pi2 and Pi5 Genes for Blast (Magnaporthe oryzae) Resistance in Rice and Field Evaluation of Introgression Lines for Resistance and Yield Traits

AbstractBlast caused by Magnaporthe oryzae is the most devastating disease causing significant loss in rice production. The destructive nature of the disease is mainly due to the genetic plasticity of M. oryzae which complicates the breeding strategies. Blast can be effectively managed by the deployment of R genes. In this study, broad‐spectrum blast resistance genes Pi2 and Pi5 were introgressed independently into popular but blast susceptible rice variety, Samba Mahsuri (BPT5204) by applying marker‐assisted backcross breeding approach. Tightly linked markers AP5930 for Pi2 and 40N23r for Pi5 gene were used in foreground selection. Background selection helped to identify the lines with maximum recovery of recurrent parent genome (RPG). The RPG recovery in Pi2 introgression lines was up to 90.17 and 91.46% in Pi5 lines. Homozygous introgression lines in BC3F4 generation carrying Pi2 and Pi5 gene were field evaluated for blast resistance, yield per se and yield‐related traits. The lines showed resistance to leaf and neck blast in multilocation field evaluation. Improved BPT5204 lines with improvement for blast resistance were on par with original BPT5204 in terms of grain yield and grain features.

The Construction of a Chinese Cabbage Marker-assisted Backcrossing System Using High-throughput Genotyping Technolog

The goal of marker-assisted backcrossing (MAB) is to significantly reduce the number of breeding generations required by using genome-based molecular markers to select for a particular trait; however, MAB systems have only been developed for a few vegetable crops to date. Among the types of molecular markers, SNPs (single-nucleotide polymorphisms) are primarily used in the analysis of genetic diversity due to their abundance throughout most genomes. To develop a MAB system in Chinese cabbage, a high-throughput (HT) marker system was used, based on a previously developed set of 468 SNP probes (BraMAB1, Brassica Marker Assisted Backcrossing SNP 1). We selected a broad-spectrum TuMV (Turnip mosaic virus ) resistance (trs ) Chinese cabbage line (SB22) as a donor plant, constructing a BC₁F₁ population by crossing it with the TuMV-susceptible 12mo-682-1 elite line. Foreground selection was performed using the previously developed trs SCAR marker. Background selection was performed using 119 SNP markers that showed clear polymorphism between donor and recipient plants. The background genome recovery rate (% recurrent parent genome recovery; RPG) was good, with three of 75 BC₁F₁ plants showing a high RPG rate of over 80%. The background genotyping result and the phenotypic similarity between the recurrent parent and BC₁F₁ showed a correlation. The plant with the highest RPG recovery rate was backcrossed to construct the BC₂F₁ population. Foreground selection and background selection were performed using 169 BC₂F₁ plants. This study shows that, using MAB, we can recover over 90% of the background genome in only two generations, highlighting the MAB system using HT markers as a highly efficient Brassica rapa backcross breeding system. This is the first report of the application of a SNP marker set to the background selection of Chinese cabbage using HT SNP genotyping technology.

Molecular stacking of wide compatibility gene, S5 n and elongated uppermost internode (eui) gene into IR 58025B, an elite maintainer line of rice

One of the most widely used and stable wild-abortive cytoplasmic male sterile (WA-CMS) line is IR 58025A. We had earlier transferred the gene (eui1) for elongated uppermost internode into the elite WA-CMS line named IR 58025eB with the help of markers. To overcome incomplete panicle exsertion in the WA-CMS line and better exploitation of indica/japonica heterosis, we have stacked a major gene for wide-compatibility (S5 n ) into IR 58025eB through marker-assisted breeding. SSR markers (RM5970) tightly linked to eui1 gene and (S5-InDel) functional maker of S5 n gene were used for foreground selection, while ninety-one microsatellite markers polymorphic between IR 58025eB and donor Dular (S5 n ) were utilized for background selection at each backcross generation and backcrossing was continued till BC3 generation. Phenotypic observation previous to background selection at each generation provided supplementary information for identifying the novel recombinants and the fast-tracked recurrent parent genome recovery which ranged from 94.50 to 96.70% in selected BC3F4 plants. At BC3F5 generation, seven stable, backcross-derived lines possessing S5 n and eui genes, high yield, and long-slender grain type have been identified; their maintainer ability was confirmed through test crosses. These improved lines are being converted to CMS lines by marker-assisted backcrossing.

