Source: WASHINGTON STATE UNIVERSITY submitted to NRP
GENOMICS ENABLED PURGING SELECTION TO DEVELOP 200 ALFALFA INBRED LINES TOWARD HIGH YIELD HYBRID PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1017114
Grant No.
2018-70005-28792
Cumulative Award Amt.
$295,000.00
Proposal No.
2018-03920
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2022
Grant Year
2018
Program Code
[AFRP]- Alfalfa and Forage Program
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Crop and Soil Sciences
Non Technical Summary
As the most widely cultivated forage legume in the world, alfalfa has been successfully developed with varieties specialized on simple traits controlled by major genes, such as winter hardiness and disease resistance. However, genetic improvements have been limited for complex traits such as forage yield, seed yield, and forage quality, which are controlled by multiple genes. Alfalfa breeders are unable to develop real inbred lines to accumulate favored alleles and create subsequent hybrids with increased vigor due to severe inbreeding depression. Natural Selection by Inbreeding Depression commonly terminates alfalfa fertility before eliminating deleterious alleles, the major cause of inbreeding depression. Our preliminary studies suggest that this hurdle can now be overcome by jointly using Bulked Segregant Analysis and Marker Assisted Selection in combination with natural selection to purge deleterious alleles before they express harmful effects. Based on the genotypes of ~5,000 SNPs on the 200 accessions of the alfalfa diversity panel, we propose to divide these accessions into two heterotic galaxies for developing inbred lines. For each galaxy starting at the third generation, DNA will be pooled for individuals with less and severe inbreeding depression, respectively. The genetic loci detected will be genotyped by using targeted amplicon sequencing for marker-assisted selection among the lines with severe inbreeding depression. The two galaxies of inbred lines are expected to be a turning point for the alfalfa industry to produce real hybrids that will boost alfalfa yield and quality.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20116401081100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1081 - Breeding;
Goals / Objectives
Our goal is to overcome the current bottleneck of inbreeding depression for developing alfalfa inbreds. This dramatic change will facilitate the accumulation of favored alleles and the production of alfalfa hybrids and, in turn, will boost forage yield and quality. We plan to achieve this goal by accomplishing the following three objectives:Objective 1: Map the genetic loci associated with inbreeding depressionPrevious studies indicate that inbreeding depression is mainly caused by recessive deleterious alleles. These alleles only express their harmful effects when they are homozygous, that is, when two (diploid) or four (autotetraploid) copies of the deleterious allele are present at each genetic locus. When one copy of a deleterious allele is combined with normal alleles to become heterozygous, the phenotypic performance is equal to the homozygous form with only normal alleles. During the process of inbreeding, such as self-cross mating, the proportion of heterozygous genes decreases by 50% and the proportion of homozygous genes with two copies of deleterious alleles increases by 50% with each subsequent generation.Natural Selection by Inbreeding Depression (NSID) is an effective way to remove the recessive deleterious alleles from self-crossing species such as rice and wheat because the number of these alleles among the survivors is low. Consequently, these species are tolerant to inbreeding. In outcrossing species such as maize and alfalfa, the proportions of heterozygous genes are much higher than self-crossing species. Deleterious alleles can hide in a heterozygous state and pass from generation to generation. Therefore, inbreeding in outcrossing species is severe. Alfalfa breeders can rarely conduct self-crossing for more than four generations before progeny lose fertility. Because of this severe inbreeding depression, alfalfa cultivars are maintained as outbreds to reduce the probability of homozygosity.Multiple deleterious loci cumulatively affect fertility. The phenotypic variation of fertility makes it possible to identify the genetic loci associated with deleterious alleles. Genotyping on the identified loci will allow breeders to eliminate individuals with heterozygous genes containing deleterious alleles before harmful effects are expressed. Our objective is to identify these genetic loci and make them publicly available to increase alfalfa breeders' probability of successfully developing inbred lines from their breeding programs.Objective 2: Develop two complementary heterotic galaxies, each comprised of 100 inbred lines with average residual heterozygosity below 5%Alfalfa breeders have invested substantial time and effort toward developing inbred lines for hybrid production with high forage yield and quality. Unfortunately, self-incompatibility and inbreeding depression do not allow low residual heterozygosity. Thus, alfalfa varieties are maintained as synthetic populations. Although individual plants within a variety are similar, varieties must retain substantial variation to prevent inbreeding depression, which precludes the generation of true hybrids that exhibit increased vigor. All