Marker assisted pedigree breeding based improvement of the Indian mega variety of rice MTU1010 for resistance against bacterial blight and blast and tolerance to low soil phosphorus

Rice production is affected by many biotic and abiotic stresses; among them, bacterial blight (BB) and blast diseases and low soil phosphorous stress cause significant yield losses. The present study was carried out with the objective of combining the BB resistance gene, Xa21, the blast resistance gene, Pi54, and the low soil phosphorous tolerance QTL/gene, Pup1, into the genetic background of the Indian mega-rice variety, MTU1010 (Cottondora Sannalu), through marker-assisted pedigree breeding. RP5973-20-9-8-24-12-7 [a near isogenic line (NIL) of MTU1010 possessing Pup1] and RP6132 [a NIL of Akshayadhan possessing Xa21 + Pi54] were crossed and ‘true’ F1s were identified, using the target gene-specific markers and selfed. F2 plants, which are homozygous for all the three target genes/QTLs, were identified using PCR based markers and were advanced further through the pedigree method of breeding, with selection based on phenotypic traits specific for MTU1010. At the F5 generation, a set of 15 promising triple positive homozygous lines were identified and screened for their resistance against BB and blast diseases and tolerance to low soil P. Among them, two lines (LPK 30-18-16 and LPK 49-15-22) showed higher yields as compared to MTU1010, along with the desirable long slender grain type in both low soil P and normal soil P plots, and also exhibited high levels of resistance against BB and blast diseases, with lesser grain shattering as compared to MTU1010. These lines are being advanced for multi-location trials for validating their performance.


Introduction
Rice is the predominant food crop of India in terms of area, production and consumption. In fact, the demand for rice is steadily increasing, as the number of rice consumers increase, especially in developing countries such as India [1]. It is predicted that the world population will exceed eight billion people in the next decade. Even though significant improvements have been witnessed in rice production following the advent of the Green Revolution, there is an imminent need to improve the productivity of rice (which has stagnated in the last decade or two), in order to meet the demands of an ever-growing population. This is indeed impeded by major biotic and abiotic stresses, causing huge yield losses in different rice growing ecosystems. Among the biotic stresses, two most devastating diseases in rice, namely blast, which is caused by the fungus Magnaporthe oryzae, and bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae, have plagued rice farmers since the beginning of rice cultivation throughout Asia and India [2]. BB and blast diseases can cause yield losses up to 74 to 81% [3] and 70 to 80% [1], respectively.
Among the abiotic stresses, low soil P stress is one of the major constraints on plant growth and yield worldwide, in many crops including rice [4,5]. It is estimated that in more than 40% of arable lands, crop productivity is limited by P deficiency, and hence farmers are applying more and more phosphatic fertilizers, which in turn increase the cost of cultivation and pollution of water bodies. Genetic improvement through the deployment of resistant/tolerant genes is the most effective approach to prevent damage due to the two major biotic stresses, viz., BB, blast [6] and also low soil P levels [7]. MTU1010 (IET15644; also known as Cottondora Sannalu) is a medium-duration Indian mega-rice variety, released by the Andhra Pradesh Rice Research Station (APRRS), Acharya N. G. Ranga Agricultural University (ANGRAU), Maruteru, Andhra Pradesh, India, during the year 2000. It is a high-yielding rice variety, adaptable to multiple rice growing ecosystems, and has a desirable medium-slender grain type. However, despite these desirable features, MTU1010 is highly susceptible to BB and has only a moderate level of tolerance to blast disease, both of which cause significant yield losses in the variety. Furthermore, MTU1010 is also highly sensitive to low soil P levels, which reduces its production in problem soils. In order to cope with these three problems, development of resistant/tolerant breeding lines and their cultivation is considered the most effective, economical and environment friendly strategy. Considering the imminent need to improve BB and blast resistance along with low P tolerance, through this study, we attempted to deploy the strategy of marker-assisted pedigree breeding (MAPB) to transfer Xa21, Pi54 and Pup1 into the genetic back ground of MTU1010.
Homozygous positive F 2 plants were identified through foreground selection, using the target QTLs/gene-specific markers mentioned above, and advanced further by selfing through pedigree breeding to F 5 generation, based on selection for phenotypic characters specific for MTU1010. Marker-assisted selection was carried out through mini-scale rapid DNA isolation protocol, described in [12]. PCR protocols described in [13] were adopted for indirect selection of Xa21and Pi54, while the PCR protocol described in [7] was adopted for indirect selection of Pup1, using the markers mentioned earlier. Fifteen promising homozygous positive F 5 lines, which closely resembled MTU1010 in terms of plant type and grain type, were selected and further evaluated for their resistance/tolerance to target stresses (i.e., BB and blast diseases and low soil phosphorous tolerance) as well as for key agro-morphological traits.

