Recurrent Parent Genome Recovery Research Articles

Development of multinutrient-rich biofortified sweet corn hybrids through genomics-assisted selection of shrunken2, opaque2, lcyE and crtRB1 genes.

Sweet corn has gained worldwide popularity. Traditional sweet corn possesses low concentration of essential nutrients such as lysine (0.15-0.25%), tryptophan (0.03-0.04%) and provitamin-A (proA 3-4 ppm), and deficiency leads to serious health problems in humans. Here, stacking of shrunken2 (sh2), opaque2 (o2), lycopene epsilon cyclase(lcyE) and β-carotene hydroxylase(crtRB1) genes were undertaken in the parents of four hybrids viz., APQH1, APHQ4, APHQ5 and APHQ7 using marker-assisted backcross breeding (MABB). Gene-linked markers (umc2276 and umc1320) for sh2, while gene-based markers for o2 (umc1066 and phi057), lcyE (5'TE-InDel) and crtRB1 (3'TE-InDel), were used for genotyping in BC1F1, BC2F1 and BC2F2. Selected backcross progenies showed high recovery of recurrent parent genome (92.4-97.7%). The reconstituted sweet corn hybrids possessed significantly high lysine (0.390%), tryptophan (0.082%) and proA (21.14 ppm), coupled with high kernel sweetness (brix 18.96%). The improved sweet corn hybrids had high cob yield (12.22-15.33 t/ha) across three environments. These newly developed biofortified sweet corn hybrids possess great significance in providing balanced nutrition. This is the first report of combining sh2, o2, lcyE and crtRB1 genes for enrichment of sweet corn hybrids with multiple essential nutrients.

Marker-Assisted Introgression and Stacking of Major QTLs Controlling Grain Number (Gn1a) and Number of Primary Branching (WFP) to NERICA Cultivars.

The era of the green revolution has significantly improved rice yield productivity. However, with the growing population and decreasing arable land, rice scientists must find new ways to improve rice productivity. Although hundreds of rice yield-related QTLs were already mapped and some of them were cloned, only a few were utilized for actual systematic introgression breeding programs. In this study, the major yield QTLs Grain Number 1a (Gn1a) and Wealthy Farmer’s Panicle (WFP) were introgressed and stacked in selected NERICA cultivars by marker-assisted backcross breeding (MABB). The DNA markers RM3360, RM3452, and RM5493 were used for foreground selection. At BC3F4 and BC3F5 generation, a combination of marker-assisted selection and phenotypic evaluation were carried out to select lines with target alleles and traits. Further, genotyping-by-sequencing (GBS) was conducted to validate the introgression and determine the recurrent parent genome recovery (RPGR) of the selected lines. The Gn1a and/or WFP introgression lines showed significantly higher numbers of spikelets per panicle and primary branching compared to the recurrent parents. In addition, lines with Gn1a and/or WFP alleles were comparatively similar to the recurrent parents (RP) in most yield-related traits. This study demonstrates the success of utilizing yield QTLs and marker-assisted selection to develop and improve rice cultivars.

Validating a segment on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety

Cadmium (Cd) contamination of rice is a serious food safety issue that has recently been gaining significant public attention. Therefore, reduction of Cd accumulation in rice grains is an important objective of rice breeding. The use of favourable alleles of Cd accumulating genes using marker-assisted selection (MAS) is theoretically feasible. In this study, we validated a segment covering OsHMA3-OsNramp5-OsNramp1 on chromosome 7 of japonica for establishing low-cadmium accumulating indica rice variety. The OsHMA3-OsNramp5-OsNramp1jap haplotype significantly decreased grain Cd concentration in middle-season indica genetic background. The improved 9311 carrying the OsHMA3-OsNramp5-OsNramp1jap haplotype with recurrent parent genome recovery of up to 91.6% resulted in approximately 31.8% decrease in Cd accumulation in the grain and with no penalty on yield. There is a genetic linkage-drag between OsHMA3-OsNramp5-OsNramp1 jap and the gene conditioning heading to days (HTD) in the early-season indica genetic background. Because the OsHMA3-OsNramp5-OsNramp1-Ghd7jap haplotype significantly increases grain Cd concentration and prolongs growth duration, the linkage-drag between OsHMA3-OsNramp5-OsNramp1 and Ghd7 should be broken down by large segregating populations or gene editing. A novel allele of OsHMA3 was identified from a wide-compatibility japonica cultivar, the expression differences of OsNramp1 and OsNramp5 in roots might contribute the Cd accumulating variation between japonica and indica variety.