available alfalfa inbred lines are semi-inbreds; consequently, the corresponding hybrids are semi-hybrids. We aim to develop 200 inbred lines with average residual heterozygosity below 5%. The low residual heterozygosity will provide alfalfa breeders with two benefits: 1) these inbred lines can be used directly to produce real hybrids and 2) residual inbreeding depression will also be low. As a result, alfalfa breeders will be able to further develop new recombinant lines.The majority of the 200 inbred lines will originate from the 200 accessions of the alfalfa diversity panel. These lines cover the majority of genetic diversity in alfalfa worldwide. Based on their relationship revealed by ~5,000 genetic markers, we aim to divide them into two groups of similar size and to maximize the distance between the groups. Development of recombinant inbred lines will be restricted within groups to develop two complementary heterotic galaxies. Greater heterosis is expected for crossings between the two galaxies. Substitutions of the 200 accessions with others, such as the elite lines donated from the alfalfa industry, will be limited to 30% to ensure the coverage of alfalfa diversity is maintained. Accomplishing this objective will provide public and private breeders with inbred lines to create hybrids from 10,000 combinations between the two complementary heterotic galaxies. Additionally, breeders will have the opportunity to create their own inbred lines to expand the combinations for superior hybrids.Objective 3: Stakeholder outreach for immediate economic and ecologic prosperityOur team maintains close connections with the alfalfa industry. One example is the project, "Marker-Assisted Breeding In Elite Alfalfa Germplasm To Enhance Biomass Productivity During Drought", funded with $500,000 from Alforex Seeds. This project is a collaboration between Co-PD Dr. Long-Xi Yu and a scientist from New Mexico State University. Alforex has already contributed multiple elite lines to our research and has expressed interest in contributing additional lines to this new research project. Outreach to companies such as Alforex will add more value to the development of inbred lines. The elite lines are more relevant to future production. The genetic markers identified by these lines are more important for MAS used by the alfalfa industry to develop inbred lines. With successful applications, we are more likely to receive additional support, including financial, from the alfalfa industry. Active engagement of the alfalfa industry, including breeders and growers during our research process, will facilitate incorporation of our results as soon as they are available. For example, both whole genome sequencing and targeted amplicon sequencing technologies are currently accessible to alfalfa breeders. Genetic markers identified in this new project can be used immediately by breeders and will be valuable for eliminating deleterious alleles from the alfalfa industry's breeding programs.
Project Methods
Experimental designOur primary objective is to develop two complementary heterotic galaxies, named Yu Galaxy and Peel Galaxy, each containing 100 inbred lines with average residual heterozygosity below 5%. The origins of these inbred lines will be the 200 accessions from the alfalfa diversity panel, with a maximum of 30% substitutions in the current breeding programs led by Co-PD Yu or Peel. The challenge of this project is to break through a bottleneck of inbreeding depression. We will use asexual propagation to enforce natural selection by inbreeding depression. Starting from the third generation, our novel approach will jointly use high-throughput phenotyping and genotyping technologies to map the genes controlling inbreeding depression and apply MAS based on Targeted Amplicon Sequencing (TAS) to eliminate deleterious alleles. Ultimately, this approach will prevent the termination of fertility.Method 1: Multiple Bulked Segregant AnalysisFor each generation and each galaxy (Yu or Peel), DNA will be extracted from the mixture of un-germinated seeds and each of the 10 lowest (bottom) and 10 highest (top) seed-producing individuals for each variety. Equal amounts of DNA will be pooled for each of the three categories (un-germinated seed procedure, bottom seed procedure, and top seed procedure), for each galaxy (Yu and Peel), and for each generation. In total, we will pool 36 DNA samples (6 generations x 2 galaxies x 3 categories) for whole genome sequencing (WGS). Multiple Bulked Segregant Analysis (BSA) will be conducted for samples within a generation and across generations. We will use a modified version of the multiple BSA statistics formula. The nominator will equal the sum of the three pairs of categories. The denominator will equal the product of the standard deviations of the three categories.Method 2: Purging selectionPurging selection refers to the reduction of deleterious allele frequency. The most common approach is NSID. Many deleterious alleles are recessive and only express their harmful effects when homozygous. During inbreeding, progeny are more likely to be homozygous. The highest level of inbreeding is self-crossing, followed by full-sib mating. The individuals with homozygous deleterious alleles are less fit and have a lower survival rate in future generations. This process of inbreeding may purge all of the deleterious alleles and reach a fitness