Pyramiding of leaf rust resistance genes in bread wheat variety DWR 162 through marker assisted backcrossing

A popular widely grown but leaf rust susceptible Indian bread wheat variety was pyramided with two leaf rust resistance genes, Lr24 and Lr28 by Marker Assisted Backcrossing (MABC). The near isogenic line of PBW 343 introgressed with Lr24 and Lr28 was used as a donor. The MABC approach employed one SSR marker, Xwmc313 and one SCAR marker SCS421 with its locus linked to Lr28 and two SCAR markers, SCS719 and SCS 1302 linked to Lr24 for foreground selection to select plants carrying Lr24 and Lr28 genes. Marker assisted background selection in BC2F2 to recover the genome of recipient parent involving 42 polymorphic SSR markers dispersed throughout the genome facilitated mean recurrent parent genome (RPG) recovery of 80.3% with a range from 67.5–94.7%. BC2F2 plants were characterized for DUS (Distinctiveness Uniformity Stability) for phenotypic identification of plants that mostly resemble DWR 162 variety. Field evaluation of BC2F2 plants for yield and its component traits to assess the effect of these two genes in the background of DWR 162 revealed high yield potential of selected plants along with leaf rust resistance and no yield penalty was apparent. This study successfully demonstrate the practical utility of MABC in wheat rust resistance breeding for developing resistant lines in the background of any elite and popular wheat cultivar with relatively higher speed and precision.

Development of advanced fragrant rice lines from MR269 × Basmati 370 through marker-assisted backcrossing

Fragrance in rice is an appealing attribute to consumers. The increasing demand for fragrant rice highlights the need to develop fragrant rice variety that suit the preference of local consumers in addition to reduce fragrant rice imports. Marker-assisted backcrossing (MABC) was employed to develop advanced fragrant rice lines from the cross between MR269 and Basmati 370. MR269 is a Malaysian high-yielding rice variety but non-fragrant and was used as recurrent parent whereas Basmati 370 is a well-known fragrant traditional rice variety and was used as donor parent for the fragrance gene. Two generations of backcrosses and a generation of selfing were conducted to introgress the fragrance gene and restore the recurrent parent genome in the backcross progenies. As a result, 14 advanced fragrant rice lines were developed. These advanced fragrant rice lines carried homozygous alleles for the fragrance gene, similar to Basmati 370. The average recovery of recurrent parent genome was 88.4%. Besides being fragrant, the advanced fragrant rice lines also had most of the morphological and agronomical traits similar to MR269. Grain quality of the advanced fragrant rice lines in terms of gelatinization temperature, amylose content and gel consistency are also similar to both parents. Besides, the advanced fragrant rice lines had 2-acetyl-1-pyrroline content similar to Basmati 370. MABC approach applied in this study has successfully introgressed the fragrance gene and accelerated the recovery of recurrent parent genome in advanced fragrant rice lines, therefore these lines can be delivered to the farmers and consumers for use in due time.

Marker-assisted introgression of broad-spectrum blast resistance genes into the cultivated MR219 rice variety.