Introgression of "QTL-hotspot" region enhances drought tolerance and grain yield in three elite chickpea cultivars.

With an aim of enhancing drought tolerance using a marker-assisted backcrossing (MABC) approach, we introgressed the "QTL-hotspot" region from ICC 4958 accession that harbors quantitative trait loci (QTLs) for several drought-tolerance related traits into three elite Indian chickpea (Cicer arietinum L.) cultivars: Pusa 372, Pusa 362, and DCP 92-3. Of eight simple sequence repeat (SSR) markers in the QTL-hotspot region, two to three polymorphic markers were used for foreground selection with respective cross-combinations. A total of 47, 53, and 46 SSRs were used for background selection in case of introgression lines (ILs) developed in genetic backgrounds of Pusa 372, Pusa 362, and DCP 92-3, respectively. In total, 61 ILs (20 BC3 F3 in Pusa 372; 20 BC2 F3 in Pusa 362, and 21 BC3 F3 in DCP 92-3), with >90% recurrent parent genome recovery were developed. Six improved lines in different genetic backgrounds (e.g. BGM 10216 in Pusa 372; BG 3097 and BG 4005 in Pusa 362; IPC(L4-14), IPC(L4-16), and IPC(L19-1) in DCP 92-3) showed better performance than their respective recurrent parents. BGM 10216, with 16% yield gain over Pusa 372, has been released as Pusa Chickpea 10216 by the Central Sub-Committees on Crop Standards, Notification and Release of Varieties of Agricultural Crops, Ministry of Agriculture and Farmers Welfare, Government of India, for commercial cultivation in India. In summary, this study reports introgression of the QTL-hotspot for enhancing yield under rainfed conditions, development of several introgression lines, and release of Pusa Chickpea 10216 developed through molecular breeding in India.

Introgression of qDTY1.1 Governing Reproductive Stage Drought Tolerance into an Elite Basmati Rice Variety “Pusa Basmati 1” through Marker Assisted Backcross Breeding

Drought stress at the reproductive stage in rice is one of the most important cause for yield reduction, affecting both productivity and quality. All Basmati rice varieties, including the popular cultivar “Pusa Basmati 1 (PB1)” is highly sensitive to reproductive stage drought stress (RSDS). We report for the first time, improvement of a Basmati cultivar for RSDS tolerance, with the introgression of a major quantitative trait locus (QTL), “qDTY1.1” into PB1. The QTL was sourced from an aus variety, Nagina 22 (N22). A microsatellite (simple sequence repeat (SSR)) marker “RM 431” located at telomeric end (38.89 mb) of chromosome 1, and located within a 1.04 mb QTL region was employed for foreground selection for qDTY1.1 in the marker assisted backcross breeding process. A set of 113 SSR markers polymorphic between N22 and PB1 were utilized for background selection to ensure higher recurrent parent genome recovery. After three backcrosses followed by five generations of selfing, eighteen near isogenic lines (NILs) were developed, through combinatory selection for agro-morphological, grain and cooking quality traits. The NILs were evaluated for three consecutive Kharif seasons, 2017, 2018 and 2019 under well-watered and drought stress conditions. RSDS tolerance and yield stability indicated that P1882-12-111-3, P1882-12-111-5, P1882-12-111-6, P1882-12-111-7, P1882-12-111-12, P1882-12-111-15 and P1882-12-111-17 were best in terms of overall agronomic and grain quality under RSDS. Additionally, NILs exhibited high yield potential under normal condition as well. The RSDS tolerant Basmati NILs with high resilience to water stress, is a valuable resource for sustaining Basmati rice production under water limiting production environments.

