DOI: 10.5061/dryad.wdbrv15mb
title: Strategies for the utilization of USDA mungbean germplasm collection for breeding outcomes

description: Global and mid-west U.S. agriculture requires diversification and new
 sources of protein for sustainable crop production. Mungbean as a legume
 crop has a long cultivation history in Asia; however, its potential lays
 hitherto underexplored in the U.S. As a first step towards exploring
 mungbean for crop diversification in northern latitudes, crop germplasm
 centers that harbor worldwide crop resources need to be utilized. This
 study explores the potential of mungbean in the U.S. northern latitudes
 through the utilization of the United States Department of Agriculture
 (USDA) germplasm resources. Complete collection of USDA mungbean germplasm
 was screened under Iowa field conditions in 2017, to shortlist 482
 accessions to create an Iowa mungbean panel. The Iowa mungbean panel was
 further characterized for field adaptability traits in 2018 and 2019 and
 genotyped using genotype-by-sequencing (GBS) to conduct association
 mapping of important traits. Genetic markers were identified for both
 quantitative traits - days to flowering (DTF), plant height (PHT),
 leaf-drop at maturity (LDMS), 100-seed weight (SDWT) and Fusarium wilt
 score (WS), and qualitative traits; seed color (SC), seed-coat texture
 (ST), hypocotyl color (HC), and pod-color (PC). We report FERONIA, a known
 flowering-pathway gene as the candidate gene for the QTL with largest
 effect on DTF. In addition, important epistatic interactions were also
 uncovered for WS and SDWT. Further, accessions with desirable magnitude of
 traits were identified as potential parents. Diversity analyses and field
 phenotypic data indicates potential for mungbean improvement to suit
 mid-western U.S. cultivation.
description: <b>Plant
 material and planting: </b>Mungbean accessions were obtained in 2017
 from the United States Department of Agriculture, Plant Genetic Resources
 Conservation Unit, Griffin Georgia. These accessions were sourced from at
 least 42 countries of the world (Supplementary Table S1). In 2017, ~3000
 accessions of mungbean were planted in an evaluation trial near Ames, IA,
 to begin germplasm evaluation with the intent to make targeted
 hybridizations to initiate a grain legume mungbean breeding program. Ten
 seeds were hand planted on June 6th, 2017 in, single-row plots with
 30" between adjacent rows, 1"1-inch deep furrows with
 seed-to-seed distance at 3". Mungbean plots were observed throughout
 the season, and data was collected on traits such as, emergence, stand
 count, days to flowering (DTF), disease susceptibility and shattering
 tendency. Due to large variations in maturity, plants were hand-harvested
 as plots matured over a period of 2-3 weeks in the Fall season. Soybean
 single-plant belt thresher (ALMACO, Nevada, US) was used to thresh pods.
 Based on DTF and other critical agronomic traits data, 482 mung lines were
 selected for further evaluation in the subsequent year. Supplementary
 Figure S1 shows the geographic distribution of these selected
 accessions. In 2018, due
 to storage issues, seeds were only available for 445 accessions. In 2018,
 these 445 mungbean accessions, were grown at two locations in Iowa near
 Ames (42.0308° N, 93.6319° W) and near Boone (42.0597° N, 93.8802° W) in a
 randomized complete block design (RCBD) with two blocks/reps per location.
 Locations were picked for their proximity to Iowa State University and
 centralized location in the state. Genotypes were mechanically planted
 using ALMACO four-row planter (ALMACO, Nevada, US) at depth of 1", in
 two-row plots (7' long) with 50 seeds per row. Row to row distance
 was 30". In Fall 2018, all accessions were genetically purified in
 the greenhouse using single-seed descent method and sent to winter-nursery
 for seed increase. Increased seeds were brought back in the spring. In
 2019, 482 accessions were grown in Iowa near Ames (42.0308° N, 93.6319°
 W), and Muscatine (41.4245° N, 91.0432° W)
 in randomized complete block design (RCBD) with two
 blocks/reps per location. Genotypes were mechanically planted using ALMACO
 four-row planter (ALMACO, Nevada, US) at depth of 1", in four-row
 plots (7' long) with 25 seeds per. Row to row distance was 15".
 Row to row distance was reduced for weed suppression under low-input
 systems particularly hand weeding to avoid any chemical injury due to
 herbicide on juvenile plants. In-season application of Select Max,
 Basagran, and Ultra-Blazer was used to control latter flushes of weeds. No
 adverse effect of herbicide was noted on the crop plants. <b>DNA
 marker development:</b> A pilot study was used to determine
 appropriate strategy for library creation. In the pilot study, we compared
 double- (<i>Btg1</i>-<i>Taq1</i>) and
 single-digest (<i>ApeK1</i>) restriction fragmentation for GBS
 library creation using 8 mungbean accessions selected on the basis of
 phenotypic diversity. For an average of two million reads-per-sample
 sequencing depth, double-digest libraries yielded 9824 polymorphic loci,
 as compared to 16675 loci yielded by single-digest. Results of pilot
 experiment showed benefits of using <i>ApeK1</i> for
 full-scale, therefore <i>ApeK1</i> was selected for library
 generation with an aim to generate 2.5 million reads per sample. Plant
 leaf tissues for all samples were collected by growing genotypes in the
 growth chamber. In total, 49 duals indexed GBS libraries were generated
 using <i>ApeKI</i>. This number included 482 USDA accessions
 and 17 additional non-USDA mungbean genotypes (commercial varieties and
 improved germplasm obtained from Asian Vegetable Research and Development
 Center, Taiwan). All libraries were combined into a single pool and
 sequenced using 2 lanes of a NovaSeq 1x100-bp run. Approx. ≥1,500M reads
 were generated for the pool. Variants were called using freebayes approach
 (Garrison and Marth, 2012). One genotype was dropped from further analyses
 due to large proportion of missing marker data. <b>Phenotypic data collection: </b> Days to flowering (DTF): First day of flowering was recorded as days-to-flowering. Plant height (PHT): Plant height was measured (in cm) from base of the main stem representing the plot to the top-most node at end-of-the-season. Leaf drop at maturity score (LDMS): LDMS was scored at the end-of-the-season. LDMS was scored on a 1 to 5 scale. Using visual estimation, a score of “1” denoted &gt; 90% leaf-drop; “2” denoted 60-90% leaf-drop; “3” denoted 30-60% leaf-drop; “4” denoted 5-30% leaf-drop; score of “5” denoted that &lt; 5 % leaves had dropped. Fusarium Wilt score (WS): Field data was recorded as wilt score (WS) 1 to 5, ranging from highly resistant (1), resistant (2), moderately resistant (3), susceptible (4), highly susceptible (5). The 1-5 scores represented the aggregate of incidence and severity. Plots were scored as “1” if no plants were found wilted, dead and when observed no stem lesions were found at the soil level; “2” if stem lesions were observed but no wilted or dead plants were present; “3” if stem lesions were observed with few (1-10 %) mildly wilted plants; “4” if both stem lesions and up to 20 % plants showed severe wilting and/or death; “5”, when 20 to 100 % of the plants were dead. 100-seed wt. (SDWT): measured as sample weight of 100 harvested seeds in grams. Qualitative traits scored on the Iowa mungbean panel were: pod color (PC), hypocotyl color (HC), seed color (SC) and seed texture (ST). The seed color was rated on a scale of 1-5, where 1 = green, 2 = yellow, 3= brown, 4 = black, and 5 = speckled. Seed texture was rated on a binary scale of 1 = matt and 2 = shiny, and hypocotyl color was rated as 1= purple and 2= green.