The rice cultivar MR219 is famous for its better yield and long and fine grain quality; however, it is susceptible to blast disease. The main objective of this study was to introgress blast resistance genes into MR219 through marker-assisted selection (MAS). The rice cultivar MR219 was used as the recurrent parent, and Pongsu Seribu 1 was used as the donor. Marker-assisted foreground selection was performed using RM6836 and RM8225 to identify plants possessing blast resistance genes. Seventy microsatellite markers were used to estimate recurrent parent genome (RPG) recovery. Our analysis led to the development of 13 improved blast resistant lines with Piz, Pi2 and Pi9 broad-spectrum blast resistance genes and an MR219 genetic background. The RPG recovery of the selected improved lines was up to 97.70% with an average value of 95.98%. Selected improved lines showed a resistance response against the most virulent blast pathogen pathotype, P7.2. The selected improved lines did not express any negative effect on agronomic traits in comparison with MR219. The research findings of this study will be a conducive approach for the application of different molecular techniques that may result in accelerating the development of new disease-resistant rice varieties, which in turn will match rising demand and food security worldwide. © 2016 Society of Chemical Industry.

Molecular Breeding of Rice Restorer Lines and Hybrids for Brown Planthopper (BPH) Resistance Using the Bph14 and Bph15 Genes.

BackgroundThe development of hybrid rice is a practical approach for increasing rice production. However, the brown planthopper (BPH), Nilaparvata lugens Stål, causes severe yield loss of rice (Oryza sativa L.) and can threaten food security. Therefore, breeding hybrid rice resistant to BPH is the most effective and economical strategy to maintain high and stable production. Fortunately, numerous BPH resistance genes have been identified, and abundant linkage markers are available for molecular marker-assisted selection (MAS) in breeding programs. Hence, we pyramided two BPH resistance genes, Bph14 and Bph15, into a susceptive CMS restorer line Huahui938 and its derived hybrids using MAS to improve the BPH resistance of hybrid rice.ResultsThree near-isogenic lines (NILs) with pyramided Bph14 and Bph15 were obtained by molecular marker-assisted backcross (MAB) and phenotypic selection. The genomic components of these NILs were detected using the whole-genome SNP (Single nucleotide polymorphism) array, RICE6K, suggesting that the recurrent parent genome (RPG) recovery of the NILs was 87.88, 87.70 and 86.62 %, respectively. BPH bioassays showed that the improved NILs and their derived hybrids carrying homozygous Bph14 and Bph15 were resistant to BPH. However, the hybrids with heterozygous Bph14 and Bph15 remained susceptible to BPH. The developed NILs showed no significant differences in major agronomic traits and rice qualities compared with the recurrent parent. Moreover, the improved hybrids derived from the NILs exhibited better agronomic performance and rice quality compared with the controls under natural field conditions.ConclusionsThis study demonstrates that it is essential to stack Bph14 and Bph15 into both the maternal and paternal parents for developing BPH-resistant hybrid rice varieties. The SNP array with abundant DNA markers is an efficient tool for analyzing the RPG recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important for rice molecular breeding.

Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21.

Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

The addition of submergence-tolerant Sub1 gene into high yielding MR219 rice variety and analysis of its BC2F3 population in terms of yield and yield contributing characters to select advance lines as a variety

ABSTRACTA cross was made between MR219 (high yielding but submergence intolerant) and Swarna-Sub1 (submergence tolerant) to produce submergence-tolerant rice variety using the marker-assisted backcrossing (MABC) method to protect the farmers of low-lying land from flash floods during rain. Knowledge of yield and yield contributing factors plays a vital role in the selection process of a variety. This experim ent was designed to determine the genetic diversity among recently produced different lines of BC2F3 population and also to compare all the lines with MR219 to find the best one. Agronomical, yield and yield contributing data were taken, while genotypic and phenotypic coefficients, variance components and heritability were estimated. Introgression of the target gene, Sub1, was done using tightly linked marker, and also background recovery was measured using simple sequence repeat (SSR) markers in different generations. The observed recurrent parent genome (RPG) recovery of BC2F2 generation was 95.37%, which indicates high-level similarity between the recurrent parent (MR219) and the resulting lines. Thirty newly developed lines of BC2F3 population, resulting backcross of MR219 and Swarna-Sub1, were planted with four replications following randomised complete block design (RCBD). Newly developed lines were grouped into four clusters based on traits with UPGMA dendrogram and cluster analysis to select the 10 best plants. This study will help the future researchers to select the best plants of a breeding programme after introgression of a gene considering phenotype performances to develop new varieties.