Drought Tolerant near Isogenic Lines (NILs) of Pusa 44 Developed through Marker Assisted Introgression of qDTY2.1 and qDTY3.1 Enhances Yield under Reproductive Stage Drought Stress

Reproductive stage drought stress (RSDS) is detrimental for rice, which affects its productivity as well as grain quality. In the present study, we introgressed two major quantitative trait loci (QTLs), namely, qDTY2.1 and qDTY3.1, governing RSDS tolerance in a popular high yielding non-aromatic rice cultivar, Pusa 44, through marker-assisted backcross breeding (MABB). Pusa 44 is highly sensitive to RSDS, which restricts its cultivation across drought-prone environments. Foreground selection was carried out using markers, RM520 for qDTY3.1 and RM 521 for qDTY2.1. Background selection was achieved with 97 polymorphic SSR markers in tandem with phenotypic selection to achieve faster recurrent parent genome (RPG) recovery. Three successive backcrosses followed by three selfings aided RPG recoveries of 98.6% to 99.4% among 31 near isogenic lines (NILs). Fourteen NILs were found to be significantly superior in yield and grain quality under RSDS with higher drought tolerance efficiency (DTE) than Pusa 44. Among these, the evaluation of two promising NILs in the multilocational trial during Kharif 2019 showed that they were significantly superior to Pusa 44 under reproductive stage drought stress, while performing on par with Pusa 44 under normal irrigated conditions. These di-QTL pyramided drought-tolerant NILs are in the final stages of testing the All India Coordinated Rice Improvement Project varietal trials for cultivar release. Alternately, the elite drought-tolerant Pusa 44 NILs will serve as an invaluable source of drought tolerance in rice improvement.

Pyramiding Ty-1/Ty-3, Ty-2, ty-5 and ty-6 genes into tomato hybrid to develop resistance against tomato leaf curl viruses and recurrent parent genome recovery by ddRAD sequencing method

Tomato leaf curl virus (ToLCV) is a begomovirus transmitted by whitefly (Bemisia tabaci) and causes Tomato (yellow) leaf curl disease (TYLCD/ToLCD) in many parts of India and rest of the world resulting in huge economic losses. Many Ty genes identified either from wild and/or cultivated tomato, which confers various levels of resistance to ToLCV. The current study aimed at stacking four important resistant genes (Ty-1/Ty-3, Ty-2, ty-5, and ty-6) in parental lines of a commercial tomato hybrid JKTH811 with the aid of the marker-assisted backcross breeding program. As a donor source, two pre-breeding lines, CLN3125K (Ty-1/Ty-3 and Ty-2) and FLA456 (ty-5 and ty-6) was used. Crossed population screened by using tightly linked Ty genes specific markers for foreground selection and attempted the fastest recovery of recurrent parental genome through NGS based ddRAD sequencing approach. Ty genes introgressed parental lines as well as the resulting F1 hybrids were tested for their bio-efficacy against ToLCNDV (New Delhi var.) under greenhouse and field conditions. Disease severity of Ty genes pyramided female lines and F1 hybrids were found as significantly lower when compared to original parental lines and hybrid. This report demonstrates the efficiency of Ty genes pyramiding from different donors in a hybrid against ToLCV and recovery of the recurrent genome using the ddRAD-seq platform in the minimum generations possible.

Recovery of Recurrent Parent Genome in a Marker-Assisted Backcrossing Against Rice Blast and Blight Infections Using Functional Markers and SSRs.

