A step-by-step protocol for crossing and marker-assisted breeding of Asian and African rice varieties
Year: 2024
Type: article
Abstract: A step-by-step protocol for crossing and marker-assisted breeding of Asian and African rice varieties Yugander Arra 1,4 §, Eliza P-I. Loo 1, B.N. Devanna 1,2, Melissa Stiebner 1 & Wolf B. Frommer 1,3, § 1 Faculty of Mathematics and Natural Sciences, Institute for Molecular Physiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 2 ICAR-National Rice Research Institute, Cuttack 753006, Odisha, India 3 Institute of Transformative Bio-Molecules, Nagoya University, Chikusa, Nagoya 464-8601, Japan 4School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram (IISER TVM), Maruthamala P.O., Vithura, Thiruvananthapuram-695551, Kerala, India § For correspondence: [email protected], [email protected] AbstractImproving desirable traits of popular rice varieties is of particular importance for small-scale food producers. Breeding is considered the most ecological and economic approach to improve yield, especially in the context of the development of pest and pathogen-resistant varieties. Being able to cross rice lines is also a critical step when using current transgene-based genome editing technologies, e.g., to remove transgenes. Moreover, rice breeders have developed accelerated breeding methods, including marker-assisted backcross breeding (MABB) to develop novel rice varieties with in-built resistance to biotic and abiotic stressors, grain, and nutritional quality. MABB is a highly efficient and cost-effective approach in accelerating the improvement of recipient variety by introgressing desirable traits, especially from landrace cultivars and wild rice accessions. Here we provide a detailed protocol including movie instructions for rice crossing and MABB to introgress target trait(s) of interest into the elite rice line. Further, we also highlight the tips and tricks to be considered for a successful crossing and MABB. Key features This protocol provides detailed information on techniques for crossing rice varieties and for breeding rice varieties with new traitsThe protocol includes instructions both for making rice crosses as well as MABBThe protocol may provide beginners with detailed instructions including troubleshooting guides Keywords: Rice, back cross-breeding, donor parent, recipient parent, gene introgression, MABB BackgroundRice is one of the most important food staples for over 3.5 billion people. Classical breeding enables the development of new varieties by trait introgression for important agronomic characters and increased tolerance/resistance to abiotic/biotic stressors. Backcross breeding uses donor and recipient parents (DP and RP, respectively); the DP carries desired traits to be introgressed into an RP, typically an elite variety. Classical breeding inadvertently introduces undesirable traits from the donor and/or the loss of beneficial RP traits. It is therefore necessary to backcross the resulting progeny, i.e., the F1generation multiple times with the RP to eliminate as much of the chromosomal segments from the DP that do not carry the trait of interest as possible. Crossovers close to trait gene loci are desirable to eliminate the chance that linked loci adversely impact the resulting variety's performance.Developing a new rice variety through breeding takes 7-8 years, (two seasons per year, maximum 16 seasons of cultivation) (Krishnan et al., 2022). New varieties must be tested in multilocation field trials to validate trait improvements relative to parental lines before new varieties are registered, which takes another 2 years. MABB reduces this timeline to three years and reduces linkage drag from the DP (Krishnan et al., 2022). MABB introgresses loci encoding desired traits into the RP and reconstitutes the genome of RP by background selection, eliminating undesirable genomic fragments, i.e. linkage drag, from the DP. MABB uses gene-based/gene-linked molecular markers for foreground; to select the desired trait/loci from DP, background selection (to determine the percentage recovery of RP genome), and recombinant selection (to determine the presence of linkage drag) (Arra et al., 2018; Singh and Krishnan, 2016). The success of MABB depends on among other things, differentiating the genome of DP from the genome of RP. Typically, simple sequence repeats (SSRs) markers are used as polymorphic molecular markers for differentiating genomic backgrounds (Mason, 2015). SSR markers are short DNA motifs (2–6 nucleotide repeats) that exhibitvariable repeat numbers in the genome. SSRs are abundant, multi-allelic, co-dominant, hypervariable, and relatively uniformly distributed in the genome. SSR markers have become important and widely used in rice breeding (Arra et al., 2018; Ellur et al., 2016; Sundaram et al., 2008). Apart