Application of marker‐assisted backcross to introgress Bph3, Bph14 and Bph15 into an elite indica rice variety for improving its resistance to brown planthopper

AbstractTo improve brown planthopper (Nilaparvata lugens Stål; BPH) resistance of an elite indica cultivar of South China, Hemeizhan (HMZ), we applied marker‐assisted backcross (MABC) to incorporate three BPH‐resistance genes (Bph3, Bph14 and Bph15) into the genetic background of HMZ. In the third backcross (BC3) generation, we obtained near‐isogenic lines (Bph3‐NIL, Bph14‐NIL, Bph15‐NIL and Bph14 + Bph15‐NIL) with more than 96% recovery of recurrent parent genome, and pyramided lines (Bph3 + Bph14‐PYL, Bph3 + Bph15‐PYL and Bph3 + Bph14 + Bph15‐PYL) with more than 89% recovery of recurrent parent genome. These lines showed stronger resistance against BPH than HMZ at seedling and booting stages. The rank of resistance gene effect was Bph3 + Bph14 + Bph15 ≥ Bph3 + Bph15 ≥ Bph3 +Bph14 ≥ Bph14 + Bph15 ≥ Bph3 ≥ Bph15 ≥ Bph14 &gt; none. Compared with HMZ, only Bph3 + Bph14 + Bph15‐PYL had a significant difference in yield per plant, and the lines carrying Bph3 had higher amylose contents, indicating that Bph3 was tightly linked to Wxa allele. These improved lines are good intermediate sources of broad‐spectrum and durable BPH resistance to improve other indica cultivars. Our results demonstrate that MABC is a very efficient approach to improve BPH resistance of elite rice cultivar.

Stacking S5-n and f5-n to overcome sterility in indica-japonica hybrid rice.

Pyramiding of S5 - n and f5 - n cumulatively improved seed-setting rate of indica-japonica hybrids, which provided an effective approach for utilization of inter-subspecific heterosis in rice breeding. Breeding for indica-japonica hybrid rice is an attractive approach to increase rice yield. However, hybrid sterility is a major obstacle in utilization of inter-subspecific heterosis. Wide-compatibility alleles can break the fertility barrier between indica and japonica subspecies, which have the potential to overcome inter-subspecific hybrid sterility. Here, we improved the compatibility of an elite indica restorer line 9311 to a broad spectrum of japonica varieties, by introducing two wide-compatibility alleles, S5-n and f5-n, regulating embryo-sac and pollen fertility, respectively. Through integrated backcross breeding, two near isogenic lines harboring either S5-n or f5-n and a pyramiding line carrying S5-n plus f5-n were obtained, with the recurrent parent genome recovery of 99.95, 99.49, and 99.44 %, respectively. The three lines showed normal fertility when crossed to typical indica testers. When testcrossed to five typical japonica varieties, these lines allowed significant increase of compatibility with constant agronomic performance. The introgressed S5-n could significantly improve 14.7-32.9 % embryo-sac fertility in indica-japonica hybrids. In addition, with the presence of f5-n fragment, S5-n would increase the spikelet fertility from 9.5 to 21.8 %. The introgressed f5-n fragment greatly improved anther dehiscence, embryo-sac and pollen fertility in indica-japonica hybrids, thus leading to improvement of spikelet fertility from 20.4 to 30.9 %. Moreover, the pyramiding line showed 33.6-46.7 % increase of spikelet fertility, suggesting cumulative effect of S5-n and f5-n fragment in seed-set improvement of inter-subspecific hybrids. Our results provided an effective approach for exploiting heterosis between indica and japonica subspecies, which had a profound implication in rice breeding.