The most vital aspect of marker-assisted backcross breeding is the recurrent parent genome recovery. This enables the selection of only parents with recovered recipient/recurrent parent genome in addition to the targeted genes. The recurrent parent genome recovery (RPGR) ensures that non-desirable genomic segments are removed while the gene of interest is sustained in the recombined progenies without further segregations. This work was aimed at quantifying the RPGR of backcross populations with introgression of bacterial leaf blight resistance genes. Putra-1, a Malaysian elite variety, high yielding with inherent resistance to blast but susceptible to bacterial leaf blight (BLB), was crossed with IRBB60 which is resistant to BLB disease. The IRBB60 has four Xoo resistance genes—Xa4, xa5, xa13 and Xa21. Tightly linked polymorphic functional and SSR markers were used for foreground selection at every stage of backcrossing to select progenies with introgressed target genes. Background selection was done to quantify the percentage of RPGR in the selected lines using 79 confirmed polymorphic microsatellites. Result obtained showed that the percentage of RPGR was 80.11% at BC1F1, 95.30% at BC2F1 and 95.9% at BC2F2. The introgression of Xa4, xa5, xa13 and Xa21 Xoo resistance genes were faster through the adopted marker-assisted backcross breeding compared to what could be obtained through conventional breeding. All the 16 selected lines displayed resistance to BLB with three lines showing high resistance to the disease. The blast resistance contained in the genetic background of Putra-1 was also sustained in all the selected lines. The newly developed lines were recommended as new rice varieties for commercial cultivation.

Enhancing β-Carotene Concentration in Parental Lines of CO6 Maize Hybrid Through Marker-Assisted Backcross Breeding (MABB).

Vitamin A deficiency (VAD) is a global health problem; many people around the world, especially children and pregnant women, are VAD deficient or insufficient. Maize is known as an important source of provitamin A for humans. Hence, enhancement of provitamin A carotenoids (pVAC) in maize varieties through breeding or biofortification is a good option for alleviating VAD in developing countries, especially India. So far, numerous maize hybrids have been developed in India. Among them, CO6, derived from UMI1200 × UMI1230, is a popular maize hybrid and adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. However, CO6 is deficient for pVAC carotenoid β-carotene. Thus, the objectives of this study were to increase the β-carotene concentration in UMI1200 and UMI1230 and generate the β-carotene enriched hybrids through marker-assisted backcross breeding (MABB). For this purpose, the maize genotype HP467-15 was used as the donor for transferring the β-carotene gene, crtRB1, into UMI1200 and UMI1230. In the MABB scheme, we used one gene-specific marker (crtRB1 3′TE) and 214 simples sequence repeat (SSR) markers for foreground and background selection, respectively. As a result, six improved lines with recurrent parent genome recovery (RPGR) ranging from 90.24 to 92.42%, along with good agronomic performance, were generated. The β-carotene concentration of the improved lines ranged from 7.056 to 9.232 μg/g. Furthermore, five hybrid combinations were generated using improved lines and evaluated in a comparative yield trial (CYT) and multi-location trials (MLT) along with the original hybrid CO6 and commercial hybrids. It was revealed that ACM-M13-002 was a superior hybrid with a 7.3-fold increase in β-carotene concentration and with a comparable yield to CO6. In summary, the improved maize inbreds can be used as possible donors for the development of β-carotene-rich cultivars in maize breeding programs and the β-carotene enriched hybrid developed in this study will hold great promise for food and nutritional security.

Marker Assisted Development and Characterization of Herbicide Tolerant Near Isogenic Lines of a Mega Basmati Rice Variety, \u201cPusa Basmati 1121\u201d