equilibrium. On the other hand, the process may terminate further inbreeding because plants may become unable to produce seeds in the later generations.In this study, we will primarily use self-cross mating with asexual propagation to maintain high selection intensity (1%) on NSID. Five full-sib crosses will be used as a backup in case all self-crosses fail. In case both self-crosses and full-sib crosses fail, the previous generation will be crossed with other lines that are in the same situation to enhance fertility. A previous study has demonstrated that lines derived from parents that have gone through purging selection are more tolerant to inbreeding depression than their parents. For the bottom half of lines ranked by seed counts and plant height (combined index with equal weight), MAS will be applied on the genetic loci identified by multiple BSA on inbreeding depression. For lines that only generate a few seeds, we will make clones through asexual propagation until obtaining at least 100 seeds. The survivors out of these seeds are expected to have less deleterious alleles than their parents through the selections against inbreeding depression. The more seeds produced from the asexual propagation, the higher selection pressure to eliminate deleterious alleles.The residual heterozygosity will be 0.015625 and 0.125 for six and three generations of self-crosses, respectively. The residual heterozygosity will be 0.18 for six generations of the full-sib crosses. Under the assumption that 75% of the lines succeed with self-crossing through six generations, 12.5% of lines that need outcrossingwith other lines at the fourth generation and continue self-crossing for three generations, and 12.5% of lines succeed with full-sib crossing through six generations, the average residual heterozygosity will be 0.05.Method 3: Whole genome and targeted amplicon sequencingCompared with the genome size of major crops such as maize (3 Gb) and wheat (15 Gb), alfalfa has a much smaller genome (800-1000 Mbp), which makes conducting WGS time- and cost-effective. WGS will allow us to pinpoint functional variants using BSA, laying the foundation for MAS to eliminate deleterious alleles. The identified genetic loci will be genotyped with TAS. Severe inbreeding depression starts with the third generation. TAS will start in the fourth generation.TAS will be conducted in Co-PD See's Lab, following a specified protocol. One flow cell will provide approximately 400 million sequence reads with an average size of 150 bp. The total number of reading bases will be set at 60G. This setting will provide 40X depth for two samples on a single flow cell.Method 4: Imagery analysisTotal seed weights will be recorded for the top ten seed producing individuals identified visually for each line/generation. About one gram of seeds will be weighed for each individual and recorded using an image capturing device, for a total of 12,000 (10 individual x 200 lines x 6 generations) images. Images will be analyzed with ImageJ software (Schneideret al.2012) to obtain seed size (length, width, area), seed shape (eccentricity), and total seed count in each image. Thousand Seed Mass (TSM) and total seeds will be calculated based on the total weight, sample weight, and the number of seeds sampled.Method 5: Asexual propagationSelection intensity is one of the four factors determining genetic gain such as eliminating deleterious alleles. For self-crosses, the selection is determined by the number of seeds. For some individuals, multiple clones are necessary to produce more seed to increase selection intensity. We will follow the stem cutting procedure to make clones for populations with less than 100 individuals. The surviving individuals with seeds will be propagated to ensure having enough seeds to generate over 100 progeny.Method 6: Extension to engage stakeholdersWe anticipate widely disseminating our results and notifying stakeholders about the value of integrating our research via several communication outlets, including:The inclusion of elite lines from stakeholders: We will include elite lines donated by stakeholdersup to 30% of the total inbred lines. We will also work with donors to make sure these donated lines are available for public access. The remaining lines will be chosen from the 200-line alfalfa accession diversity panel, with the aim of including the existing diversity of alfalfa.Access to genetic markers: The genetic markers associated with inbreeding depression in alfalfa will be published in peer-reviewed journals. Breeders will be able to use these markers to develop their inbred lines through MAS and eliminate deleterious alleles in their own alfalfa populations.Access to seed: Seed of the 200 newly developed inbred lines will be deposited in the USDA-ARS National Plant Germplasm System for public free accessLocal, regional, and national meetings: The project investigators will attend and present findings at local, regional, national, and international conferences.Social media: Research results will be made available to the alfalfa community, industry partners, and other alfalfa-focused stakeholders. Activity updates will be provided to the general public through press articles, radio and television interviews, newsletters, oral or poster presentations, written reports and flyers, Facebook and Twitter, and postings on our project website, hosted by PD Zhang (http://zzlab.net).