Recurrent parent genome recovery in different populations with the introgression of Sub1 gene from a cross between MR219 and Swarna-Sub1

Recurrent parent genome (RPG) recovery is the most important step of marker assisted backcross breeding. This breeding approach is used to develop new varieties by inserting a particular gene of interest into the background of a modern variety, and removing undesirable segments linked with the target allele, which can survive after many generations of backcrossing. The marker assisted backcrossing technique is the most effective way to minimize the limitation of conventional breeding, and recover the parental genome within 2–3 generations. MR219, A modern rice variety of Malaysia (susceptible to submergence) was crossed with a high-yielding submergence tolerant variety Swarna-Sub1 (donor) to produce a new submergence tolerant rice variety. Foreground selection for the sub1 gene was done using tightly linked markers. Estimation of RPG recovery was conducted in earlier generations with previously screened polymorphic SSR markers. A total of 385 SSR markers were tested to identify the polymorphism between the parents and 88 of them found to be polymorphic. Background analysis revealed 65.55–77.8 and 78.79–95.5 % recovery in BC1F1 and BC2F1 generations, respectively. In the BC2F2 generation the average RPG recovery was 95.37 % and that for the selected plant was 96.3 %. This study revealed the usefulness of marker assisted backcrossing for the quick recovery of a parental genome in a backcrossing population.

Improvement of Basmati rice varieties for resistance to blast and bacterial blight diseases using marker assisted backcross breeding

Marker assisted backcross breeding was employed to incorporate the blast resistance genes, Pi2 and Pi54 and bacterial blight (BB) resistance genes xa13 and Xa21 into the genetic background of Pusa Basmati 1121 (PB1121) and Pusa Basmati 6. Foreground selection for target gene(s) was followed by arduous phenotypic and background selection which fast-tracked the recovery of recurrent parent genome (RPG) to an extent of 95.8% in one of the near-isogenic lines (NILs) namely, Pusa 1728-23-33-31-56, which also showed high degree of resemblance to recurrent parent, PB6 in phenotype. The phenotypic selection prior to background selection provided an additional opportunity for identifying the novel recombinants viz., Pusa 1884-9-12-14 and Pusa 1884-3-9-175, superior to parental lines in terms of early maturity, higher yield and improved quality parameters. There was no significant difference between the RPG recovery estimated based on SSR or SNP markers, however, the panel of SNPs markers was considered as the better choice for background selection as it provided better genome coverage and included SNPs in the genic regions. Multi-location evaluation of NILs depicted their stable and high mean performance in comparison to the respective recurrent parents. The Pi2+Pi54 carrying NILs were effective in combating a pan-India panel of Magnaporthe oryzae isolates with high level of field resistance in northern, eastern and southern parts of India. Alongside, the PB1121-NILs and PB6-NILs carrying BB resistance genes xa13+Xa21 were resistant against Xanthomonas oryzae pv. oryzae races of north-western, southern and eastern parts of the country. Three of NILs developed in this study, have been promoted to final stage of testing during the ​Kharif 2015 in the Indian National Basmati Trial.

Marker-assisted introgression of bacterial blight and blast resistance into DRR17B, an elite, fine-grain type maintainer line of rice

DRR17A is a stable wild-abortive cytoplasmic male sterile line with medium-slender grain type. DRR17A and its maintainer line DRR17B are highly susceptible to two of the major rice diseases, bacterial blight (BB) and blast. To improve DRR17B for resistance against BB and blast, we have introgressed a major dominant gene each conferring resistance against BB (Xa21) and blast (Pi54) into the maintainer line through marker-assisted backcross breeding using RP-Bio-Patho-2 (a near-isogenic line of Samba Mahsuri possessing Xa21 and Pi54) as the donor parent. PCR-based molecular markers tightly linked to Xa21 and Pi54 were used for foreground selection of the resistance plants at each backcross generation, while molecular markers tightly linked to the major fertility restorer genes, Rf3 and Rf4, were used for negative selection (i.e. selection of plants possessing non-fertility-restoring alleles at the two loci) at BC1 generation. After foreground selection for the target genes at each backcross generation, the ‘positive’ plants were screened with parental polymorphic markers for identifying backcross plants possessing maximum recovery of DRR17B genome. Marker-assisted backcrossing was continued till BC3 generation, and a single BC3F1 plant possessing the target genes with ~94 % recovery of recurrent parent genome was identified and selfed to generate BC3F2s. A total of six homozygous BC3F2 plants were identified and advanced. At BC3F5, six promising, stable, backcross-derived lines possessing high level of resistance against BB and blast, high yield, short plant stature, fine-grain type, have been identified; their maintenance ability and heterotic potential validated through test crosses and these lines are being converted to CMS lines through marker-assisted breeding.