BackgroundDirect-seeded rice (DSR) is a potential technology for sustainable rice farming as it saves water and labor. However, higher incidence of weed under DSR limits productivity. Therefore, there is a need to develop herbicide tolerant (HT) rice varieties.ResultsWe used marker assisted backcross breeding (MABB) to transfer a mutant allele of Acetohydroxy acid synthase (AHAS) gene, which confers tolerance to imidazolinone group of herbicides from the donor parent (DP) “Robin” into the genetic background of an elite popular Basmati rice variety, Pusa Basmati 1121 (PB 1121). Foreground selection was done using the AHAS gene linked Simple Sequence Repeat (SSR) marker RM6844 and background selection was performed using 112 genome-wide SSR markers polymorphic between PB 1121 and Robin. Phenotypic selection for agronomic, Basmati grain and cooking quality traits in each generation was carried out to improve the recovery of recurrent parent phenome (RPP). Finally, a set of 12 BC4F4 near isogenic lines (NILs), with recurrent parent genome (RPG) recovery ranging from 98.66 to 99.55% were developed and evaluated. PB 1121-HT NILs namely 1979-14-7-33-99-10, 1979-14-7-33-99-15 and 1979-14-7-33-99-66 were found superior to PB 1121 in yield with comparable grain and cooking quality traits and herbicide tolerance similar to Robin.ConclusionOverall, the present study reports successful development of HT NILs in the genetic background of popular Basmati rice variety, PB 1121 by introgression of mutated AHAS allele. This is the first report on the development of HT Basmati rice. Superior NILs are being evaluated in the national Basmati trials, the release of which is likely to provide a viable option for the adoption of DSR technology in Basmati rice cultivation.

Biofortification of sweet corn hybrids for provitamin-A, lysine and tryptophan using molecular breeding

Traditional sweet corn is poor in provitamin-A, lysine and tryptophan, deficiency of which causes serious health problems. Here, parental lines of two shrunken2 (sh2) -based sweet corn hybrids viz., ASKH-1 and ASKH-2 were targeted for introgression of crtRB1 and opaque2 (o2) genes through marker-assisted backcross breeding. Gene-based markers; umc1066 (SSR) and 3′TE-InDel were utilized for foreground selection of o2 and crtRB1, respectively in BC1F1, BC2F1 and BC2F2 generations. Background selection employing 102–113 polymorphic SSRs led to >90% recovery of recurrent parent genome. Reconstituted hybrids recorded high mean provitamin-A (18.98 μg/g) with a maximum of 7.7-fold increase over original hybrids (3.12 μg/g). High mean lysine (0.39%) and tryptophan (0.10%) with an average enhancement of 1.71- and 1.79-fold, respectively was recorded among reconstituted hybrids over original versions (lysine: 0.23%, tryptophan: 0.06%). Improved hybrids exhibited high phenotypic resemblance with their original hybrids. The average cob yield (11.82 t/ha) and brix (17.66%) of improved hybrids was at par with their original versions (cob yield: 11.27 t/ha, brix: 17.04%). These biofortified sweet corn hybrids rich in provitamin-A, lysine and tryptophan hold immense significance as multinutrient-rich balanced food. This is the first report to stack sh2, crtRB1 and o2 genes to improve nutritional quality in sweet corn.

Marker aided introgression of \u2018Saltol\u2019, a major QTL for seedling stage salinity tolerance into an elite Basmati rice variety \u2018Pusa Basmati 1509\u2019

Marker assisted backcross breeding was used to transfer Saltol, a major QTL for seedling stage salinity tolerance from the donor FL478 to Pusa Basmati 1509 (PB 1509), a high yielding and early maturing Basmati rice variety. Foreground selection was carried out using three markers namely, AP3206f, RM3412b and RM10793, linked to Saltol. In addition, 105 genome-wide SSR markers polymorphic between FL478 and PB 1509 were used in background selection. Among the BC3F4 near isogenic lines (NILs) developed, recurrent parent genome recovery ranged from 96.67 to 98.57%. Multi-season evaluation identified some of the NILs showing significantly higher yield with grain and cooking quality comparable to PB 1509. All the NILs exhibited tolerance to salinity with significantly higher relative water content, membrane stability index and proline content as compared to PB 1509. The root and shoot concentration of Na+, K+ and Na+/K+ in NILs was at par with FL478 under stress conditions. The gene OsHKT1;5 located in the Saltol region showed higher expression levels under stress indicating its role in conferring salinity tolerance. Salt tolerant NILs of PB 1509 will be useful in stabilizing production in salt affected areas.