from its application in plant breeding, crosses are an essential step for eliminating transgenes introduced via genome editing. Genome editing offers the potential to target multiple loci for providing broad-spectrum resistance, nutritional fortification, and yield improvement in crops (Eom et al., 2019; Oliva et al., 2019; Schepler-Luu et al., 2023; J. Y. Wang and Doudna, 2023). Notably, the term ‘new breeding methods’ frequently used in the context of genome editing is not an optimal choice, since it is rather an alternative for generating genetic variation, in particular, targeted modifications compared to the use of naturally occurring mutations or chemically or radiation-induced mutagenesis, while the breeding process remains in the hands of breeders and requires the technologies and experience of breeders. In countries with appropriate regulations, genome-edited crops are treated as equivalent to crops generated by classical breeding (Buchholzer and Frommer, 2023). Material and reagents Biological material:Two rice parents; DP and RP, to develop new desirable rice varieties. A. Choice of DP:Reproductive isolation can limit the ability to cross diverse rice varieties or landraces (Kumar and Virmani, 1992; Wang et al., 2024). Incompatibility between DP and RP cross leads to sterility in subsequent crosses (Sitch, 1990). Therefore, choose a DP that is genetically compatible with the RP which produces fertile F1 seeds after cross. In rice, crosses involving indica and japonica subspecies often produce sterile seeds. Similarly, crosses between O. sativa spp. indica (RP) and the DP from a wild rice species/Oryza glaberrima are reported to show embryo abortion/sterility.The DP should possess at least a few characters for Distinctness, Uniformity, and Stability (DUS) to the RP. DUS characters play a key role during varietal nomination and evaluation (Pourabed et al., 2015). MABB-derived lines with at least 1-2 DUS characters to the RP are desirable. The choice and variety of releases also depend on DUS characters.Choose a DP that provides a novel resource for pest/disease resistance, stress tolerance, yield attributes, agronomic characters, or grain quality.In most cases DP is either a landrace or a wild accession. Note that it is challenging to make a successful cross between some rice species due to crossing barriers (e.g. Oryza sativa and Oryza glaberrima). B. Choice of RP:Choose an elite RP that possesses desirable characteristics including plant phenotype, protection against biotic/abiotic factors, or utilization traits (grain and cooking quality attributes).The improved RP with higher gain yield and better economic returns tends to have higher acceptance among large-scale farmers. Laboratory supplies Murashige and Skoog basal salt mixture (Duchefa, catalog # M0254)Sucrose (Sigma-Aldrich, catalog # S0389)Phytagel (Sigma-Aldrich, catalog # P8169)peqGOLD Plant DNA Mini Kit (VWR, catalog # 13-3486-02)GoTaq G2 Green Master Mix (Promega, catalog # M7823)Agarose (Sigma-Aldrich, catalog # A6013)GeneRuler 1 kb Plus DNA Ladder, ready-to-use (Thermo Scientific, catalog # SM1334) Equipment Analytical and Precision Balances (Precisa Gravimetrics AG, Series 321LS, model: LS 2200C)Laboratory reagent bottles with screw cap, 1,000 ml (Duran®, catalog # 218015455)Benchtop pH meter, WTWTM inolab TM 7110 (Fisher Scientific, catalog # 11731381)Magnetic stirring bars, cylindrical 25x8 mm (VWR, catalog # 442-0483)Autoclave (Systec, model # 3850 EL)Sterile workbench (Thermo Scientific, HeraguardTM Eco)Petri dishes (Sigma-Aldrich, catalog # P5606-400EA) Tissue culture vessel, Magenta™GA-7 (VWR, catalog # SAFSV8505-100EA)Hand-held wooden cereal dehuskerCentrifuge tubes with screw cap, 15 ml (Thermo Scientific, catalog # 339650)Permanent marker pen Precision tweezers (VWR, catalog # 232-1220)Precision forceps, extra sharp, curved (VWR, catalog # BOCH1940)Growth chamber with 27 °C, 16/8 day/night photoperiod (Percival, model # CU-41L5)Head-band Magnifying Glasses (may be advantageous for proper emasculation)Seed paper bag (11.5 x 6 cm)Retro floor lamp with E27 Bulb including 3 lights tree floor lampPlastic pots, 11x11x12 cm (Mayer shop, catalog # 722009)Plastic tray to keep plastic pots, 50x32x6 cm (Mayer shop, catalog #749112)Whatman filter paper (VWR, catalog # 516-0593)Height adjustable stool, useful during emasculation Spray bottle for water, 1000 ml (Roth, # IC8T.1) Nanodrop (Thermo Fisher, catalog #13-400-519)Thermocycler (Bio-Rad, model: T100TM, catalog #1861096)EasyPhor Medi gel electrophoresis system 3GT 15x10 cm (Biozym, catalog # 615162)Gel documentation unit (Vilber E-BOX CX5 TS)Drying and heating cabinets with mechanical adjustment (Binder, catalog # E28) Procedure A. Germination and growth conditionsDehusk the rice seeds using a small hand-held wooden cereal dehusker. Transfer