Progress 09/01/18 to 08/31/22

Outputs
Target Audience:Alfalfa breeders, geneticists, and farmers Changes/Problems:The only major change wasthat self-pollination was one generation shorter than planned due to the government shutdown in 2019 and COVID19 in 2020-2022. What opportunities for training and professional development has the project provided?The project provided support for the training and professional development at both graduate and postdoc levels. A graduate student and a postdoc were trained for research and career development.Two people were supported for workshops and conferencesto present their discoveries to reach stakeholders. Zhou Tang, High Throughput Phenotyping using UAV images. WSU Molecular Plant Science Retreat, March 5, 2022. Yang Hu, Entrepreneur network at WSU, July 25-29, 2022. How have the results been disseminated to communities of interest?The research results have been disseminated through multiple platforms, including publications, extension articles, websites, and conferences.Our project has generated 16 peer publications, including one in 2018, two in 2019, three in 2020, and ten in 2022. The USDA's support was acknowledged in all these publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We advanced one more generation of self-pollination last year. The last generation has gone through five generations of self-pollination in total, which is only one generation shorter than planned (six generations of self-pollination for 200 lines) due to the government shutdown in 2019and the COVID-19 restrictions in 2020-2022. In total, we have conducted self-pollination on 4,693 plants originating from 275 initial lines. Some of these lines were terminated, and some were divided into new lines. The final generation has 231 lines. Currently, we are in the process of seed multiplication to deposit to Western National Germplasm Bank. We collected DNA samples of 534 plants individually. The individual DNA samples were pooled into 121 bulk samples based on growth vigor (strong and weak) for exome target capture sequencing with 112,626 DNA sequences. The next-generation sequencing achieved a total of 4.26 GB reads, resulting in 10X sequencing depth on average. We obtained a total of 611,183 SNPs with high quality (a missing rate of over 50%, sequencing depth of over 20X, and minor allele frequency over 5%). A genome-wide association study identified 47 genetic loci associated with alfalfa plant growth vigor. The GO analysis generated 30 GO terms, of which 20 GO terms were significantly enriched. The associated SNPs were near genes involved in stress response, defense responses against pathogens, and plant reproduction. These identified SNPs benefit the development of alfalfa inbred lines by purging the deleterious alleles and via biomass improvement through marker-assisted selection.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Medina CA, Kaur H, Ray I, Yu L-X* Strategies to Increase Prediction Accuracy in Genomic Selection of Complex Traits in Alfalfa (Medicago sativa L.). Cells 2021, 10, 3372. https://doi.org/10.3390/cells10123372.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Medina CC, Samac DA, Yu L-X* (2021) Pan-Transcriptome Identifying Master Genes and Regulation Network in Response to Drought and Salt Stresses in Alfalfa (Medicago sativa L.). Sci Rep 11, 17203 (2021). https://doi.org/10.1038/s41598-021-96712-x).
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Medina CA and Yu L-X* (2021) Developing SNPs and strategies for genomic analysis in alfalfa. In Yu and Kole (Eds.) The Alfalfa Genome. https://doi.org/10.1007/978-3-030-74466-3.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lin S, Medina CA, Norberg S, Combs D, Wang G, Shewmaker G, Fransen S, Llewellyn D, Yu L-X* (2021) Genome-Wide Association Studies Identifying Multiple Loci Associated with Alfalfa Forage Quality. Frontiers Plant Sci. 12:648192. doi: 10.3389/fpls.2021.648192.
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Yu, L., C. Medina, and M. Peel. 2021. Genetic and genomic assessments for improving drought resilience in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. p.235-253. https://doi.org/10.1007/978-3-030-74466-3_14.