Marker-Assisted Backcrossing to Develop an Elite Cytoplasmic Male Sterility Line in Rice.

Cytoplasmic male sterility (CMS) is a cornerstone of hybrid production in many crops. In three-line hybrid systems, use of CMS, maintainer, and fertility restorer lines is necessary for production of hybrid seeds. Limited resources of CMS and low variation of CMS lines cause genetic vulnerability to pathogens. Therefore, diversifying the CMS sources is indispensible for a sustainable production system of hybrid seed. In this study, we attempted for the first time to transfer CMS into maintainer line Yosen B in restricted generations using the marker-assisted backcrossing (MABC) method. The resultant F1 hybrid of IR68897 A/Yosen B cross was backcrossed to Yosen B, and CMS plants in each backcross generation (from BC1 F1 to BC3 F1 ) were selected based on phenotyping test and MABC. Molecular assessment of backcross progenies was conducted using a mitochondrial CMS-specific marker and 34 polymorphic nuclear simple-sequence repeat (SSR) markers in early generations (from BC1 F1 to BC2 F1 ) and was continued using 9 additional SSRs and 82 inter-simple sequence repeat (ISSR) markers in BC3 F1 . A MABC strategy could successfully recover the recurrent parent genome (RPG) in BC3 F1 generation, and decreased heterozygosity of final CMS plants. Restorability test with known wild-abortive restorer lines (viz. IR36 and IR24) showed that combination of Yosen A × IR24 could produce highly fertile F1 hybrid. Evaluation of some important agronomic traits of the final CMS line (BC4 F1 ) at field condition showed that it was comparable to the original maintainer fertile counterpart. Phenotypic and marker-assisted selections could considerably decrease the time needed for full recovery of RPG so that final CMS line could show a high similarity to original fertile counterpart.

Introgression of blast resistance genes into the elite rice variety MR263 through marker-assisted backcrossing.

Blast caused by the fungus Magnaporthe oryzae is a significant disease threat to rice across the world and is especially prevalent in Malaysia. An elite, early-maturing, high-yielding Malaysian rice variety, MR263, is susceptible to blast and was used as the recurrent parent in this study. To improve MR263 disease resistance, the Pongsu Seribu 1 rice variety was used as donor of the blast resistance Pi-7(t), Pi-d(t)1 and Pir2-3(t) genes and qLN2 quantitative trait locus (QTL). The objective was to introgress these blast resistance genes into the background of MR263 using marker-assisted backcrossing with both foreground and background selection. Improved MR263-BR-3-2, MR263-BR-4-3, MR263-BR-13-1 and MR263-BR-26-4 lines carrying the Pi-7(t), Pi-d(t)1 and Pir2-3(t) genes and qLN2 QTL were developed using the simple sequence repeat (SSR) markers RM5961 and RM263 (linked to the blast resistance genes and QTL) for foreground selection and a collection of 65 polymorphic SSR markers for background selection in backcrossed and selfed generations. A background analysis revealed that the highest rate of recurrent parent genome recovery was 96.1% in MR263-BR-4-3 and 94.3% in MR263-BR-3-2. The addition of blast resistance genes can be used to improve several Malaysian rice varieties to combat this major disease.

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6%), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.