the dehusked seeds into a 15 ml centrifuge tube into which add 7-8 ml of 70% ethanol. Incubate the seeds for two minutes at room temperature with shaking at 80 rpm. Discard the ethanol and add 7-8 ml of 4% sodium hypochlorite into the tube followed by 5 minutes of incubation at room temperature with shaking at 80 rpm. Discard the bleach solution and rinse the seed repeatedly with sterile water until no traces of bleach are noticeable. Finally, air-dry the sterilized seeds on sterilized filter papers on a clean workbench for 15-20 mins.Transfer the dry sterilized rice seeds onto Petri dishes containing autoclaved half-salt strength Murashige and Skoog salt (½ MS) media supplemented with 1% sucrose (Table 1). Germination in Petri dishes generally improves germination efficiency. To start seed germination, incubate the seeds (on the petri dishes) in a plant growth chamber at 27 ℃ under dark conditions for three days. After three days, transfer germinated seeds into a tissue culture vessel, Magenta™GA-7 filled with 50 ml of ½ MS media and incubated at 27 ℃ with 16 hours light/8 hours darkness for six days. After six days, transfer the seedlings to 11x11x12 cm (L x B x H) pots filled with soil and grow under greenhouse conditions (28 ± 1 ℃; relative humidity between 60-80%) until the booting stage. Water regularly and apply fertilizers as required (Luu et al., 2020). Remove old and/or dried leaves, if present, to avoid hindrance during the crossing process. B. Emasculation:The ideal criteria for emasculation are, i) plants reached the post-booting stage, ii) show healthy panicles, iii) have no obvious signs of anthesis yet, and iv) floral organs (stigma and stamen) are still located inside the lower half of the spikelet (Figure 1). Day 1: Spikelet opening leading to anthesis is triggered by high light intensity and humidity. Therefore, the plant growth conditions, including light intensity, temperature, and humidity should be taken into consideration when deciding the appropriate time for emasculation. It is recommended to perform emasculation either in the early morning hours (5-7 am) or early evening hours (5-7 pm). Carefully inspect rice plants for panicles with more than 50% exsertion (panicle exsertion: the distance between the leaf cushion of the flag node and the neck-panicle node; Figure 2). Correct selection of panicles is key to avoiding self-pollination. Remove the leaves surrounding the panicle, except the flag leaf, to prevent interference during the crossing. Remove young spikelets at the base of the panicle and flowered spikelets in the upper part of the panicle to achieve the optimum number (50-60) of spikelets per panicle (Movie 1). For emasculation, it is recommended to cut individual spikelets at a 35-40 degree angle just above the anthers with a pair of sterile scissors. Be careful not to damage the stigma while cutting the spikelet (Movie 2). Carefully remove each stamen (six stamens in total) from a rice spikelet using sharp tip forceps. Do not damage or break the anthers in the spikelet to avoid pollen contamination, hence self-fertilization. It is important to emasculate all the flowers in the panicle- a single anther is sufficient to self-pollinate the entire panicle. C. Dusting and bagging:The DP plants with 30-50% of flowering panicles should be used as a source of anthers for dusting.While carrying out the crosses in greenhouse conditions, care should be taken to position the recipient and the donor plants in such a way that the flowers of these plants are at similar heights for ease of crossing. The inflorescence or panicle of DP plants close to flowering was exposed to high light intensity (>400 µE m-2 s-1) by lowering the height of the overhead light source for 15-30 minutes to encourage flowering. Exposure to high light intensity also increases the microclimate temperature of the panicle to 30-40℃. Inflorescence with flowers with anthers at the stage of dehiscence should be used for dusting on emasculated panicles of the RP (Figure 3; Movie 3). It is important to begin dusting from the top of the emasculated panicle and slowly slide the DP panicle down the RP panicle with a gentle stroke. Immediately after dusting, cover the panicle with pre-labeled white paper bags, (important information on labels: name of the cross, date, and filial (F) generation). Depending on the availability of anthers, dusting can be repeated with the same panicle on the same or the next day to increase the percentage seed set. Tip: use a flag leaf to support the paper bag as florets are not supposed to be folded after the crossing. Day 2-6: Provide plants with optimal growth conditions.Day 7: If desired, one can observe the seed set one week after dusting. Gently remove the paper bag from the panicle. seeds are The percentage seed set can be by a of filled to in to