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Parajuli A., L.X Yu, M. Peel, D. See, S. Wagner, S. Norberg, and Z. Zhang. 2021. Self-incompatibility, Inbreeding Depression, and Potential to Develop Inbred Lines in Alfalfa. In: Yu LX., Kole C. (eds) The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. pp 255-269. https://doi.org/10.1007/978-3-030-74466-3_15
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang Hu, and Zhiwu Zhang*, GridFree: a python package of imageanalysis for interactive grain counting and measuring. Plant Physiology, 2021, https://doi.org/10.1093/plphys/kiab226.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhou Tang, Atit Parajuli, Chunpeng James Chen, Yang Hu, Samuel Revolinski, Cesar Augusto Medina, Sen Lin, Zhiwu Zhang*. Validation of UAV-based alfalfa biomass predictability using photogrammetry with fully automatic plot segmentation. Scientific Reports, 2021, doi: 10.1038/s41598-021-82797-x.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Jiabo Wang* and Zhiwu Zhang*, GAPIT Version 3: Boosting Power and Accuracy for Genomic Association and Prediction. Genomics, Proteomics and Bioinformatics, 2021, https://doi.org/10.1016/j.gpb.2021.08.005.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Matthew T. McGowan, Zhiwu Zhang & Stephen P. Ficklin, Chromosomal characteristics of salt stress heritable gene expression in the rice genome. BMC Genomic Data, 2021, https://doi.org/10.1186/s12863-021-00970-7.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Alfalfa breeders, geneticist, and farmers. Changes/Problems:The government shutdown in 2019 and COVID19 in 2020 and 2021 severely delayed our progress of self-pollination. We only completed four generation of selfing pollination, which is two generations shorter than planned. An extension of one year (September 1, 2021 to August 31, 2022) was approved to continue self-pollination and genomic analyses on the sequencing data.? What opportunities for training and professional development has the project provided?The project provided support for the training and professional development at both graduate and postdoc levels.A graduate student and a postdoc were trained for research and career development.Two people were supported for workshops and conferencesto present their discoveries to reach stakeholders. James Chen, Decoding Images in minutes with GRID. WSU Plant Science Retreat, March 6, 2021. Yang Hu, Becoming an Entrepreneurial Woman at WSU, April 22, 2021. How have the results been disseminated to communities of interest?The research results have been disseminated through multiple platforms, including publication, extension articles, website, and conferences. Three articles on the method and software development has been published. What do you plan to do during the next reporting period to accomplish the goals? Selfing Genomic analyses with sequencing data. Publications Training for graduate and postdoc Outreach stakeholders

Impacts
What was accomplished under these goals? Our experiment was conducted at the USDA facilities in Prosser, WA, and in Logan, UT. Each location aimed to develop 100 inbred lines, named WA galaxy (led by CoPD Dr. Longxi Yu) and UT galaxy (led by CoPD Dr. Mike Peel). In parallel, the Corteva alfalfa breeding team also joined the project to develop Corteva Galaxy by contributing data and DNA samples. The Corteva galaxy started with 15 clones and currently maintains 58 inbred lines with three generations of self-pollination. Even with thegovernment shutdown in 2019andthe COVID-19 restrictions in 2020 and 2021, we completed four generations of self-pollination with 4,125 plants self-pollinated. We published a review on alfalfa inbreeding depression. Our experiment is the largest on the number of self-pollination plants. The WA galaxy starts with 114 accessions and UT with 146 breeding lines. The 114 accessions in the WA Galaxy were selected from the alfalfa core collection as the alfalfa diversity association population. The selection was based on the relationship among the 114 accessions to maximize the coverage of genetic diversity. The selection of the 146 breeding lines in the UT galaxy was based on the comments of the grant review panel to emphasize breeding lines. The UT galaxy represents three distinct germplasm pools. The first pool is derived from a type of sativa grown commercially throughout the alfalfa-producing regions of the US. This material has gone through multiple cycles of recurrent selection for persistence under abiotic stress, particularly drought and saline conditions. Its productivity is comparable to commercial alfalfa with dormancy ranging from 2 to 5. The second pool is derived from tetraploid falcata alfalfa, primarily from the Semipalatinsk region of Kazakhstan, and was introduced into the US by N.E. Hansen during the early 20thcentury. The third pool, also falcata, is derived from the Don region of Russia and is distinct from the other two pools. Theoretically, using material genetically distinct from current US commercialized alfalfa increases the potential