Evaluation of BB, blast resistance, and low soil phosphorous tolerance in the improved breeding lines phenotypic screening for BB resistance
Screening for BB resistance was carried out among the selected F 5 lines, along with the parents and check lines in the field at ICAR-IIRR, Hyderabad, during wet season (i.e., kharif season, 2018). The bacterial cultures of a virulent isolate of Xanthomonas oryzae pv. Oryzae (collected from Hyderabad, Telangana State, India), viz., DX-020 were maintained as per the protocols described by [14], and inoculation was done at maximum tillering stage by following the leaf clipping method described in [15]. The lesion length on the leaves were measured after 15 days of inoculation, and scoring was done as per the standard evaluation system (SES) of the International Rice Research Institute (IRRI), Philippines (IRRI, 2013).

Phenotypic screening for blast resistance
The selected F 5 lines along with the parents and checks-MTU1010 NIL (RP5973-20-9-8-24-12-7), Akshayadhan NIL (RP6132), susceptible check (HR12) and resistance check (Tetep) -were screened for blast resistance in the uniform blast nursery (UBN) at ICAR-IIRR, during wet season 2018. A local virulent isolated of the blast pathogen, Magnaporthe grisea, SPI-40, originally collected from Hyderabad, Telangana, India, was cultured and stored, as described by [16]. The young seedlings at the four-leaf stage were inoculated with the blast spores, as described in [13], and scored 15 days after inoculation, based on a 0 to 9 scale, as per IRRI-SES (IRRI, 2013).

Phenotypic screening for low soil P tolerance
During wet season 2018, the selected F 5 lines along with the parents and checks-MTU1010 NIL (RP5973-20-9-8-24-12-7), Akshayadhan NIL (RP6132), susceptible check (ISM) and resistance check (Swarna) were transplanted in the low soil P plot of ICAR-IIRR in a randomized block design of spacing 15cm x 20cm, in three replications. Except for the application of phosphorus, all the other agronomic practices including recommended doses of nitrogen, potassium and micro nutrients (i.e., Iron and zinc) were followed.

Evaluation of agro-morphological traits in the improved breeding lines of MTU1010
During wet season 2018, the selected F 5 lines possessing Pup1, Xa21 and Pi54, and the parents, MTU1010 NIL (RP 5973-20-9-8-24-12-7) and Akshayadhan NIL (RP 6132), were transplanted into the experimental farm of ICAR-IIRR, in 2m 2 plots in a randomized block design of spacing 15cmx20 cm in soil with optimum levels of macro and micro-nutrients. Data was recorded for the selected agro-morphological traits, i.e., days to 50% flowering (DFF), plant height (PH), number of productive tillers per plant (NPT), panicle length (PL), number of grains per panicle (GPP,nos.), dry shoot weight (DSW), dry root weight (DRW), root to shoot ratio (RSR), grain yield per plant (GY) and root volume (RV).

Statistical analysis
Data was recorded for all the parameters in both normal soil P and low soil P plots, and statistical analysis was conducted using the SAS 9.2 (SAS version 9.2 software packages; SAS Institute, Inc.; Cary, NC) software for analysis of coefficient of variation (CV), critical difference (CD), standard error (SE) and analysis of variance (ANOVA).