Development of lysine and tryptophan rich maize (Zea mays) inbreds employing marker assisted backcross breeding

The molecular markers for mutant opaque2 allele and modifier loci (endosperm/amino acid) have provided opportunity to apply marker assisted backcross breeding to enhance essential amino acids (a.a.), lysine (lys) and tryptophan (trp) in maize endosperm. In present study, SSR phi057 that lie within opaque2, SSRs flanking opaque2, modifier loci and genic SSRs unlinked to opaque2 were used for molecular characterization in backcrosses between opaque2 donor, CML142 and the recurrent parents, BML2 and BML15 (parental lines of DHM119). Two double recombinant individuals, with highest recovery of recurrent parent genome (RPG) each from BML2-BC2F1 (91.25% RPG) and BML15-BC2F1 (91.77% RPG) with <25% kernel opacity were advanced (BC2F2 and BC2F3). Two individuals from BML2-BC2F2 (P117 and P268) and 3 individuals from BML15-BC2F2 populations (P27, P59 and P287) with highest trp and lys content (0.96 and 3.96% in BML2-BC2F2; 0.98 and 3.92% in BML15-BC2F2) were selfed and rigorous phenotyping was performed in BC2F3 populations. The normal and converted lines (CBML2 and CBML15) were statistically significant for all the phenotypic characters. The trp and lys content in converted inbreds was significantly higher than the normal lines.

Marker assisted conversion of a high oleic maintainer line into a high oleic CMS line in sunflower (Helianthus annuus L.)

In order to diversify the CMS lines for a sustainable production system of hybrid sunflower seeds with enhancedoleic acid content, the present study was attempted for the first time in India to transfer cytoplasmic male sterility into a newly developed high oleic maintainer line, COSFHO 7B using marker-assisted backcross breeding (MABB) approach. This MABC strategy successfully recovered the recurrent parent genome in BC3F1 generation and decreased heterozygosity of newly developed high oleic CMS plants. Field evaluation of few important agronomic traits of the high oleic CMS line in BC4F1 generation showed that it was comparable to the fertile maintainer counterpart, COSFHO 7B. The present study implies that marker assisted backcross breeding strategy could considerably decrease the time needed for complete recovery of recurrent parent genome.

Introgression of QTLs determining sorghum downy mildew (SDM) resistance into elite maize line UMI 79 through marker-assisted backcross breeding (MABC)

Sorghum downy mildew (SDM), caused by Peronosclerospora sorghi is one of the important diseases in maize. Here, we illustrate the transfer of two QTLs for SDM on chromosome 3 and 6 from UMI 936 (w) into an elite maize inbred line UMI 79 through marker-assisted backcross breeding (MABC). For foreground selection, plants carrying heterozygote allele for the QTLs for SDM were selected from F1 and three back cross (BC1-BC3) generations using simple sequence repeat (SSR) markers linked to the targeted QTLs region of SDM. Consequently, foreground positive plants from BC3F1 generation selfed to produce BC3F2 and plants carrying the favorable alleles for the QTLs for SDM were selected followed by stringent phenotyping against SDM and advanced to BC3F3. As a result, two improved lines (79/936–1–7-7-7-46-46 and 79/936–1–7-7-10-80-80) possessing QTLs for SDM were obtained. Background selection using 146 SSR markers distributed evenly across the maize genome revealed 92.45% and 89.68% recovery of recurrent parent genome (RPG) in two improved lines. Phenotypic characterization of the improved lines showed more than 90% of recovery of the recurrent parents in morphological traits and a resistance to SDM. In conclusion, the newly developed SDM resistant inbred lines are a potential source for improving SDM resistance in maize breeding programs.

Marker-assisted selection of qMrdd8 to improve maize resistance to rough dwarf disease.

