Performing Department
(N/A)
Non Technical Summary
Soybean is a major crop in US agriculture, and producing higher-quality soybean lines is imperative to growing the soybean industry. Hybrid crops benefit from hybrid vigor, a phenomenon in which the offspring of genetically unique parents exhibit increased growth. The creation of these hybrids is difficult in soybean since it is a hermaphroditic plant that self-pollinates before it can be pollinated by a different individual plant. The major goal of this project is to improve the ease with which hybrid soybean crosses can be made by soybean breeders.Sex chromosomes have evolved in plants repeatedly, conferring femaleness or maleness to individual plants within a species. Such plants must cross-pollinate, which leads to the creation of more vigorous hybrid offspring. In this project, I propose to engineer sex chromosomes in soybean, thus creating unique male and female soybean plants. I will accomplish this by using molecular cloning and transformation techniques to genetically manipulate soybean plants based on our knowledge of what constitutes sex chromosomes in plants. Again using molecular cloning and transformation techniques, I will also create a soybean line in which sex determination will be controllable by shining a red light on the plants during a particular developmental phase--this is a tool called optogenetics. This will yield plants that remain hermaphrodites by default, making it possible for them to be deployed in fields without the need to be monitored. This project will thus produce soybean lines that will enable soybean breeders to create stronger hybrid lines, thus bolstering the soybean industry.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Goals / Objectives
Hybrid crop lines benefit from hybrid vigor, a phenomenon in which the offspring of genetically unique parents exhibit increased growth. The creation of these lines is difficult in soybean since it is a hermaphroditic plant that self-pollinates. The major goal of this project is to improve the ease with which hybrid soybean crosses can be made, which will bolster the soybean industry. The objectives of this goal are as follows:?Design and insert a two-gene sex-determining cassette into a female-sterile soybean line to create a ZW sex chromosome system.Design and implement an optogenetic system into soybean. Then, apply that system to inducibly convert genetically hermaphroditic plants into male-sterile plants, thus generating a conditionally gynodioecious soybean line.
Project Methods
The first aim of this project is to engineer sex chromosomes in soybean. Based on work that my lab has done to identify conserved single-copy genes that are critical for ovule development, I will select a female-sterile soybean line from the USDA-ARS Soybean Genetics and Genomics Database without any serious pleiotropic effects to transform with a two-gene sex-determining cassette. I will design and create this cassette using well established molecular cloning techniques. One gene will code for an ovule-promoting gene that will restore female fertility. The second gene will code for an artificial small RNA that will knock out a single-copy gene critical for anther development, thus suppressing male fertility. I will utilize the Wisconsin Crop Innovation Center's Agrobacterium-mediated soybean transformation service to transform plantlets of my female-sterile cultivar of choice with my two-gene construct. Up to six individual transformation events will be returned in the form of T1 seed.To evaluate the effect of transformation with my two-gene cassette, I will plant the T1 seed returned from the transformation service and grow the plants in grow rooms with tightly controlled temperature, light, and humidity. I will then perform RNAseq in triplicate using samples from leaf, ovule, and primordial anther tissues from the wild-type Williams 82 cultivar as well as each of the returned transformed lines to compare RNA profiles and determine whether any of the transformants display the expected results. Upon successful transformation of a working construct, I expect to observe plants exhibiting female fertility and male sterility.The second aim of this project is to design and implement an optogenetic system into soybean and then to use that system to create an inducibly gynodioecious soybean line (one having female and hermaphroditic individuals). To do this, I will adapt the PULSE optogenetic system to soybean; this system has not, to our knowledge, been adapted to plants other than Arabidopsis thaliana and Nicotiana benthamiana, and only in whole plants in the former. I will isolate some of the necessary components from genomic soybean DNA and will purchase plasmids containing some of the other components from Addgene. Then, I will create new constructs using well established molecular cloning techniques that will allow reconstitution of this system in soybean.Before testing the optogenetic system in whole soybean plants, I will first evaluate it in soybean protoplasts, which can be easily isolated and transformed, by using it to drive conditional expression of a GFP reporter. Upon validation of this system in protoplasts via confocal microscopy, I will utilize the Wisconsin Crop Innovation Center's Agrobacterium-mediated soybean transformation service to transform plantlets of the wild-type cultivar Williams 82 with my PULSE optogenetic constructs. The center will return T1 seed from at least six independent transformation events. I will again use confocal microscopy on T1 plantlets grown from seed to confirm the presence of GFP, thus validating the system.Upon validation of the PULSE system in soybean protoplasts via confocal microscopy, I will apply the system to induce sex determination in soybean. Soybean plants that are treated with red light in the absence of blue light will effectively become females that are incapable of self-pollination, while untreated plants will remain hermaphroditic. In order to create a female plant, "maleness" must be repressed. To accomplish this, I will use the PULSE system to conditionally express an artificial sRNA, either an amiRNA or a syn-tasiRNA, to knock out a single-copy gene critical for anther development. It will be necessary to identify the precise developmental stage at which red-light treatment should occur, as well as how often and for what duration. To answer these questions, I will attempt treatment at different stages of normal anther development and will also test how many treatments and for what duration are necessary to induce male sterility.To determine whether treated plants exhibit male sterility, I will compare pollen production in untreated transformants with that in unmodified plants. To confirm the presence of the artificial sRNA, I will prepare sRNA sequencing libraries from promising transformants and sequence them on an Illumina Novaseq X. These libraries will be prepared using leaf, ovule, and primordial anther tissues to ensure successful artificial sRNA expression. I will also generate and sequence mRNA libraries to ensure effective knockout of the target gene.All results will be published in peer-reviewed scientific journals, and molecular constructs and soybean lines created through this project will be made publicly available. I will also communicate my findings at conferences, for example at the National Association of Plant Breeders (NAPB) annual meeting.