of identifying distinct heterotic groups. A similar process occurred during the development of US hybrid maize, which largely originated by crossing distinct early flowering Northern flints with late flowering Southern dents. Severe inbreeding depression was observed in the first two cycles, which is consistent with the findings that haploids have the same level of inbreeding depression as diploid on germination and survival. In the WA galaxy, the first generation of SP produced an average of 200 seeds out of selfing per individual, which is about 50-fold less than open pollination (~10,000 seeds per plant).The average seed number decreased 65% from generation 1 to 2. Intensive selections were conducted against inbreeding depression, including planting a large number of seeds and selecting individuals on growth. Consequently, the pace of inbreeding depression was dramatically reduced. Compared to generation 2, the average seed number was decreased by only 7% in generation 3. Currently, generation 4 is in the process of harvesting. We selected 112,626 DNA sequences to design capture probes. Between an array-based capture or an in-solution capture, the latter was selected. These probes are labeled with beads that can be pulled down after hybridization with the sample DNA. After removal of the beads, the fragments that have been pulled down can be sequenced using Next-Generation sequencing platforms. The probe sequences were generated from de novo transcriptome assembly of two alfalfa subspecies, Medicago sativassp.sativa and Medicago sativassp.falcata, using Illumina RNA-seq technology. These transcripts were taken from multiple parts of the plant tissue, such as roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes. We recorded seed production, observed growth vigor, and collected DNA from leaf samples for extreme individuals to map deleterious alleles. The seed production was classified into three categories: high yield, low yield, and no yield. The growth vigor was classified into two categories: strong and weak. Both DNA for individual plants and pooled DNA from ~4 (1 to 9) individual plants within the same categories and same locations were barcoded. CoPD See conducted exome capture sequencing on the Illumina sequencing platform with 10X sequencing depth. Currently, we are in the process of genomic analyses with the sequencing data to identify genetic markers associated with inbreeding depression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 3. Wei Huang, Ping Zheng, Zhenhai Cui, Zhuo Li, Yifeng Gao, Helong Yu, You Tang, Xiaohui Yuan, and Zhiwu Zhang*. MMAP: A Cloud Computing Platform for Mining the Maximum Accuracy of Predicting Phenotypes from Genotypes. Bioinformatics, 2020. https://doi.org/10.1093/bioinformatics/btaa824
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 2. Zhou Tang, Atit Parajuli, Chunpeng James Chen, Yang Hu, Samuel Revolinski, Cesar Augusto Medina, Sen Lin, Zhiwu Zhang*. Validation of UAV-based alfalfa biomass predictability using photogrammetry with fully automatic plot segmentation. Scientific Reports, doi: 10.1038/s41598-021-82797-x.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang Hu, and Zhiwu Zhang*, GridFree: a python package of imageanalysis for interactive grain counting and measuring. Plant, Physiology, 2021, https://doi.org/10.1093/plphys/kiab226


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Alfalfa breeders, geneticist, and farmers Changes/Problems:The first challenge is still maintaining lines. We start with 258 lines at generation 0 and ended up with 215 lines at the first generation, and 210 at the second generation. As there is still substantial variation within lines, we are dividing original lines into the ones producing round seeds and producing wrinkle seeds as sublines. If the wrinkle sublines can produce seeds, we will gain another 100 sublines.The second challenge is to gain more cycles per year. We obtain only one cycle per year partially due to factors such as government shutdown in the first year and COVID19 in the second year. The other factor is the diversity among the original lines makes the long stretch for the What opportunities for training and professional development has the project provided?A graduate student and a postdoc were trained for alfalfa research and proposal development. How have the results been disseminated to communities of interest?We presented our results at the International Conference of Plant and Animal Genome in January 2020. We developed a software package, GRID (http://zzlab.net/GRID), to conduct high throughput phenotyping for alfalfa experiments. The software has been used for plant breeding community. What do you plan to do during the next reporting period to accomplish the goals?Selfing is entering a difficult period. To minimize the loss of lines, we will increase the number of plants per line and split original lines into sublines. With the 100,000 probes developed in the last period, we will genotype the 40 pairs of samples to conduct BSA to map genes related to inbreeding depression.