Maize rough dwarf disease (MRDD) is caused by viruses in the Fijivirus genus in the family Reoviridae. MRDD resistance can be improved by a combination of marker-assisted selection (MAS) and conventional breeding strategies. In our previous study, we fine-mapped a major QTL qMrdd8 and developed the functional Indel marker IDP25K. In the present study, qMrdd8 from the donor parent X178 was introgressed into elite inbred lines derived from the three corn heterotic groups using multi-generation backcrossing and MAS. Recipient lines included Huangzao4, Chang7-2, Ye478, Zheng58, Zhonghuang68, B73, and Ji846. Markers used for foreground selection included IDRQ4, IDRQ47, IDP25K, and IDP27K. Background selection was carried out in the BC3 or BC4 using 107 SSR markers to select lines with the highest rate of recovery of the particular recurrent parent genome. Plants from BC4F2 and BC3F2 that carried the shortest qMrdd8 interval from X178 and those with the highest rate of recovery of the recurrent parent genome were then selected to create converted homozygous inbred lines. In 2017, seven converted inbred lines and five hybrids exhibited enhanced resistance to MRDD, while other agronomic traits were not affected under nonpathogenic stress conditions. Thus, the MRDD resistance allele at the qMrdd8 locus, or IDP25K, should be valuable for maize breeding programs in China.

Marker-Assisted Introgression of Multiple Resistance Genes Confers Broad Spectrum Resistance against Bacterial Leaf Blight and Blast Diseases in PUTRA-1 Rice Variety

Bacterial leaf blight caused by Xanthomonas oryzae pv oryzae (Xoo) and blast caused by Magnaporthe oryzae are major diseases responsible for significant yield loss in rice production across all rice growing regions. Host plant resistance has been advocated as a sustainable means of guarding against the diseases. This experiment was conducted with the aim to introgress multiple resistance genes against bacterial leaf blight and blast diseases through marker-assisted backcross breeding. Two dominant (Xa4 and Xa21) and two recessive (xa5 and xa13) Xoo resistance genes were introgressed into a high yielding Malaysian rice variety Putra-1 with genetic background of three blast resistance (Piz, Pi2 and Pi9) genes. Eight polymorphic tightly linked functional and SSR markers were used for foreground selection of target genes. Seventy nine polymorphic SSR markers were used in background selection. The plants were challenged at initial stage of breeding and challenged again at BC2F2 with the most virulent Malaysian pathotypes of Xoo (P7.7) and Magnaporthe oryzae (P7.2) to test their resistance. Results obtained from foreground marker segregation analysis at BC1F1 and BC2F1 showed that the marker polymorphism both fitted into the Mendel’s single gene segregation ratio of 1:1 for both Xoo and blast resistance. At BC2F2, results indicated that foreground marker polymorphism fitted into the expected Mendelian ratio of 1:2:1 for blast resistance only. Marker-assisted background selection revealed high percentage of recurrent parent genome recovery (95.9%). It was concluded that the inheritance of blast resistance in the introgressed lines was mainly due to single gene action while the inheritance of Xoo resistance was substantially due to single nuclear gene action. The incorporation of four bacterial leaf blight and three blast resistance genes (Xa4 + xa5 + xa13 + Xa21; Pi9 + Pi2 + Piz) in the newly developed lines would provide for broad spectrum and durable resistance against the two major diseases studied.

INTROGRESSION OF BACTERIAL BLIGHT RESISTANCE GENES; Xa33 AND Xa38 INTO RICE GENOTYPE ADT 47 THROUGH MARKER ASSISTED SELECTION

Bacterial blight is one of the serious diseases of rice. Utilization of resistant varieties is considered to be the most effective method of control. ADT 47 rice genotype has nearly been improved with the blight resistance genes xa5, xa13 and Xa21, through marker assisted selection (MAS). As the objective of this study, marker assisted backcrossing breeding (MABB) was adopted for targeted introgression of the broad spectrum resistance genes, Xa33 and Xa38 to the Improved ADT 47 rice variety. The rice genotypes FBRI-15, PR114 were used as donors of Xa33 and Xa38 genes, respectively. The marker RMWR7.1 linked to Xa33 and the sequenced-tagged site marker Os04g53050-1 specific to Xa38 were validated in parents who were used for foreground selection. True F1 hybrids were selected using the polymorphic markers. Out of one hundred and thirty seven SSR markers, forty-two were polymorphic for Improved ADT 47 x FBRI-16 which was used for background selection. Foreground selection revealed that a single F1 plant was heterozygote in Improved ADT 47 x FBRI-15 and it was used to develop BC1 generation. At BC1F1, one plant was found to be heterozygous for Improved ADT 47 x FBRI-15. At BC2F1, a single plant was found heterozygous as well from the cross. In the final BC3F1 population, one heterozygous plant was validated from the cross, the plant was subjected to background selection for selecting plants having the genetic background similar to that of the recurrent parent (i.e., confirmation of recurrent parent genome recovery) using the forty-two dominant SSR markers. In BC3F1, there was a 92.80% parental genome recovery in the Improved ADT 47 x FBRI-15. This high parental genome line was selfed to produce BC2F2 for selection of homozygous lines for xa5, xa13, Xa21 and Xa33/Xa38. The study recommends that the BC2F2 plants will be selfed using pedigree method and then advance to preliminary yield trial (PYT), then Advanced yield trial (AYT) and finally to on farm assessment for release.