Combining BB, blast, and low P resistance/tolerance genes/QTLs into the genetic background of MTU 1010
A total of 96 F 1 seeds were generated from the cross between MTU1010 (RP5973-20-9-8-24-12-7, i.e., the female parent) and a NIL of Akshayadhan (RP6132, i.e., the male parent). Foreground selection was carried out with the target gene-specific markers, viz. K20-2, pTA248 and Pi54MAS, which are specific for Pup1, Xa21 and Pi54, respectively. A total of 87 true F 1 s (heterozygous plants) were identified. They were then selfed to generate the F 2 population consisting of 1267 plants. Phenotypic screening for BB resistance revealed a total of 980 resistant plants, which were further genotyped with Xa21 gene-specific marker, pTA248, among which 315 plants were identified to be homozygous for the gene. They were then subjected to a screening using Pi54 as the specific marker Pi54-MAS, and the Pup1 specific co-dominant CAPS marker, K20-2. Among the 315 plants, 15 were found to be triple positive (i.e., triple homozygous for all the three target genes, i.e., Pup1Pup1, Xa21Xa21, Pi54Pi54) (Fig 2). These 15 plants were the advanced through pedigree breeding till F 5 generation. In each generation of selfing, phenotypic selection was deployed for identification of promising plants, resembling MTU1010, based on agro-morphological traits.

Assessment of the improved breeding lines under BB, blast and low P stresses
Phenotypic screening was carried out for fifteen homozygous F 5 lines against three target stresses viz., low soil P, BB and blast diseases during wet season, 2018, at ICAR-IIRR, Hyderabad.

Evaluation of improved breeding lines for agro-morphological traits
Under normal soil P condition, 15 breeding lines (possessing Pup1, Xa21 and Pi54) derived from the cross of RP5973-20-9-8-24-12-7 x RP6132 were evaluated for 13 agro-morphological traits associated with low soil P tolerance at F 5 generation. The ANOVA test revealed significant differences between the tolerant and sensitive parents and also the breeding lines for all the traits ( Table 2).