the panicle with the paper the spikelets seeds develop (Figure and to the light or dark of the seeds care should be taken to keep the and from the seeds develop a light (Figure with a maximum of et al., are for (Figure seeds to be dried at ℃ for 2 to the of the seeds to leads to seed damage due to of which seed and The dried seeds can be either used for germination or at ℃ for backcross breeding MABB is an application of molecular markers to and choose the target loci and to the RP genome recovery during (Arra et al., MABB was to develop lines with than classical MABB the recovery of the RP genome and reduces the number of backcross plants carry the target loci in the genetic background of RP, and the recovery of the RP genome can be in these lines and F1 seeds from the cross at ℃ for 2 to the F1 seeds as in A. the i.e., selection, and of healthy leaf from plants and both DP and with sterilized from into 2 ml tubes containing This protocol the Plant DNA Mini genomic DNA isolation from leaf DNA can be the genomic DNA using Nanodrop DNA markers to target from the genomic DNA the of the F1 plants on the compared to RP and DP. or for the target of interest are in the F1 (Figure the F1 plants for backcross breeding (Figure selection to the linkage drag and to the breeding lines with maximum RP genome recovery In the we a of two polymorphic SSR markers the DP and for each of the rice we that the markers at a distance the for the better recovery of the RP genome. For genome recovery we breeding that was for the presence of target through selection and using polymorphic SSR Further, to the linkage drag, we a set of SSR close to the target the polymorphic SSRs differentiating the RP and the DP, and used to the breeding in the for better RP genome recovery and linkage drag using the of simple sequence repeats (SSRs) than SSR markers have reported for rice for the SSR is on the SSR markers distributed uniformly on all rice at least distance between each for of DP and RP. genomic DNA from DP and RP using a DNA isolation or and DP and RP with SSRs using and markers between DP and at least polymorphic SSRs between DP and RP for each rice polymorphic SSRs to F1 to to lines with maximum recovery of the RP genome lines to for or nomination and varietal for or background leaf with sterilized cm from individual breeding plants in 2 ml genomic DNA from leaf using an plant DNA isolation or the of genomic DNA with Nanodrop and the to with 1 of DNA of each of each 5 of Green Master Mix (Promega, and 3 of a with the at for 3 followed by of at for at for and at for 1 The is at for 5 the presence or of targeted via a of the gel with a gel documentation and the for the presence of the gene and also the of these on the background selection, use the SSR marker generated for each breeding and the RP genome recovery using the and start with a grain to rice and anthers before a crossing It is more challenging to remove anthers from grain and the to for better to remove at least the top spikelets from each and remove anthers with to avoid anthesis and self-pollination. to cut the spikelet with an from both of the spikelet as it the opening of the spikelet and rice DP plants with flowering for Spray the panicles with water before to high light intensity to dusting, it is to clean and remove dried leaves from the base of the leaves as a for and which damage the developed after a successful panicles regularly for and both on the panicle or inside the bag the to keep plants in a clean and the pots or tray with water to the maximum for seed rice use for emasculation and with two flowered was by from the & the Research under and to by the and of New no of and developed the and MS and the have to the of the of the novel resistance gene into the genetic background of elite rice variety and Frommer, B. number of countries genome editing in New J. and J. of plant DNA from A. of a novel resistance in and of its resistance with J. 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A of genome editing is the and A. of rice to and by of the Figure The rice panicle the floral organs inside of the and stamen are located inside the lower half of the Figure of rice panicles from the booting stage to from flag panicle panicle panicle emasculated cross cutting of spikelet Figure at and of individual Figure of seed on recipient after 15 after after the of rice seed after Figure of in F1 by markers 1 kb Plus parent, parent, for both parents Figure for marker-assisted backcross maximum recipient genome recovery into improved lines by molecular markers number of number of for the by ½ salt strength Murashige and Skoog and Skoog basal salt
Source: BIO-PROTOCOL
DOI: 10.21769/p2665
Author: Wolf Frommer
Language: English
Subfield: Genetics
Domain: Life Sciences
SDG: Zero hunger
Open Access status: diamond
OpenAlex ID: W4396726081