Basmati rice lines development carrying multiple bacterial blight resistance genes pyramided using the marker-assisted backcross breeding approach

Basmati rice is treated as one of the world’s best rice due to its extra-long grain and unique aroma,. Over the last decade, Pakistan has earned more than US$2 billion through basmati export. Unfortunately, basmati rice is being threatened with bacterial leaf blight (BLB) disease that leads to poor production and quality. We attempted pyramiding of BLB genes through marker-assisted backcross breeding using Super Basmati as the recipient and IRBB60 as the donor parent. BLB resistance genes Xa4, xa5, and Xa21 were used for introgression. Molecular markers MP1 and MP2, RM122, and pTA248 were used for Xa4, xa5, and Xa21 genes respectively for foreground selection. The 85 SSR polymorphic molecular markers were used for background selection, which showed a more than 90% recurrent parent genome recovery. Phenotypic traits for grain dimensions were also considered while selecting backcross female parents from BC1F1 to BC5F1, which resulted in saving the resources from background selection before the selfing of backcrossed lines. Multiple lines carrying BLB resistance genes combinations Xa4 + xa5 and Xa4 + xa5 + Xa21 were developed. The BC5F4 uniform lines showed resistance against BLB disease at hotspot locations. One of the developed lines, PKBB15–116, which has a yield advantage of more than 10%, tolerant to lodging, and has basmati quality. These pyramided lines in basmati background will also be helpful for breeders to incorporate resistance genes in a shorter period of time, in order to develop new basmati rice varieties in future.

Identification of a novel locus, BPH38(t), conferring resistance to brown planthopper (Nilaparvata lugens Stal.) using early backcross population in rice (Oryza sativa L.)

Rice is the most important staple food crop, and it feeds more than half of the world population. Brown planthopper (BPH) is a major insect pest of rice that causes 20–80% yield loss through direct and indirect damage. The identification and use of BPH resistance genes can efficiently manage BPH. A molecular marker-based genetic analysis of BPH resistance was carried out using 101 BC1F5 mapping population derived from a cross between a BPH-resistant indica variety Khazar and an elite BPH-susceptible line Huang–Huan–Zhan. The genetic analysis indicated the existence of Mendelian segregation for BPH resistance. A total of 702 high-quality polymorphic single nucleotide polymorphism (SNP) markers, genotypic data, and precisely estimated BPH scores were used for molecular mapping, which resulted in the identification of the BPH38(t) locus on the long arm of chromosome 1 between SNP markers 693,369 and id 10,112,165 of 496.2 kb in size with LOD of 20.53 and phenotypic variation explained of 35.91%. A total of 71 candidate genes were predicted in the detected locus. Among these candidate genes, LOC_Os01g37260 was found to belong to the FBXL class of F-box protein possessing the LRR domain, which is reported to be involved in biotic stress resistance. Furthermore, background analysis and phenotypic selection resulted in the identification of introgression lines (ILs) possessing at least 90% recurrent parent genome recovery and showing superior performance for several agro-morphological traits. The BPH resistance locus and ILs identified in the present study will be useful in marker-assisted BPH resistance breeding programs.