Impacts
What was accomplished under these goals? We have completed the second generation of selfing on 1400 plants spanning 215 lines. There are 500 plants producing seeds with an average of 70 seeds per plant. The number of remaining lines is 210. Among these lines, we identified 40 lines with paired types of plants (healthy and unhealthy). Leaf samples were collected, and DNA was extracted from the paired plant to map genes controlling inbreeding deficiency using bulked segregation analyses (BSA). We assembled 100,000 biotinylated probes. These probes will be used for hybridization with the BSA DNA samples to extract the probe regions by magnetic pulldown for sequencing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhenhai Cui, H Dong, A Zhang, Y Ruan, S Jiang, Y He, and Zhiwu Zhang*. Denser Markers and Advanced Statistical Method Identified More Genetic Loci Associated with Husk Traits in Maize. Scientific Reports, 2020, https://doi.org/10.1038/s41598-020-65164-0.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: James Chen and Zhiwu Zhang. GRID: A Python Package for Field Plot Phenotyping Using Aerial Images. Remote Sensing, 2020, https://www.mdpi.com/2072-4292/12/11/1697 . IF=4.118 (1, 2, 3, 5, 6).
  • Type: Websites Status: Published Year Published: 2020 Citation: https://zzlab.net/research/alfalfa


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:Alfalfa breeders, geneticist, and farmers Changes/Problems:The biggest challenge is to maintain lines. We start with 258 lines at generation 0 and ended up with 215 lines at generation 1. The missing lines were replanted to produce more seeds. 1. Venomization treatment (4 °C) to increase the germination rate of seeds. 2. Cloning the individuals without enough seeds 3. Add third selection (marker-assisted selection) to purge deleterious alleles. What opportunities for training and professional development has the project provided?A graduate student and a postdoc were trained for alfalfa research and proposal development. How have the results been disseminated to communities of interest?We developed a software package, GridFree (http://zzlab.net/GridFree) to characterize alfalfa seeds through imaging, including the number of seeds. Counting seeds can be conducted with pictures from a cellphone. The software has been used for plant breeding community. What do you plan to do during the next reporting period to accomplish the goals?Currently we are assembling three types of targets. The first type is the reduced genome sequences. The second type is the alfalfa genome reference contigs which includes contigs developed through de novo transcriptome assembly using Illumina RNA-seq technology from two subspecies as Medicago sativa ssp sativa andMedicago sativa ssp falcata. These two subspecies are important parents in USDA-ARS alfalfa breeding programs. The transcripts for the assembly were generated from roots, nitrogen-fixing root nodules, leaves, flowers, elongation stem internodes, and post-elongation stem internodes. The assembled transcripts of 112626 sequences makes the Medicago sativa Gene Index 1.2. The third type is gene exomes that can be assembled by Expressed Sequence Tags (ESTs) of Alfalfa (Medicago sativa). The Ests were collected from public DNA databases that included DNA Data Bank of Japan and National Center for Biotechnological Information. We have downloaded 479,842 EST sequences in total from public databases. The selected sequences will be used to create biotinylated probes. Through hybridization, the regions of interest in sample DNA will be isolated by magnetic pulldown for sequencing.

Impacts
What was accomplished under these goals? A total of 258 lines were selected for selfing to develop inbred lines, including 114 lines from the diverse alfalfa association panel and 144 lines from breeding lines for drought resistance. These lines were planted (diverse lines) or cloned (breeding lines) as generation zero in December of 2018. Each of the 114 diverse lines were planted in seven cells on 98-cell seeding tray. The germination was visible at day 3. At day 44, plants were transplanted to 6-inch pots. Each of the 144 breeding lines, single clone taken for selfing. Bamboo stakes were added to support the standing plants. At the third month, screen cages were applied before flowering. Self-pollination was conducted by tripping flowers. At week 12 during bloom, seed were harvested for each individual plants. There were 101 diverse lines and 114 breeding lines with seed. There are a total of 215 lines remaining. The seed from the diverse lines were counted and the seeds from the breeding lines were weighted. The distributions of seeds counts and weight were similar to each other. The harvested generation 1 (S0) seeds were placed on Petri dishes for germination tests. Multiple selections were enforced to purge deleterious alleles underlying inbreeding depression. The first selection was conducted by choosing the individual with the most seeds in each line. The second selection was based on germination tests. The best germinated seeds were transplanted on 98-well seeding trays. For the lines that had low germination rate, seeds were treated for venomization at 4 °C. The harvest of generation 2 (S1) is expected at end of 2019. ?

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang M., Liu X., Zhou Y., Summers R. M., Zhang Z., 2019 BLINK: A package for the next level of genome-wide association studies with both individuals and markers in the millions. Gigascience: giy154.