Discussion
MTU1010 (also known as Cottondora Sannalu) is a mega-rice variety of India, released by APRRS, ANGRAU, Maruteru, Andhra Pradesh, India. It is of medium-duration maturity (120 days), has good grain quality (i.e., long slender grain type) and is suitable for planting in both wet and dry seasons, with a high yielding capacity of six to seven tons/ha. The variety is estimated to be cultivated in >12% of Indian rice areas, due to its wider adaptability along with good level of tolerance to BPH [17]. However, the variety is highly susceptible to BB and has only moderate level of tolerance to blast disease, both of which cause significant yield losses in many states of India. Further, MTU1010 is also highly sensitive to low soil P conditions [18]. We had earlier developed a NIL of MTU1010 (RP5973-20-9-8-24-12-7) through the targeted transfer of the low soil P tolerance gene Pup1 [18] and NIL of Akshayadhan (RP6132; slender grain type rice, similar to MTU1010 in terms of grain quality and yield), having resistance against BB (conferred by Xa21) and blast (conferred by Pi54) [8].
Using these two lines as parents, we improved MTU1010 for tolerance to low soil P conditions along with improvement of resistance against BB and blast, in the present study. In the present-day scenario of emergence of BB and blast as major production constraints in rice, coupled with increasing cost of P fertilizers, along with low availability of soil P in many rice growing areas, development of multiple stress resistant/tolerant varieties/breeding lines has assumed significance in rice breeding. Despite significant increase in production and productivity of different rice varieties, over the last five decades, in recent years, the yield and productivity levels of Indian rice varieties have stagnated. One of the main reasons for this is the susceptibility of most of the popularly cultivated mega-rice varieties, like Samba Mahsuri, Swarna, MTU1010, etc., to pests and diseases and various abiotic stresses [7], such as low soil P levels. In order to sustain the yield levels of these elite and popular rice varieties, it is imperative to develop disease resistant and low P tolerant versions. Resistance breeding is considered the most economical and eco-friendly strategy for management of biotic stresses [19].
While resistance breeding is the only option for management of BB [14], in case of blast disease, even though many effective fungicides are available, they are often costly and cannot be afforded by small and medium farmers. In such a scenario, selection of resistance genes/gene combinations for deployment is important to sustain rice cultivation in areas prone to blast and BB diseases. With this objective, in the present study, breeding lines possessing resistance to blast and BB diseases, along with tolerance to low soil P, and having yield and grain quality similar to or better than MTU1010, have been developed through the strategy of MAPB, coupled with phenotype-based selection for agro-morphological traits.
Pi54 is a major blast resistance gene derived from a Vietnamese rice line, Tetep. The gene is known to be very effective against the blast pathogen isolates prevalent in India [20,21]. Along with Pi54, we also considered deployment of another major dominant BB resistance gene, Xa21, which confers broad-spectrum resistance against Indian isolates of Xoo [22,23]. Xa21 was originally introgressed from an accession of African wild rice, Oryza longistaminata. In addition to Pi54 and Xa21, a major QTL/gene associated with tolerance to low soil P, Pup1 (Phosphorus uptake1), was also deployed in this study for enhancing the performance in low soil P conditions. Pup1 was identified on 12th chromosome of rice of the Aus variety, Kasalath, fine-mapped, cloned, and characterized [24][25][26]. The QTL/gene was introgressed through marker-assisted breeding into the Indica rice varieties, IR64, IR72, Dodokan, Batur, and Situ [9]. Phenotypic evaluation of the introgressed lines suggested that Pup1 is effective in different genetic backgrounds, in both irrigated and rain-fed environments. Hence, in this study, even though MTU1010 is a variety released for irrigated ecosystem, we deployed Pup1 in the variety, so that the breeding lines could perform well in soils with low available P.
We had earlier developed improved versions of elite rice varieties and hybrid rice parental lines, which showed excellent resistance against BB and blast [21][22][23]. We selected one major gene each conferring resistance against BB (i.e., Xa21) and blast (i.e., Pi54), as there are no reports of large-scale break down of resistance conferred by these resistance genes in India [24,25].
We had earlier transferred Pup1 QTL into the genetic background of elite rice varieties [7]. To date, Pup1 is the only major QTL/gene available for improvement of low soil P tolerance. Based on the above-mentioned points, the present study was formulated in order to combine tolerance to low soil P and resistance against BB and blast into the genetic background of NIL of MTU1010, by crossing RP5973-20-9-8-24-12-7 x RP6132 through MAPB (i.e., for Pup1, Xa21, and Pi54), coupled with phenotypic selection for recovery of superior traits present in MTU1010 (i.e., for high yield, good plant type and a desirable long-slender grain type).
Analysis of variance among the F 5 lines revealed significant differences between the parents and breeding lines for days to fifty percent flowering, plant height, number of productive tillers, panicle length, root length, dry root weight, thousand grain weight and grain yield per plant under low soil P condition, but significant differences were not observed for dry shoot weight. Notably, most of the improved breeding lines (possessing Pup1, Xa21, and Pi54) in normal soil P and low soil P plots were found on par, and few lines were found to be superior with respect to yield, as compared to MTU1010 NI L (Tables 2 and 3).
Four breeding lines (LPK4-20-4, LPK30-18-16, LPK49-15-22, and LPK42-18-26) showed significantly higher single plant yield as compared to MTU1010NIL in normal soil Pcondition, while three breeding lines (LPK30-18-16, LPK49-1-21, LPK49-15-22) showed significantly higher single plant yield in low soil P condition. All the breeding lines were observed to be higher in terms of single plant yield as compared to the male parent RP613. In most of the    improved breeding lines (i.e., F 5 lines), there was no apparent yield penalty associated with the presence of BB (Xa21) and blast (Pi54) resistance genes, along with low P (Pup1) tolerance. Two breeding lines (LPK30-18-16 and LPK49-15-22) (possessing Xa21, Pi54, and Pup1) in F 5 generation displayed high yield under both low soil P and normal soil P conditions, possessed long slender grain type, and showed high level of resistance to blast and BB diseases. This indicates that stringent marker-assisted selection in the early generation coupled with stringent phenotypic selection in the later generations can result in identification of superior lines/segregants. The two lines mentioned above are expected to perform well in areas prone to the two diseases, and also in upland areas of rice cultivation, where there is low availability of soil phosphorous [26]-for instance, in the nascent state of Telangana-during both wet and dry seasons, due to their short duration, similar toMTU1010.
Further, the breeding lines of MTU1010 NIL possessing BB and blast resistance along with Pup1 can also serve as good donors for targeted transfer of the major genes/QTL to other elite rice varieties cultivated in the Telangana state. The promising breeding lines developed in this study will be advanced through the pedigree method of breeding for possible multiplication and evaluation through multi-location trials in the state of Telangana, and also across the country through All India Coordinated Research Improvement Project (AICRIP). Further, these lines can also serve as excellent donor for resistance against BB, blast and tolerance to low soil P, as they possess, Xa21, Pi54 and Pup1, respectively.